Heavy Metals in Brown Bears from the Central European Carpathians

âelechovská O. , I . Li terák, S. Ondru‰, Z. Pospí‰ i l : Heavy Metals in Brown Bears from the Central European Carpathians. Acta Vet. Brno 2006, 75: 501-506. The aim of the present study was to assess heavy metal load in the brown bear (Ursus arctos) living in the central European Carpathians. Between 2002 and 2004, fifteen bears were examined to evaluate the distribution of cadmium, lead, mercury and copper in the animals’ muscles (diaphragm), spleen, liver and kidney. The highest content of cadmium, lead and mercury was found in the kidney (17.4 ± 5.2 mg·kg-1, 1.16 ± 0.39 mg·kg-1, 0.39 ± 0.25 mg·kg-1), whereas the lowest content of the metals was observed in the muscles (0.017 ± 0.009 mg·kg-1, 0.299 ± 0.308 mg·kg-1, 0.013 ± 0.011 mg·kg-1). Second highest concentration of cadmium, lead and mercury was detected in the liver (0.83 ± 0.24 mg·kg-1, 0.99 ± 0.61mg·kg1, 0.11 ± 0.05 mg·kg-1). Copper distributions and concentrations in bear tissues were as follows (in descending order): liver (23.9 ± 6.7 mg·kg-1), > kidneys (9.0 ± 3.3 mg·kg-1), > muscles (1.9 ± 1.6 mg·kg-1) and > spleen (1.0 ± 0.2 mg·kg-1). As compared with heavy metal load observed in bear tissues between 1988 and 1990, the concentration of cadmium in the muscles and liver was significantly lower (p < 0.01). On the contrary, mercury values were significantly higher in the liver (p < 0.05), kidney and muscle (p < 0.01). Lead and copper tissue concentrations did not change substantially. Cadmium, Lead, Mercury, Copper, AAS, the Low Tatras Heavy metals are a part of the Earth’s crust and their biosphere incidence, distribution and form are influenced by anthropogenic activities rather than natural processes (Nriagu 1988, 1991; Nriagu and Pacyna 1988). Once released in the biosphere, metals persist for a long time while they may change their forms and dissolve (MacDonald et al. 2000). Immissions caused by burning fossil fuels and waste, car exhausts and residua of widely used pesticides can result in the environment contamination by inorganic and organic compounds. Chemical contamination of the environment impairs ecological balance. Sensitive plant species are affected, large forest areas are destroyed and toxic substances enter the food chain (Yaaqub et al. 1991; Gnamus et al. 1995; 2000). Even an insignificant concentration of these substances can, in the long run, endanger the health of the flora and fauna (Yaaqub et al. 1991; Massanyi et al. 2000ab; Beiglblock et al. 2002; Hell et al. 2005; Massanyi et al. 2005). Some wild animal species, especially predators representing the last component of the food chain, can be seen as bioindicators of environmental contamination by inorganic and organic pollutants (Gufler et al. 1997; Dietz et al. 2000; Hoekstra et al. 2003; MacDonald et al. 2005). One of the top predators with a typical food strategy is the brown bear (Ursus arctos), living in Eurasian and North American areas that have been almost untouched by human activities. The bears of Central Europe live mainly in the Carpathians. Although inorganic and organic tissue contaminants in polar bears from various polar areas have been described relatively sufficiently (Dietz et al. 2000; Muir et al. 1999; Hoekstra et al. 2003; et al. 2001ab), heavy metal tissue concentrations of the brown bear have been studied only sporadically. Heavy metals in brown bear tissues were detected in the Karelian area of ACTA VET. BRNO 2006, 75: 501–506; doi:10.2754/avb200675040501 Address for correspondence: Doc.Ing. Olga âelechovská, Ph.D. Department of Biochemistry, Chemistry and Biophysics Faculty of Veterinary Hygiene and Ecology University of Veterinary and Pharmaceutical Sciences Palackého 1-3, 612 42 Brno, Czech Republic Phone + 420 541 562 606 E-mail: celechovskao@vfu.cz http://www.vfu.cz/acta-vet/actavet.htm Russia (Medvedev 1999) and in the western Carpathians of Slovakia (Chudík and MaÀkovská 1989; Îi l inãar et al. 1992). Heavy metal contamination in the Slovak Carpathians can be influenced by industrial plants affecting the local environment for the past several decades. They are mainly thermal power stations consuming brown coal (Keegan et al. 2006), copper and aluminium works (Wilcke et al. 1998, 1999), or extractive industry (Miadoková et al. 2000). A direct connection between air pollution and contamination of animals was reported in previous studies (Tataruch and Onderschek 1991; Chudík and MaÀkovská 1987). The aim of our study was to determine the content and distribution of cadmium, lead, mercury and copper in tissues of brown bears living in the western Carpathians of central Slovakia between 2002 and 2004, to compare heavy metal concentrations with the values observed in the