## 2 Material and Methods
### Tooth Sampling and Analysis  
This study was conducted on 69 molar teeth of red deer (_Cervus elaphus_) from \"Zielonka\" Landscape Park (Greater Poland voivodeship, Poland) and 10 molar teeth of roe deer (_Capreolus capreolus_) from forest divisions located in Drawskie Lakeland (West Pomeranian voivodeship, Poland). The animals were culled by hunters as per hunting limits during the hunting season. The age of the animals was identified in the presence of a representative of the Polish Hunting Association on the basis of dental development and condition. In total, 41 teeth of young and 28 teeth of mature roe deer, and 10 teeth of mature red deer were obtained for the analyses (Table 1). The teeth were cleaned of residues, and the material was obtained by separation with a diamond disc on enamel, dentin, and cement. Samples were stored in plastic tubes until further analysis. The tooth tissues were then ground into powder. All samples were transferred to 1.5 mL microtubes and stored in a freezer (\(-\)80 \({}^{\circ}\)C). Then, the samples were analyzed using inductively coupled plasma optical emission spectrometry (ICP-OES, ICAP 7400 Duo, Thermo Scientific, Waltham, MA, USA) equipped with a concentric nebulizer and cyclonic spray chamber to determine Al, Ba, Cd, Fe, K, Li, Mg, Na, Ni, Pb, Sr, Zn. Samples were analyzed in radial and axial modes. The samples were thawed and placed in a laboratory oven (70 \({}^{\circ}\)C). The mass of dental tissue to be analyzed was at least 0.1 g. The samples were transferred to clean polypropylene \begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Species**} & \multicolumn{5}{c}{**The Number of Samples**} \\ \cline{2-5}  & **Total** & **Enamel** & **Dentin** & **Cementum** \\ \hline Young roe deer & 41 & 41 & 15 & 28 \\ (\(<\)1 year) & 28 & 27 & 24 & 28 \\ (\(>\)1 year) & 10 & 9 & 10 & 9 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: The number of molar samples per species. Figure 1: Study area. tubes. Then, 2 mL of 65% HNO3 (Suprapur, Merck, Darmstadt, Germany) was added to each vial, and each sample was then given a 30 min pre-reaction time under a clean cap. After the initial reaction time, 1 mL of unstabilized 30% H\({}_{2}\)O\({}_{2}\) solution (Suprapur, Merck, Darmstadt, Germany) was added to each vial. After the addition of all reagents was completed, the samples were placed in special Teflon vessels and heated in a microwave digester for 35 min at 180 \({}^{\circ}\)C (15 min to 180 \({}^{\circ}\)C and held at 180 \({}^{\circ}\)C for 20 min). At the end of digestion, all samples were removed from the microwave and allowed to cool to room temperature. In a clean fume hood, samples were transferred to 15 mL acid-washed polypropylene sample tubes. Another 80-fold dilution was performed before ICP-OES measurement. A volume of 125 \(\mu\)L was taken from each digestion. An internal standard was added to the samples to give a final concentration of 0.5 mg/L yttrium, 1 mL of 1% Triton (Triton X-100, Sigma, Livonia, MI, USA), and diluted to a final volume of 10 mL with 0.075% nitric acid (Suprapur, Mercka, Darmstadt, Germany). Until analysis, samples were stored in a monitored refrigerator at a nominal temperature of 8 \({}^{\circ}\)C. Preparation of blank samples was performed by adding concentrated nitric acid (80 \(\mu\)L) to tubes without sample and subsequent dilution as described above. Multi-element calibration standards (multi-element ICP IV standard solution, Merck, Al, Ba, Cd, Fe, K, Li, Mg, Na, Ni, Pb, Sr, Zn, were prepared with different concentrations of inorganic elements in the same way as in the blanks and deionized water (Direct Q UV, Millipore, Darmstadt, Germany, approximately 18.0 M\(\Omega\)) was used to prepare all samples. The wavelengths (nm) were Al 396.152, Ba 233.527, Cd 226.502, Fe 259.940, K 766.490, Li 670.784, Mg 285.213, Na 589.592, Ni 231.604, Pb 220.353, Sr 407.771, Zn 213.856. The results were developed with Statistica 13.1.