THE INCIDENCE OF HEAVY METALS AND OTHER TOXIC ELEMENTS IN ROE DEER (Capreolus capreolus) TISSUES

Levels of lead (Pb), cadmium (Cd), arsenic (As), mercury (Hg) and copper (Cu) in the liver, kidney and muscle of 11 individual roe deer (Capreolus capreolus) were determined. Th e samples were prepared by microwave wet digestion. Content of investigated elements was determined by the method of coupled plasma with mass spectometry. Th e lead concentrations ranged from <0.001 (liver) to 8.455 mg/kg (meat), Cd concentrations ranged from 0.004 (muscle) to 0.818 mg/kg (kidney) and As concentrations ranged from 0.002 (liver) to 0.031 mg/kg (kidney). Concentrations of Hg in examined tissues (liver, kidney, muscle) were under limit of detection (<0.001 mg/kg). Th e concentration of copper in liver ranged from 3.913 to 104.08 mg/kg. Th e results of this study showed that no samples exceeded maximum allowed levels for Cd, Hg, As and Cu. Pb concentrations in muscle samples ranged from 0.008 to 8.455 mg/kg. High concentrations of Pb in two muscle samples are most likely due to the proximity of hunting wound area, as lead was not detected in organ samples. Th e presence of some elements in the tissues of roe deer suggests the necessity of further research aimed at identifying the source of contamination in order to preserve the health of both humans and animals.


INTRODUCTION
Contamination of the environment with hazardous compounds and elements of anthropogenic origin is of increasing concern because of its eff ect on the entire biosphere, i.e., the micro-fl ora and -fauna of soils, plants and higher life, including humans and animals (Selenius et al., 1996).Many wild animals are exposed to diverse toxic substances by consuming contaminated plants and animals, or water, soil and air (Živkov Baloš et al., 2015).Th e accumulation of toxic heavy metals in plants and soil may increase the risk of transfer to herbivorous wild mammals and game animals or to livestock (Bilandžić et al., 2010).As animals can move freely and fi nd their own food, the game is a link in the chain that accumulates pollutants from the environment.Monitors have recently been defi ned as organisms in which changes in known characteristics can be measured to assess the extent of environmental contamination, so that conclusions on the health implication for other species of the environment as a whole can be drawn.Monitors may provide information about environmental concentrations of essential and toxic metals of importance for life, displaying defi ciency and toxicity, respectively (Selinus et al., 1996).
In order to fully understand the exposure of animals to many pollutants originating from the environment and to assess the harmful eff ect and estimate the risk, it is necessary to carry out a systematic study and gather data on degree and type of pollution, as well as distribution of hazardous chemicals in nature.Nowadays, a number of studies have been based on the determination of chemical contaminants in animal tissues and organs.As the result of these fi ndings, it is possible to estimate the level of human exposure to negative eff ects of these pollutants.Th e monitoring and control of game meat safety should include control measures for live animals, control measures during hunting and aft er shooting, guidelines for offi cial meat inspection, control measures for carcass processing and surveillance of chemical residues (Petrović et al., 2014).
Since it fulfi ls numerous criteria (e.g.widely geographic distribution, relatively small home range, territorial living and browsing nutrition strategy, huge availability of basic data, relatively simple sampling procedure) roe deer has been oft en mentioned in the literature as a good or even excellent monitor of toxic elements burdens on the environment (Pokorny, 2000).
Th e purpose of this study was to evaluate the concentration of environmental contaminants lead (Pb), arsenic (As), mercury (Hg), cadmium (Cd) and copper (Cu) in tissues of free-living roe deer in Serbia, as important information in performing assessments of the risk for both wildlife and humans.Th e obtained results were compared with relevant data reported from other countries.

MATERIAL AND METHODS
Samples of liver, kidney and meat of roe deer (Capreolus capreolus) shot by hunters were collected from hunting ground of Begeč settlement, municipality Novi Sad.Sampling of wild animal organs was performed during the 2013/2014 hunting season.Animals were selected according to neither sex nor age.Th us, liver and kidney samples were collected from each animal (total of 11 animals).Upon collection, all samples were placed into labeled plastic bags and stored at -18° C to avoid tissue degradation prior to analysis.
Th e samples (1g) were prepared applying the microwave (Ethos, Labstation Microwave, Milestone), digestion method (14) with the use of the mixture H 2 O 2 /HNO 3 (1:4, v/v).Aft er this process, the samples were transferred to 50 mL volumetric fl asks and diluted with deionized water.Analyses of Pb (NoG-M, IT 0.1 s/P), Cd (NoG-M, IT 1 s/P), As (He-M, IT 1 s/P), Hg (NoG-M, IT 1 s/P) and Cu (He-M, IT 0.1 s/P) were conducted by ICP-MS 7700 mass spectrometer (Agilent Technologies).Solutions used for calibration were prepared from commercial stock standard solutions with 1000 mg/l of each element (Accustandard).To calculate the recovery percentage, 6 samples of meat have been spiked with known amounts of Cd, As, Hg, Pb and Cu analytical standards.Th e obtained results are presented in Table 1.

