**2. Materials and methods**

#### **2.1 Study area**

The research area is located in the Tatra National Park (**Figure 1**). The park covers an area of 211.64 km<sup>2</sup> , of which 149.84 km<sup>2</sup> is under strict protection, 34.69 km<sup>2</sup> is under active protection, and 27.14 km<sup>2</sup> under landscape protection. In 2019, the TNP was visited by 3.9 million people. The Tatras are the only alpine mountains in Poland, where mainly alpine relief is protected, as well as valuable species of plants and animals (including endemics and relics). The research area is located in the Polish part of the Central Western Carpathians, in the northern part of the Tatra Range macroregion [15] and it is the highest part of the entire Carpathians. The specificity of this area is the complex geological structure [16–18], land relief heterogeneity (fluvial-denudation, karst, and glacial) [19–21], climatic conditions changing with the increase in altitude above sea level (air temperature, total precipitation, etc.). The specificity of the climate of the Tatra Range is determined by the incidence of different air masses. Arctic maritime air masses (PPm) have the largest share in the formation of weather, i.e., 65% of days a year, while continental polar air masses (PPk) approximately 20% of days a year [22, 23]. The above elements determine the specificity of water circulation (spatially diversified possibility of water retention, the volume of runoff, water chemistry, etc.). The soil cover of the Tatra Mountains is strongly related to, among other features, their geological substrate, morphogenetic processes, and climatic conditions, and its

#### **Figure 1.**

*Location of the study area on the background of the map of Poland and Tatra National Parks.*

characteristic feature is openwork, as well as poorly developed soils (i.e., initial soils) [24]. All the physico-geographical zones, characteristic of high mountain areas, have developed in the Tatra Mountains [25]. Two test areas in the Tatra National Park in Poland, on the northern slope of the Tatra Mountains, were selected for the study. These areas were selected owing to the diversity of the natural environment, including the physico-geographical location, landscape zone, and geological structure. The test areas were given working names—Kasprowy Wierch (KW) and Morskie Oko (MO).

#### *2.1.1 Kasprowy Wierch*

The test area covers two physico-geographical mesoregions, i.e., the Reglowe Tatras (sampling points 1–4) and the Western Tatras (sampling point 5) [26], and ranges from the forest level to the alpine level (**Table 1**). The geological structure is strongly diversified in terms of lithology and tectonics. This affects, among other things, the incompatibility of the topographic watershed with the underground watershed. The area belongs to the Bystra catchment (with the sub-catchment of the Potok Jaworzynka) and the Sucha Woda Gąsienicowa catchment, which is part of the Dunajec basin. Depending on the altitude, the mean annual air temperature ranges from 0–6°C [27], the annual total of precipitation ranges from 800 mm to 1,800 mm, and the length of the snow cover deposition ranges from 100 to 200 days a year [28]. The soil cover is varied and dominated by the following soils:


#### Polytrichum formosum *and* Vaccinium myrtillus *as Phytoindicators of Pollutants… DOI: http://dx.doi.org/10.5772/intechopen.99962*

**Table 1.**

 *Characteristics of sampling points in the Kasprowy Wierch (KW) test area - samples No. 1–5, and the Lake Morskie Oko (MO) test area*

 *- samples No. 6–10.*

Fluvisols, Rendzic Leptosols, Folic Rendzic Leptosols, Cambic Rendzic Leptosols, Haplic Cambisols (Eutric), Haplic Podzols (Skeletic), Entic Podzols, Leptic Podzols, and Folic Leptosols [29].

#### *2.1.2 Morskie Oko*

The area is located within the High Tatras, in the Białka catchment (the Dunajec river basin) drained by the Rybi Potok, the Roztoka, and the Białka (**Table 1**). With regard to the zonation of the environment, it is entirely located within the forest level. It is part of one of the largest post-glacial grooves in the Tatras (a U-shaped valley). Depending on the altitude, the mean annual air temperature ranges from 2–4°C [27], the annual total of precipitation ranges from 1,000 mm to 1,400 mm, and the length of snow cover deposition ranges from 120 to 160 days a year [28]. The dominant soils in this part are, among others: Haplic Podzols (Skeletic), Haplic Cambisols (Dystric, Skeletic), Lithic Leptosols, and Regosols (Hyperskeletic) [29].

#### **2.2 Sampling and analysis**

#### *2.2.1 Sampling*

Plant samples (two species: the moss *Polytrichum formosum* Hedw. (green parts) and the European blueberry *Vaccinium myrtillus* L. (leaves) were sampled in the area of the Tatra National Park, from the Kasprowy Wierch (KW) test area and the Lake Morskie Oko (MO) test area. The samples were taken every 100 meters of altitude, starting from an altitude of: 1,100 m above sea level for KW and from 1,000 m above sea level for MO. Owing to the limited range of occurrence at higher altitudes, the plants were sampled up to 1,550 m above sea level for KW. The geographical coordinates of the sampling sites and the designations adopted are presented in **Table 1**.

#### *2.2.2 Chemical analysis*

According to the suggestions of the following authors: Maňkovska et al. [30] and Sawidis et al. [31] regarding the sample preparation procedure, the plant material was left unwashed. The samples were dried in an electric drier at a temperature of 400°C for 72 h. Needles were separated from branches. Equal amounts of biomass from primary samples from the same plot were combined. Dry and homogenized samples were pulverized in an electric grinder. Portions of 1 g dry weight material were placed in Teflon vessels. 5 cm3 of 65% HNO3 and 3 cm3 of 36% H2O2 were added to each vessel. The mixture was mineralized in a Berghof Speed Wave microwave at a temperature of 200°C and at a pressure of 4 MPa. After processing, the samples were diluted with deionized water to a total volume of 50 cm3 and filtered through a hard paper filter. The final solutions were analyzed for heavy metals (Cd, Cr, Cu, Ni, Pb and Zn) using the inductively coupled plasma mass spectrometry (ICP-MS) method in the Bureau Veritas laboratory. Such standards and reference materials (for plants) were used. The detection limits (μg/g dm) were as follows: for Cd: 0.01, Cr: 0.1, Cu: 0.01, Ni: 0.1, Pb: 0.01 and Zn: 0.1.

#### *2.2.3 Statistical study*

Statistical analyses were performed using the IBM SPSS program. Owing to the different conditions and differences in the altitude of sampling, the analysis was carried out in two groups depending on the location of the tests (MO Lake Morskie Polytrichum formosum *and* Vaccinium myrtillus *as Phytoindicators of Pollutants… DOI: http://dx.doi.org/10.5772/intechopen.99962*

Oko and KW Kasprowy Wierch). In order to assess the compliance of the distributions with the normal distribution, the Kolmogorov-Smirnow tests were performed. As the distributions differed from the normal distribution, non-parametric methods were used for further analyses. Spearman's coefficients were used to assess the relationship between the variables. In order to assess the significance of differences between the two groups, descriptive statistics were calculated and Mann–Whitney tests were performed. P = 0.05 was assumed as the limit of statistical significance below which the results were considered significant.
