Individuals of bank (M. glareolus, n = 24) and (M. arvalis, n = 15) common voles were obtained by means of standard teriological methods and procedures from wood ecosystems (Jančová et al. ²⁰⁰⁶) in early spring 2007. The voles were trapped near the coal power station in Nováky (Prievidza district, Slovakia), which is considered to be a strongly polluted region (sources of possible environmental contamination include Nováky chemical plant, coal power station in Nováky, and Handlová–Cígeľ mines) and near the water pond in Kolíňany (Nitra district, Slovakia), which is located approximately 100 km from the town of Nováky and is considered to be a relatively polluted region (sources of possible environmental contamination include Calmit Žirany stone pit and limestone pit, aluminium production at Žiar nad Hronom, factories near the Nitra region, and intensive agricultural production). The coal power station is located west of Nováky, and the wind blows predominantly northwest and west. These two biotopes are separated by the Tribeč mountains (Fig. 1). All animals (males) used in the experiment were adult (4 months old), in good physical condition, and without anatomic pathologic conditions. We compared 14 bank voles from Kolíňany with 10 from Nováky and 8 common voles from Kolíňany with 7 from Nováky. Our research was focused on 78 femora taken from the adult voles. We compared bone length, bone weight, and histological structure of the femora between the animals from different types of environment. Values for macroscopical analysis were expressed as mean ± SD. Unpaired Student t test was used to distinguish possible differences in bone length and bone weight between examined small mammals.

The concentrations of selected heavy metals (Pb, Cd, Ni, Fe, Cu, and Zn) were determined in left femora of investigated mammals (n = 39) with the method of atomic absorption spectrophotometry (Perkin Elmer 4100 ZL) in a graphite furnace (Stawarz et al. ²⁰⁰³). The tissue samples were kept at −18° C until analysis. In the laboratory, the samples were dried at 105°C until dry mass was obtained. Then the bones were weighed (minimum 2 g) and digested in concentrated nitric acid at 90°C for 10 hours. Before analysis, the samples were diluted to 25 ml with distilled water. Detection limits were as follows: Cd = 0.005 ppm, Ni = 0.12 ppm, Fe = 0.02 ppm, Cu = 0.01 ppm, Zn = 0.13 ppm, and Pb = 0.15 ppm. The recovery of the method was 96–98%, and reproducibility was >1.0%. All metal concentrations were expressed on a dry-weight basis in mg kg⁻¹. From the final data, basic statistical characteristics were calculated (mean, SD, minimum, maximum, and median). Because the distribution of observed levels of heavy metals was normal according to Shapiro–Wilk test, parametric Student t test was used for group comparisons employing the Statistica 7.0 software program.

For histological analysis, each right femur (n = 39) was sectioned at the midshaft of its diaphysis. In total, 39 transversal sections of the femur diaphysis were cut. The bone rings were washed in running water for 5 days to remove soft tissues in bone marrow. The bones were then defatted in a mixed solution of chloroform and methanol for 7 days and bleached in 2% H₂O₂ solution for 1 day (Martiniaková et al. ²⁰⁰⁵). After dehydrating through graded alcohols (Martiniaková et al. ²⁰⁰⁶), the samples were embedded in epoxy resin (Biodur; Günter von Hagens, Germany). Transverse thin sections (70–80 μm) were prepared with a sawing microtome (Leitz 1600, Germany), mounted on glass slides with Eukitt (Merck, Germany), and examined by light microscope (Jenaval, Germany) at 225×. The qualitative histological characteristics of the compact bone microstructure were determined according to Enlow and Brown’s (¹⁹⁵⁶) and Ricqlés et al.’s (¹⁹⁹¹) classification systems; the quantitative ones were assessed using the computer software Scion Image (Scion, MD) in anterior, posterior, medial and lateral views of thin section(s). We measured area, perimeter, and the minimum and maximum diameter of primary osteons’ vascular canals. The osteons were outlined using the software Scion Image on a microphotograph at a magnification of 225×. The measured values (n = 135 for M. glareolus and n = 95 for M. arvalis) were expressed as mean ± SD. Unpaired Student t test was used to distinguish differences in the quantitative histological characteristics of compact bone between investigated voles. Correlations between heavy metals and bone structure were determined using the SPSS 8.0 statistical program (SPSS, Chicago, IL).