same location between 1988 and 1990 (Îil inãar et al. 1992), and to comment on the overall dynamism. Materials and Methods We examined tissue samples of 15 brown bears (Ursus arctos) from the orographic area of the Low Tatras mountain range. The Low Tatras, located in central Slovakia, make a central part of the western Carpathians. Tissues were collected from bears that were shot down during regulatory shooting (approved by the Ministry of the Environment of the Slovak Republic) in the autumn of 2002 (6 bears), 2003 (2 bears) and 2004 (7 bears). Sex distribution, body weight and age data are presented in Table 1. Samples of muscle (diaphragm), liver, spleen and kidneys were collected immediately after the shooting (no later than 12 hours after the death). Each sample was stored in a separate plastic bag at -18°C in a freezing box until the analysis was performed. Mercury, cadmium, lead and copper tissue concentrations were estimated. Mercury concentration was determined using dedicated AMA 254 mercury analyzer (Altech Ltd., Czech Republic). Cadmium, lead and copper detection was performed using flameless Atomic absorption spectroscopy (AAS) on Z-5000 appliance (Perkin Elmer Corp, USA). Detection limits of individual elements (3σ) and certified reference materials (SRM 1577b bovine liver, BCR No 184 bovine muscle, BCR No 186 pig kidney) used for the verification of accuracy and suitability of the methods are described in Table 2. Pre-analytic preparation was based on the dissolution of samples by a mixture of nitric acid and hydrogen peroxide in a high-pressure laboratory microwave autoclave Uniclever (Plasmatronika, Poland). Means, standard deviations, Pearson correlation and separate variance t-test were carried out using statistical package Unistat 5 version 5.1. 502 Table 1. Characteristics of examined bears Sex n Weight (kg) Age (year) m ± s.d. min max y ± s.d. F 10 108 ± 26 67.5 158 5.7 ± 1.6 M 5 117 ± 41 53 175 5.7 ± 2.4 Table 2. Limit of detection of method (LOD) and element concentration ± standard deviation in standard reference material of bovine liver (SRM 1577 b) and bovine muscle (BCR No 184). Element LOD SRM 1577b BCR No. 184 Found Certified Found Certified μg·kg-1 mg·kg-1 mg·kg-1 Cd 1.02 0.511 ± 0.039 0.500 ± 0.030 0.012 ± 0.003 0.013 ± 0.002 Pb 9.31 0.133 ± 0.005 0.129 ± 0.004 0.234 ± 0.011 0.239 ± 0.011 Cu 70.4 163 ± 6 160 ± 8 2.30 ± 0.08 2.36 ± 0.06 Hg 0.01* 0.0031± 0.0003 0.0026 ± 0.0006 * Limit of detection of AMA-254 Results and Discussion Mean concentrations in native tissues, standard deviations, medians and concentration ranges of cadmium, lead, mercury and copper in male and female bears are presented in Table 3. Significant differences between the males and females and individual elements (Cd, Pb, Hg, and Cu) were not found. This fact corresponds with the result described by Medvedev (1999). Table 4 shows the distribution of individual elements in various tissues. Metal tissue distribution was assessed as an average representation (in %) of concentration of each element in the native tissue as compared with the tissue showing the highest concentration of the same element in each individual animal (± standard deviation). Kidney basic concentration (100%) was selected for cadmium, lead and mercury; copper distribution was assessed in liver. Cadmium Cadmium showed the most marked, highly significant differences (p < 0.001) in concentration among individual tissues (Tables 3 and 4). The highest cadmium concentration was found in the kidney, the lowest concentration (1000 × lower) was observed in the muscle (diaphragm). Lower concentration was determined in the liver and spleen. Cadmium accumulation in the kidney and liver has been reported also in other mammals (Braune et al 1999; Muir et al. 1999; Kottferová and Koréneková 2000; Beiglbock et al. 2002; Ga‰parik et al. 2004; Kramarová et al. 2005). 503 Table 3. Mean element concentrations (c ± standard deviation), range and median in native tissues of bears Element Tissue n c ± s.d. cmin cmax Median mg·kg-1 Cd muscle 9 0.017 ± 0.009 0.009 – 0.042 0.015 spleen 12 0.072 ± 0.024 0.031 – 0.112 0.073 liver 12 0.829 ± 0.240 0.58 – 1.24 0.782 kidney 7 17.36 ± 5.22 9.08 – 27.32 16.44 Pb muscle 9 0.299 ± 0.301 0.068 – 0.932 0.162 spleen 12 0.165 ± 0.062 0.10 – 0.34 0.142 liver 12 0.990 ± 0.609 0.41 – 2.62 0.767 kidney 7 1.158 ± 0.391 0.65 – 1.73 1.235 Cu muscle 9 1.91 ± 1.57 0.92 – 4.88 1.13 spleen 12 0.97 ± 0.22 0.70 – 1.47 0.88 liver 12 23.9 ± 6.72 12.2 – 39.3 24.3 kidney 7 9.0 ± 3.29 4.62 – 14.01 7.82 Hg muscle 9 0.013 ± 0.011 0.0003 – 0.04 0.010 spleen 12 0.017 ± 0.012 0.001 – 0.025 0.014 liver 12 0.107 ± 0.048 0.031 – 0.172 0.098 kidney 7 0.386 ± 0.250 0.103 – 0.699 0.251 Table 4. Proportional representation of elements in tissues

