Black chernozem was used as an object of the study. These soils are considered to be the most fertile ones in the world. The soil for model studies was selected from the virgin soil top layer (0–10 cm) on the territory of the Persianovskaya Steppe nature monument (Russia, Rostov Region, Persianovskiy, 47°30'33.23"N, 40°9'18.21"E).
The investigated chernozem was characterized by high humus content in the upper horizon − 4.0%, neutral reaction of the medium - pH 7.6, heavy-loamy granulometric composition, good condition, high biological activity: total bacterial count − 3.5 bln/g of soil, catalase activity − 8.4 ml O2/g of soil for 1 min, dehydrogenase activity − 14.5 mg SSF/10 g of soil for 24 hours, an abundance of Azotobacter bacteria − 100% of the fouling lumps.
Pollution of chernozem with thallium was simulated in laboratory conditions. Thallium was introduced into the soil in the form of thallium oxide (III) — Tl2O3. Tl (III) was used since it was more toxic than Tl (I) (Ralph, Twiss, 2002; Lan, Lin, 2005). The use of oxides eliminates the influence of the accompanying anions on soil properties, as it occurs at introducing metal salts.
Thallium oxide (III) is a water insoluble compound. To uniformly distribute thallium in the soil, thallium oxide was first ground with a small volume of soil and then mixed with the rest of the soil. After that, the soil was moistened with water.
The content of thallium in the soil is usually up to 1 ppm (from 0.01 to 3.0 ppm) (Fergusson, 1990; Hofer et al., 1990; Chen et al., 1991; Tremel et al., 1997; Il'in, Konarbaeva, 2000; Salminen, 2005; Martínez-Sanchez et al., 2009; Nygard et al., 2012; Stafilov et al., 2013; Alekseenko, Alekseenko, 2013). However, there can also be higher content of thallium in the soil: 5–15 ppm in the pyrite deposit areas (Yang et al., 2005), 40–124 ppm in the thallium sulfide deposits areas (Xiao et al., 2004b), and up to 20,000 ppm near coal mines (Baceva et al., 2014).
The content of thallium in chernozem, used in the study, before pollution is 0,98 ppm (the thallium content in the soil was determined by inductively coupled plasma mass spectrometry using an ELAN-DRC-e instrument). Accordingly, NAC was set to 3 ppm. Thallium was introduced into the soil in the amount of 3, 30 and 300 ppm. It was assumed that the metal toxicity was manifested with its concentrations in the soil from 3 backgrounds (Kolesnikov et al., 2010).
Soil (1 kg) was incubated in plastic vessels in triplicates at room temperature (20–22 °C) and optimal moistening (60% of water field capacity).
The biological activity of the soil was studied since it reacted first to external influences. It was much more sensitive and informative than other chemical and, especially, physical soil properties (Kolesnikov et al., 2000).
The biological activity of chernozem was determined 10, 30 and 90 days after the contamination.
After this period, the entire mass of the soil was removed from the vegetation vessel and mixed, thereby obtaining an "average sample" from which samples were taken to determine biological indices − 3 samples from each vessel.
Laboratory-analytical studies were performed using conventional methods. The total bacterial count, the abundance of the Azotobacter bacteria, the activity of catalase and dehydrogenases, and phytotoxic properties of soil were determined. The total bacterial count in the soil was taken into account using luminescent microscopy (n = 720: 3 incubation vessels with soil x 3 soil samples x 4 square centimetres on slides x 20 fields of vision), Azotobacter - by the lumps fouling in the Ashby medium (n = 241: 3 incubation vessels with soil x 3 soil samples in Petri dishes x 25 fouling lumps), the catalase activity was determined by the decomposition rate of hydrogen peroxide (n = 36: 3 incubation vessels with soil x 3 soil samples x 4 analytical replicates), dehydrogenase activity - by the rate of conversion of triphenyltetrazolium chloride into triphenylformazan (n = 36: 3 incubation vessels with soil x 3 soil samples x 4 analytical replicates), and soil phytotoxicity was judged by the germination of radish seeds (n = 241: 3 incubation vessels with soil x 3 soil samples in Petri dishes x 25 radish seeds).
The choice of biological indicators is due to the following reasons. The total bacterial count in the soil characterizes the state of the decomposers in the ecosystem. The Azotobacter bacteria are traditionally used as an indicator of chemical soil contamination. The activity of catalase and dehydrogenases reflects the intensity of mineralization processes in the soil. Among enzymes, oxidoreductases are the most sensitive to chemical contamination. Enzyme activity is an indicator of the potential biological activity of the soil, and the decomposition rate of the bed characterizes the actual biological activity of the soil.
Based on the above biological indicators, the integral biological state indicator (IBSI) of the soil was determined (Kolesnikov et al., 2000). The presented set of indicators gives an informative picture of the biological processes taking place in the soil and its ecological state.
To calculate IBSI, the value of each of the above indicators for control (in the unpolluted soil) was taken as 100%, and the values in the remaining experiment options were expressed in relation to it as a percentage (in the contaminated soil). Then, the average value of the five selected indicators for each experiment option was determined. The value obtained (IBSI) was expressed as a percentage of the control (to 100%). The technique used allowed integrating the relative values of different indicators, the absolute values of which could not be integrated since they had different units of measurement.
To verify the reliability of the data obtained, the variance analysis was carried out, followed by the determination of the least significant difference (LSD).