Concentrations of selected heavy metals (Pb, Cd, Fe, Ni, Cu, and Zn) in the femora of M. glareolus from different types of polluted environments are listed in Table 1. Higher concentrations of Pb, Fe, Cu, and Zn were detected in the bones of bank voles from Kolíňany. A significant difference was observed only for Fe (p < 0.05). In contrast, concentrations of Cd and Ni were higher in the femora of individuals from the area of the power station in Nováky; however, these differences were not statistically significant.

Similarly, we observed higher concentrations of Pb, Fe, Cu, and Zn in bones of M. arvalis from Kolíňany (Table 2). Significant differences were detected for Fe and Zn (p < 0.05). Concentrations of Cd and Ni were also higher, although not significantly so, in individuals from the area of the power station in Nováky.

The measured values for bone weight and bone length were higher in bank and common voles from Nováky. The differences were statistically significant (Table 3).

Histological observation of thin sections of a shaft of femora from investigated voles showed an outer and inner nonvascular lamellar layer around a poorly developed reticular layer containing unorganized vascular canals (Figs. 2, 3). We rarely found several secondary osteons in femoral bone microstructure of bank and common voles, showing that the remodeling process of this rodent species is not as intensive compared with nonrodent mammals. In general, we did not identify demonstrable changes in qualitative histological characteristics of the femora in M. glareolus and M. arvalis (separately) from different biotopes.

With the quantitative histological characteristics, 135 vascular canals of primary osteons and 95 primary osteons’ vascular canals were measured in bank and common voles, respectively. The results are listed in Table 4. We found that all measured values for the vascular canals of primary osteons were higher in both species from Nováky. In contrast, no statistically significant differences were observed.

Correlation analysis in M. glareolus showed a strong positive relation between Cd and Ni (r = 0.52), Pb and bone weight (r = 0.53), Fe and bone weight (r = 0.52), and Fe and size of the perimeter of primary osteons’ vascular canals (r = 0.55). Results of correlation analysis are listed in Table 5. In common voles, a strong positive relation between Fe and Cu (r = 0.60) and between Fe and perimeter size of vascular canals of primary osteons (r = 0.55) was found (Table 6).

There is a significant relation between the amount of toxic elements in soil, water, food, and mammal organs, including bone. The results obtained by Jančová et al. (²⁰⁰⁶) suggest that concentrations of Cu, Fe, and Cd in selected organs (kidney, testis, and uterus) of yellow-necked mouse (Apodemus flavicollis) from Nováky is significantly higher in all cases compared with those from Mochovce (a part relatively clean region in Nitra), respectively. It demonstrates an increase in pollution (mainly in the soil) due to the coal power station in Nováky resulting from mine work and/or from road traffic. According to Roberts and Johnson (¹⁹⁷⁸) and Ieradi et al. (²⁰⁰³), one of the most important sources of environmental contamination from toxic elements is the coal industry. The dust emitted contains Zn, Cu, Pb, and Cd, and this contamination may increase the content of these elements in the tissues of mammals inhabiting polluted areas. In addition, it is known that concentrations of selected toxic elements (e.g., Cd, Pb, Cu, and Zn) in soft tissues and whole-body of M. glareolus are higher compared with Apodemus flavicollis (Sawicka-Kapusta et al. ¹⁹⁸⁷; Metcheva et al. ²⁰⁰¹). Therefore, we supposed that significantly higher concentrations of these elements would also be detected in the femora of M. glareolus from Nováky. Surprisingly, higher concentrations of Pb, Fe, Cu, and Zn were detected in bones of bank voles from Kolíňany, for which Fe concentration differences were significant. Similarly, we observed higher concentrations of Pb, Fe, Cu, and Zn in the femora of common voles trapped at the same biotope, with both Fe and Zn differences being significant. These results can be explained by intensive agricultural production and subsequent contamination of soil, water, and food by road traffic pollution as well as by various factories in industrial zones in Nitra. Intensive agricultural production and the use of chemicals are characteristic for the whole region of Nitra. It is generally known that an application of agricultural chemicals can lead to a higher accumulation of specific elements, including Fe and Zn, into the soil. In addition, there is heavy road traffic near the capture locality, which is also considered to be a significant source of heavy metals that have a potential ability to be transported by air flow over large distances. There is also a possibility of falling dust being transported in the air from large industrial regions, such as Bratislava, Vienna, Budapest, or factories near Nitra. This hypothesis may be supported by studies indicating the possibility of long-range transportation of heavy metals (Coggins et al. ²⁰⁰⁶).