Heavy metals are a part of the Earth's crust and their biosphere incidence, distribution and form are influenced by anthropogenic activities rather than natural processes (Nriagu 1988(Nriagu , 1991;;Nriagu and Pacyna 1988).Once released in the biosphere, metals persist for a long time while they may change their forms and dissolve (MacDonald et al. 2000).Immissions caused by burning fossil fuels and waste, car exhausts and residua of widely used pesticides can result in the environment contamination by inorganic and organic compounds.Chemical contamination of the environment impairs ecological balance.Sensitive plant species are affected, large forest areas are destroyed and toxic substances enter the food chain (Yaaqub et al. 1991;Gnamus et al. 1995;2000).Even an insignificant concentration of these substances can, in the long run, endanger the health of the flora and fauna (Yaaqub et al. 1991;Massanyi et al. 2000ab;Beiglblock et al. 2002;Hell et al. 2005;Massanyi et al. 2005).Some wild animal species, especially predators representing the last component of the food chain, can be seen as bioindicators of environmental contamination by inorganic and organic pollutants (Gufler et al. 1997;Dietz et al. 2000;Hoekstra et al. 2003;MacDonald et al. 2005).
One of the top predators with a typical food strategy is the brown bear (Ursus arctos), living in Eurasian and North American areas that have been almost untouched by human activities.The bears of Central Europe live mainly in the Carpathians.Although inorganic and organic tissue contaminants in polar bears from various polar areas have been described relatively sufficiently (Dietz et al. 2000;Muir et al. 1999;Hoekstra et al. 2003;et al. 2001ab), heavy metal tissue concentrations of the brown bear have been studied only sporadically.Heavy metals in brown bear tissues were detected in the Karelian area of Russia (Medvedev 1999) and in the western Carpathians of Slovakia (Chudík and MaÀkovská 1989;Îilinãar et al. 1992).Heavy metal contamination in the Slovak Carpathians can be influenced by industrial plants affecting the local environment for the past several decades.They are mainly thermal power stations consuming brown coal (Keegan et al. 2006), copper and aluminium works (W ilcke et al. 1998, 1999), or extractive industry (Miadoková et al. 2000).A direct connection between air pollution and contamination of animals was reported in previous studies (Tataruch and Onderschek 1991;Chudík and MaÀkovská 1987).
The aim of our study was to determine the content and distribution of cadmium, lead, mercury and copper in tissues of brown bears living in the western Carpathians of central Slovakia between 2002 and 2004, to compare heavy metal concentrations with the values observed in the same location between 1988 and 1990 (Îilinãar et al. 1992), and to comment on the overall dynamism.

