**2. Material and methods**

#### **Study areas**

The study was performed in the model towns of Krompachy and Jelšava that have important emission sources, smelters Kovohuty JSC Krompachy and Slovak Magnesite Works Jelšava (SMZ JSC). Krompachy has been known for its processing of iron and copper ore since the 14th century, and copper has been distributed all over Europe. The magnesite industry in Jelšava has flourished since the end of the 19th century. Soil contamination in both areas is caused by the fallout of solid particles containing various elements during the mining and processing of the ores [16]. More than an estimated 1500 tonnes of solid particles yearly containing accessorial elements (Cd, Cr, Cu, Pb, and Ni) from Kovohuty JSC Krompachy, and as many as 165, 000 tonnes of emissions containing magnesium in total from SMZ JSC were emitted to the atmosphere between the 1960s and the 1990s [17].

#### **Sampling and data analysis**

Soil samples were collected in April 2010 for Krompachy and in May 2009 for Jelšava at four sampling sites each along transects downwind of the emission sources (Figure 1). Four 1 kg replicates (each consisting of four subsamples) were collected from the surface horizons (0-20 cm) of permanent grasslands at each sampling site by quadrat sampling. Each replicate was stored in a plastic bag at 4 °C until analysed. The soil samples were analysed for geochemical properties and nematode community structure.

#### **2.1. Geochemical properties and heavy metals**

The soil samples were processed as follows:


Steps 1-5 were performed by the Laboratory of the Central and Testing Institute in Agriculture in Košice, Slovakia according to certified methods [18].


\* - by Slovak Soil Science and Conservation Research Institute

**Table 1.** Study sites characteristics [1, 53].

**2. Material and methods**

26 Emerging Pollutants in the Environment - Current and Further Implications

**Sampling and data analysis**

at 105 °C [18].

+

**3.** NH4

**4.** NO3 -

The study was performed in the model towns of Krompachy and Jelšava that have important emission sources, smelters Kovohuty JSC Krompachy and Slovak Magnesite Works Jelšava (SMZ JSC). Krompachy has been known for its processing of iron and copper ore since the 14th century, and copper has been distributed all over Europe. The magnesite industry in Jelšava has flourished since the end of the 19th century. Soil contamination in both areas is caused by the fallout of solid particles containing various elements during the mining and processing of the ores [16]. More than an estimated 1500 tonnes of solid particles yearly containing accessorial elements (Cd, Cr, Cu, Pb, and Ni) from Kovohuty JSC Krompachy, and as many as 165, 000 tonnes of emissions containing magnesium in total from SMZ JSC were

Soil samples were collected in April 2010 for Krompachy and in May 2009 for Jelšava at four sampling sites each along transects downwind of the emission sources (Figure 1). Four 1 kg replicates (each consisting of four subsamples) were collected from the surface horizons (0-20 cm) of permanent grasslands at each sampling site by quadrat sampling. Each replicate was stored in a plastic bag at 4 °C until analysed. The soil samples were analysed for geochemical

**1.** Soil moisture was measured gravimetrically by drying the replicates to a constant weight

**6.** The total and mobilisable concentrations of heavy metals (As, Cd, Cu, Cr, Mg, Ni, Pb, and Zn) were determined by inductively coupled plasma mass spectrometry on an Agilent 7500 C (Agilent Technologies, USA) following the manufacturer's instructions. The replicate samples were air-dried before analysis in an oven at 30 °C for 48 h or until constant weight, ground, and sifted through a 0.2-mm sieve. Metals were extracted by 2M HNO3 (10 g of soil in 50 mL of HNO3) for 6 h for total content and by 0.05M Na2EDTA

neutral solution (5 g of soil in 50 mL of Na2EDTA) for mobilisable content [19].

Steps 1-5 were performed by the Laboratory of the Central and Testing Institute in Agriculture

**2.** Organic matter content (Corg) was determined by titration with K2Cr2O7/H2SO4 [18].

was determined spectrophotometrically using Nessler reagent [18].

was determined by an ion-selective electrode [18].

emitted to the atmosphere between the 1960s and the 1990s [17].

properties and nematode community structure.

**2.1. Geochemical properties and heavy metals**

**5.** pH was determined in a solution of CaCl2 [18].

in Košice, Slovakia according to certified methods [18].

The soil samples were processed as follows:

**Study areas**

Loamy Cambisols with intense stony skeletons dominated in both areas. Despite some variations in soil properties, the basic characteristics of the soils were similar (Table 1), with soil moistures ca. 20% and inorganic nitrogen contents (Ninorg.) between 10 and 20 mg kg-1. The Krompachy soils were richer in Corg and were more acidic than the Jelšava soils (soil pH >9).

#### **2.2. Analysis of nematode community structure**

Nematode communities were isolated by extraction from 100 g of each replicate soil sample using a modified Baermann procedure [20]. The extracted nematodes were fixed in Ditlevsen's solution (formalin/acetic acid/alcohol) [21] and were counted using a microscope. All nemat‐ odes in the samples were classified to order, family, and genus. The nematode communities were analysed for (i) total abundance (number of individuals per 100 g of soil), (ii) trophic structure (bacterial feeders; fungal feeders; plant feeders; omnivores; and predators) [22], and (iii) the ecological indices (1) MI2-5 [23] with Nemaplex c-p values, (2) the Shannon-Weaver index (H´) for generic diversity [24], (3) generic richness, expressed as mean number of genera at a site, (4) EI based on the proportion of opportunistic bacterivorous and fungivorous nematodes, (5) SI based on the relative weighted abundance of guilds representing the structure of the food web, and (6) the channel index (CI) [11].


abcd Means followed by the same letters on the same rows are not statistically different according to Least Significant Difference Test (P<0.05)

Limit - limits posted by The Decree of the Ministry of Land Management of the Slovak Republic No. 531/1994-540 on the admissible values of harmful substances in uncontaminated soil. n.a. – not available

**Table 2.** Total and mobilisable concentration of trace elements in sampling sites from Krompachy (mg.kg-1) [1].

#### **2.3. Data analysis**

Spearman's nonparametric correlation coefficient (rs) was calculated using STATISTICA v. 9.0 to test the relationships between the characteristics of a nematode community and the concentrations of mobilisable heavy metals at a site [25]. Correlations at *P*<0.05 and *P*<0.01 were considered significant. Differences in the mean traits and indices of a community amongst sites were tested by Duncan's tests. We used a constrained ordination redundancy analysis (RDA) in CANOCO 5 to analyse the ecological distances between sites (nematode community and soil parameters). The significance of an axis was tested by Monte Carlo permutation [25].

The effects of contamination on soil ecosystems can be categorised as direct or indirect. Alterations in the soil communities near Kovohuty JSC Krompachy were likely due to direct toxicity from the high levels of heavy metals in the soil samples. The contamination acted mostly indirectly near SMZ JSC, altering the basic soil properties. We will present and discuss the results from these two areas separately.

**Figure 2.** Redundancy analysis (RDA) performed on physicochemical soil properties, trace elements and selected indi‐ ces in relation to sampling sites with data explained 73.1 % of the variation in the first two axes; F=10.9; P=0.002 (Krom‐ pachy) [1].