Animals of both vole species trapped at Nováky had higher concentrations of Cd and Ni in their bones. However, the differences were not significant. Our results demonstrate a slightly increased accumulation of other heavy metals in the bones of M. glareolus and M. arvalis from Kolíňany and thus provide evidence of environmental contamination.

We observed higher concentrations of Cd and Zn in the femora of M. glareolus (from both Nováky and Kolíňany) compared with the data reported by Milton et al. (²⁰⁰³). These investigators analyzed Pb (203 ± 13.1 μg g⁻¹ dry weight), Zn (173 ± 5.1 μg g⁻¹ dry weight) and Cd (0.6 ± 13.1 μg g⁻¹ dry weight) concentrations in the femora of bank voles trapped at the contaminated, unused Pb mine at Frongoch in west Wales. Accordingly, the Pb concentration was lower in our study for both biotopes. In contrast, our values (Pb concentrations in the femora of bank voles from Kolíňany and Nováky) were higher than the reference value mentioned by Milton et al. (²⁰⁰³) (Pb concentration 3.2 ± 0.8 μg g⁻¹ dry weight). Concentrations of Cd and Pb were also higher in our study (from both Kolíňany and Nováky) compared with the value reported Milton and Johnson (¹⁹⁹⁹). These investigators analyzed concentrations of Pb (0.99 ± 0.14 μg g⁻¹ dry weight), Zn (261.1 ± 7.4 μg g⁻¹ dry weight), and Cd (0.15 ± 0.04 μg g⁻¹ dry weight) in femora of laboratory-bred bank voles exposed to increased levels of dietary Zn. The Zn concentrations in the femora of bank voles from Kolíňany and Nováky were lower in our study. In contrast, it was not possible to compare our results of heavy-metal concentration in the bones of M. arvalis with published data because of their absence in the literature.

We also found significant changes related to femoral length and weight between M. glareolus and M. arvalis from different types of polluted biotopes. The voles trapped at Nováky had higher values for both of these variables. Because all individuals were adult and of the same sex, and came from similar biotopes, we speculate this fact could indicate a growth-stimulating effect of some heavy metals. Some investigations based on the experimental exposures of individuals to risk elements have shown evidence of associations between body (bone) length and body (bone) weight and increased intake of Pb, Zn, or Cd. According to Osweiler et al. (¹⁹⁸⁵), Pb affects the metabolically active growth centers of long bones in young animals, having a positive effect on bone growth. Supplementation with Zn results in stimulation of skeletal growth and maturation (Moser-Veillon ¹⁹⁹⁵). In addition, it has been demonstrated that long-term accumulation of Cd leads to decreased body weight and weight of some digestive organs in rats and rabbits (Toman et al. ¹⁹⁹⁹, ²⁰⁰²). Our results could be explained by the above-noted findings only partially because femoral length and weight were higher in M. glareolus and M. arvalis with a higher concentration of Cd but not those of Pb and Zn. However, it is known that low levels of the elements taken up from the natural environment may have a somewhat different effect on bone structure than exposure occurring under artificial conditions. These differences may be related to the differences in food quality and availability, the level of toxic elements in the ground, or differences in locomotive activity. According to the study by Velickovic (²⁰⁰⁷), black-striped mice (Apodemus agrarius, rodents) trapped at a contaminated site in Serbia had decreased body size and poorer body condition compared with mice caught in an unpolluted reference site. Decreased body size means that an individual also has decreased bone size. We observed lower values for bone length and weight in bank and common voles from Kolíňany along with higher contents of Pb, Fe, Cu, and Zn in the femora.