Materials and Methods
We examined tissue samples of 15 brown bears (Ursus arctos) from the orographic area of the Low Tatras mountain range.The Low Tatras, located in central Slovakia, make a central part of the western Carpathians.Tissues were collected from bears that were shot down during regulatory shooting (approved by the Ministry of the Environment of the Slovak Republic) in the autumn of 2002 (6 bears), 2003 (2 bears) and 2004 (7 bears).Sex distribution, body weight and age data are presented in Table 1.Samples of muscle (diaphragm), liver, spleen and kidneys were collected immediately after the shooting (no later than 12 hours after the death).Each sample was stored in a separate plastic bag at -18°C in a freezing box until the analysis was performed.
Mercury, cadmium, lead and copper tissue concentrations were estimated.Mercury concentration was determined using dedicated AMA 254 mercury analyzer (Altech Ltd., Czech Republic).Cadmium, lead and copper detection was performed using flameless Atomic absorption spectroscopy (AAS) on Z-5000 appliance (Perkin Elmer Corp, USA).Detection limits of individual elements (3σ) and certified reference materials (SRM 1577bbovine liver, BCR No 184 -bovine muscle, BCR No 186 -pig kidney) used for the verification of accuracy and suitability of the methods are described in Table 2. Pre-analytic preparation was based on the dissolution of samples by a mixture of nitric acid and hydrogen peroxide in a high-pressure laboratory microwave autoclave Uniclever (Plasmatronika, Poland).Means, standard deviations, Pearson correlation and separate variance t-test were carried out using statistical package Unistat 5 version 5.1.

Results and Discussion
Mean concentrations in native tissues, standard deviations, medians and concentration ranges of cadmium, lead, mercury and copper in male and female bears are presented in Table 3. Significant differences between the males and females and individual elements (Cd, Pb, Hg, and Cu) were not found.This fact corresponds with the result described by Medvedev (1999).
Table 4 shows the distribution of individual elements in various tissues.Metal tissue distribution was assessed as an average representation (in %) of concentration of each element in the native tissue as compared with the tissue showing the highest concentration of the same element in each individual animal (± standard deviation).Kidney basic concentration (100%) was selected for cadmium, lead and mercury; copper distribution was assessed in liver.

Cadmium
Cadmium showed the most marked, highly significant differences (p < 0.001) in concentration among individual tissues (Tables 3 and 4).The highest cadmium concentration was found in the kidney, the lowest concentration (1000 × lower) was observed in the muscle (diaphragm).Lower concentration was determined in the liver and spleen.Cadmium accumulation in the kidney and liver has been reported also in other mammals (Braune et al 1999;Muir et al. 1999;Kottferová and Koréneková 2000;Beiglbock et al. 2002;Ga‰parik et al. 2004;Kramarová et al. 2005).Cadmium concentrations determined in bear kidneys shot down between 2002 and 2004 were not statistically different from values described in bears examined between 1988 and 1990 (Îilinãar et al. 1992).However, the cadmium content in the liver and muscle (diaphragm) was significantly lower (p < 0.01).