RESULTS AND DISCUSSION
Average values of toxic and trace elements obtained in this study for the livers, kidneys and meat of all investigated roe deer are summarized in Table 2. Th e obtained values were compared with highest permissible hygienic limits for risk elements according to the maximum allowed levels (MAL) of particular contaminant in food in the Republic of Serbia (Offi cial Gazette, 2011).
Th e results were compared with the results reported by other authors from our and other countries.Beside that comparison, a collation with some other countries could be interesting as well.An overview of some previous articles addressing toxic elements levels in tissues of free living roe deer (Capreolus capreolus) and red deer (Cervus elaphus) is presented in Table 3. Concentrations of As, Cd and Hg in liver, kidney and muscle samples of roe deer did not exceed the MAL in either of the examined samples (Table 2).
Th e highest Cd-contamination (average value 0.465 mg/kg) was recorded in the kidneys of roe deer.Somewhat lower Cd levels were found in the liver (0.0527 mg/kg), while muscles were the least contaminated (0.005 mg/kg).Th e lowest and highest average concentrations of As were measured in liver (average value 0.008 mg/kg) and kidney samples (0.031 mg/kg), respectively.Mercury (Hg) was not detected in any of the investigated samples.Kidneys of roe deer revealed higher Pb-contamination than liver (average values 0.094 mg/kg in kidney and 0.077 mg/kg in liver).Th e Pb content in two muscle samples of roe deer was very high.High Pb concentrations measured in muscle samples are probably due to the proximity of gunshot wound, especially since such enormously high level of Pb in the examined muscle sample show a strong discrepancy with respect to Pb values in the liver and kidney (lead was not detected in samples of these organs).Th e maximum permissible level for Cu is prescribed only for liver (80 mg/kg).Th e average Cu level detected in the liver was 28.071±34.026mg/kg, while the value exceeding MAL (104.08 mg/ kg) was recorded in only one sample.Similar results for Cd and Pb distribution were obtained by other authors, but our results for Cd concentrations are markedly lower as compared with other investigations (Table 3).Since the age was not taken into account in data interpretation in the majority of cited studies (Table 3), the levels of toxic element could not be directly compared with our research.Secondly, the bioindicative approach was emphasized in our investigation, but many of cited studies were chosen in the vicinity of local pollution sources (smelters, industrial and mining areas).Finally, some diff erences between species must be outlined.Red deer (Cervus elaphus) whose anatomy of the digestive tract is adjusted to the intake of wide spectrum of feed, receives more wood species (35% of the feed in summer, 30% of the feed amount in winter).Roe deer whose digestive tract is only poorly adjusted to the digestion of cellulose, prefers young summer plants, herbs, fruits (they have narrow leaves and retain less falling particles) but more wide-leaved feed (herbs, summer plants which retain more deposited pollutants) than the red deer.Th is fact is likely to be the main reason of the inter-species diff erences (Kottferová and Koréneková B., 1998).

CONLUSIONS
Average as well as maximum concentrations of toxic elements in the majority tissue samples examined during our investigation did not exceed the maximum permissible levels and the levels measured in tissues of European free-living roe deer.Th e levels of investigated toxic elements are well below the concentrations considered dangerous for animal's health.However, high concentrations of some toxic elements still confi rm the imperative of imposing relevant control program that will include veterinary offi cials as well as hunters and other subjects involved in game meat chain.

Table 1 :
Isotopes, limit of detection (LOD) and recovery rates for monitored elements Statistical analysis was performed by the STATISTICA 12 soft ware package, version 16.0.Data were grouped according to tissue and presented as mean ± standard error, minimum and maximum values.

Table 2 .
Toxic elements concentrations (mg/kg) in diff erent tissues of roe deer

Table 3 .
Content of toxic elements (mg/kg) in diff erent tissues of roe deer and red deer according to various authors Legend: n.i.-not investigated; *(dry weight)