Our results from the qualitative histological analysis correspond with the data published by Enlow and Brown (¹⁹⁵⁸) and by Martiniaková et al. (²⁰⁰⁵) for compact bone microstructure of rodents. We rarely found several secondary osteons in our bone samples. It is generally known that aged rats and mice (rodents) lack true Haversian cortical bone remodeling but not cancellous bone remodeling activity (Erben ¹⁹⁹⁶). Therefore, some secondary osteons can be observed in their long bones (near the endosteal border). The presence of several secondary osteons in our voles might also be related to biomechanics, body size, or phylogenetic origin of rodent species. In general, we did not identify demonstrable changes in qualitative histological characteristics between bank and common voles from different polluted biotopes. In addition, histomorphometry showed no statistically significant differences for measured variables of primary osteons’ vascular canals between the animals. Therefore, we suppose that environmental factors (including heavy metals in the environment) influence the macroscopic structure of the bones in M. glareolus and M. arvalis trapped at different types of polluted environments in Slovakia. The voles from Nováky had significantly higher femoral length and weight compared with those from Kolíňany in our study. In contrast, heavy metals did not affect compact bone morphology. According to Havill (²⁰⁰³), bone microstructural morphology is also genetically mediated and provides the direction for further exploration into this phenomenon. Beamer et al. (²⁰⁰¹) mentioned that environmental factors are important contributors to variability in bone microstructure, but genetics still play a substantial role, and estimates of heritability range from 40 to 93%.

It has been reported that essential and nonessential metals may interact with each other, thus affecting uptake, bioaccumulation and toxicity. The results of such interactions are highly variable and range from antagonism to synergism depending on the metal, its external concentration and exposure scenario, length of exposure, species, and specific organs (Norwood et al. ²⁰⁰³; Komjarova and Blust ²⁰⁰⁸). We observed a strong positive relation between Cd and Ni in the femora of bank voles. It is known that Cd as well as Ni and Zn interact actively with Ca channels (Paquin et al. ²⁰⁰²), which is typical for bone cells. According to Komjarova and Blust (²⁰⁰⁸), Cd, Ni, and Zn uptake rates are closely related to each other. A high correlation coefficient of 0.62 between Ni and Cd has also been observed in bones of people working in steel mills (Brodziak-Dopierala et al. ²⁰⁰⁹). In our study, a strong positive interaction between Fe and Cu in the femora of common voles was identified. Cu forms a synergetic system with Fe (Cu–Fe), which advantageously influences the course of enzymatic processes. This interaction (r = 0.83) has been noted in trabecular bone of humans (Brodziak-Dopierala et al. ²⁰⁰⁹). In addition, research by Hisanaga et al. (¹⁹⁸⁸) performed on bone samples taken from human ribs showed interactions between Fe and Cu that were characterized by a high correlation coefficient (r = 0.52). In both vole species, a strong positive relation between Fe and the perimeter size of primary osteons’ vascular canals was found. This indicates that a higher content of Fe in bones significantly influences some variables of vascular canals of primary osteons in bank and common voles.

In conclusion, our contribution is a pilot study about the relation between femoral bone structure and accumulation of some heavy metals in the bones of bank and common voles from different types of polluted environments in Slovakia. Further research in this direction must extend the number of analyzed skeletal elements and verify the results that were obtained from our skeletal samples, especially in other species of small mammals.

This study was supported by grant KEGA 3/7338/09 (Ministry of Education, Slovak Republic). The authors gratefully acknowledge revision of the English text by Kim Dammers (Konyang University).

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