Lead
The highest lead concentrations were found in the kidney.Concentration values in the liver were not significantly different from the kidney values.Highly significant difference in lead concentrations was observed between the kidney and muscle (diaphragm) (p < 0.001), kidney and spleen (p < 0.001), liver and muscle (diaphragm) (p < 0.005), and liver and spleen (p < 0.001).We found a significant correlation between the liver and kidney (r = 0.70, p < 0.05).
Changes of lead concentration in the muscle (diaphragm), liver and kidney as compared to values reported between 1988 and 1990 were not significant.

Mercury
A correlation between the body weight and mercury content was found in the kidney (r = 0.89, p < 0.01), which is in accordance with the cumulative function of mercury in the organism.Mercury cumulated in the tissues in the following order: kidney > liver > spleen > muscle (diaphragm).A significant difference in the mercury concentration was observed between diaphragm and spleen (p < 0.05) and liver and kidney (p < 0.05), while a highly significant difference was determined between the muscle (diaphragm) and liver, spleen and liver, muscle (diaphragm) and liver (p < 0.001), and spleen and kidney (p < 0.01).
It should be emphasized that mercury concentration in the liver was significantly higher (p < 0.05) compared to values determined between 1988 and 1990; the difference between the kidney and diaphragm was even higher (p < 0.01).

Copper
A significant correlation between the copper concentration in the spleen and the age of bears (r = 0.88, p < 0.01) was estimated.Copper is an essential element that is a part of many enzymes.The highest concentrations were observed in the liver, followed by the kidney, spleen and muscle (diaphragm), respectively.Highly significant concentration differences were observed between the liver and spleen, spleen and kidney, liver and kidney, muscle (diaphragm) and liver (p < 0.001), and kidney and muscle (diaphragm) (p < 0.005).Copper concentration in the spleen was not significantly different from the muscle (diaphragm) values.
The copper content in the kidney and muscle (diaphragm) was identical with the values published by Îilinãar et al. (1992).The concentration in the liver was significantly higher (p < 0.01) compared to Îilinãar et al. (1992) but identical with values reported by Medvedev (1999).
It is alarming that cadmium and lead concentrations determined in the kidney and liver of examined bears are still too high, mercury levels have even increased compared to values determined between 1988 and 1990.Relatively high concentrations of toxic elements were found also in tissues of foxes, boar (Piskorová et al. 2003) anddeer (Ga‰parik et al. 2004).However, these levels are lower than those observed in bears and are in accordance with the assumption that the heavy metal load of omnivores is higher than that of ruminants living in the same area (Chudík and MaÀkovská 1987).Although emissions of toxic elements have been decreasing since 1990 (Keegan et al. 2006), they are more acid and the mean soil acidification (Schwartz et al. 1999) allows for easier infiltration of certain metals into the soil extract and thus into plants.Thus metals become a part of the food chain of bears and other animals.This is also supported by recent reports on high heavy metal concentrations in mushrooms growing in the Spi‰ area, Slovakia (Zimmermannová et al. 2001).Indeed, an increasing synanthropy of the western Carpathians bears can affect the heavy metal content in their tissues (Langgemacht et al. 1995).

Table 1 .
Characteristics of examined bears

Table 2 .
Limit of detection of method (LOD) and element concentration ± standard deviation in standard reference material of bovine liver (SRM 1577 b) and bovine muscle (BCR No 184).
* Limit of detection of AMA-254

Table 3 .
Mean element concentrations (c ± standard deviation), range and median in native tissues of bears