Ecological and Functional Properties of Steppe Soils under Moderate Anthropogenic Impact

Steppe soils of a small industrialized city with moderate anthropogenic impact for example Krasny Kut, Saratov region were analysed to ascertain their ecological and functional state. In the course of this work, the concentration of heavy metals (Zn, Cu, Pb, Ni, Cr and Cd) was determined in the soil samples, including the hazard coefficient (Kо) and the total contamination coefficient (Zc). Magnetic susceptibility, magnetic coefficient (Kmag), thermomagnetic effect (dk) of the soil samples were analysed together with the activity of soil enzymes (dehydrogenases, catalases, peroxidases and invertases). Using ecological and geochemical analytical methods, a widespread excess of maximum permissible concentration (MPC) of mobile forms Ni, Pb, Cu and Zn was recorded in the soil samples of Krasny Kut, and a single excess of MPC was observed for Cr and Cd. According to Zc indicator values, 4 samples were classified as soils with moderately dangerous levels of contamination and 2 samples with dangerous levels of contamination. Using petromagnetic analysis, a few samples were observed to contain a moderate amount of introduced technogenic magnetic particles and one sample with a hazardous amount of introduced technogenic magnetic particles. Medium, high and very high levels of dehydrogenase, catalase, peroxidase and invertase activities were recorded in the soil samples, indicating the absence of ecotoxicants inhibiting the enzymes. The observed peculiarities in the ecological and functional state of soils, representative of the steppe zone of the Eastern part of the European territory of Russia will be required for monitoring, reducing and forecasting the anthropogenic burden on soil ecosystems.


Introduction
Due to an exponential growth in industrial production and an increase in anthropogenic impact on the environment, researches on the assessment of soil contamination with dangerous xenobiotics, primarily heavy metals (HMs) and hydrocarbons (HCs), which have mutagenic, teratogenic and carcinogenic properties and pose an immediate threat to human health, have become particularly relevant in recent decades [1] [2] [3] [4]. Problems of soil contamination with HMs and HCs in large cities are given much attention both in Russia [5] [6] and in other countries [4] [7]. For a comprehensive assessment of the integral impact of HMs and HCs on abiotic and biotic components of urban soils, comprehensive studies of ecological, functional, biological, geochemical and physical-chemical properties of soils, including magnetic properties have to be conducted [8] [9].
Many researchers have shown that magnetic susceptibility values of urban soils increase with an increase in their technogenic magnetite content, which is capable of absorbing different HMs [10] [11] [12]. Therefore, to identify sources of HM contamination of urban soils, an assessment of the magnetic susceptibility of soils is used while an assessment of thermomagnetic effect is used to search for hydrocarbon contamination [13] [14].
Under the influence of increased concentrations of HMs and HCs, many researchers have established changes in soil biological activities: reduction in the number of certain groups of soil microorganisms [15] [16] [17] [18] and an inhibition of soil enzyme activities (dehydrogenases, catalases, ureases, amylases, invertases, etc.) [19] [20] [21]. Based on these facts, the number of microorganisms of certain ecological and physiological groups and the activity of soil enzymes are widely used as indicators in soil-ecological monitoring of urban soils [22] [23] [24]. At the same time, there has been very little research conducted on the biological, ecological, geochemical and magnetic properties of soils in small industrialized cities.
The authors show that the size of a locality (the number of inhabitants and the associated amount of waste introduced into the landscape), as well as the profiles of enterprises operating in the locality, determine the technogenic migration of elements into the soils. Also, the degree of soil degradation under the influence of xenobiotics and methods of rehabilitation is largely dependent on the properties of the soil itself and the climatic features of the area. For Saratov region, located in the steppe zone of the Eastern part of the European territory of Russia, steppe soils are predominant, which are characterized by a certain vulnerability to pollutants due to the arid climate and excessive accumulation of carbonates, chlorides, and sulfates in the soil profile or in the subsurface layers [25]. The purpose of this research is to assess the ecological and functional state of steppe soils with moderate anthropogenic impact using a small industrialized city-Krasny Kut in Saratov region as an example.

Materials and Methods
The objects of the study were chestnut saline and chestnut carbonate soils sampled from a moderately industrialized city Krasny Kut, Saratov region, located in the southern part of the Saratov Volga region in the zone of dry tipchak-kovyl steppes on a low-level plain. These soils were formed under conditions of unstable and insufficient moisture precipitation.  [25] taking into account regional features [28].

Study Area Description and Sampling
The City Krasny Kut with an area of 8 km 2 and a population of 14,800 people is specialized in and represented by enterprises of regional significance in machine-building, metalworking, electricity and food industries. Relatively unfavorable environmental conditions were found in areas of garage and industrial development, in areas of municipal solid waste landfills and filtration fields that have a negative impact on atmospheric air, soil and vegetation cover, surface and underground water. In the course of this research on the territory of Krasny Kut, 24 soil samples were studied ( Figure 1).
Sampling and preparation of samples for analysis were carried out in accordance with GOST 17.4.4.02-84 [29]. Soil sampling took into account the wind rose, the features of the microrelief, the layout of buildings and communications.
According to GOST, the upper part of the soil horizon "A" was tested to a depth of 5 -10 cm. The size of the test sites ranged from 2 -3 to 10 m 2 . Samples were taken using the "envelope" method. The weight of the combined sample was from 0.5 to 1.0 kg.

Determination of Heavy Metal Concentrations
Concentrations of HMs in the soil samples were determined by atomic absorption spectrometry with flame atomization on a "KVANT-2AT" spectrophotometer.
Mobile acid-soluble forms of HMs (Cu, Zn, Ni, Cd, Pb) were determined in extracts of 1M HNO 3 [30]. To identify environmentally dangerous levels of HM concentrations in the soil, the actual concentration of each HM was compared with its MPC, calculating the hazard coefficient (Ko) using the formula [30]: where Ci is the content of the HM form in the sample, mg•kg −1 ; MPC is the maximum permissible concentration of the HM, mg•kg −1 .
Mobile forms of HMs were used to assess the extent of soil geochemical transformation by determining the total rate of pollution (Z c ) using the formula [30]: where n is the number of the identified elements; Ko-hazard coefficient of HM determined in the sample. When calculating Z c , we used an excess over the MPC (Ko) [31].

Determination of Magnetic Susceptibility and Thermomagnetic Effect
The specific magnetic susceptibility of the soil samples was measured in the laboratory using a serial KT-10 kappameter at a low frequency (KLF, 976 Hz) and  [13]. To assess the extent to which technogenic magnetic particles were introduced into soils, we used the coefficient of magnetism (K mag ), which was calculated based on measurements of magnetic susceptibility using the formula [13]: where K i is the average value of magnetic susceptibility in the soil, and K fon is the average value of magnetic susceptibility in the background area.
When evaluating thermomagnetic effect (dk), the increase in magnetic susceptibility values of each samples after heating to 500˚C in an oxidizing medium was determined using the formula [13]: where k is the magnetic susceptibility, and k t is the magnetic susceptibility after heating.

Determination of Soil Enzyme Activities
The total activities of soil dehydrogenases (AD), catalases (AC), peroxidases (AP) and invertases (AI) were determined according to methods in soil enzymology. The activity of dehydrogenases was determined photometrically on the iMARK microplate photometer through a reduction of the colorless substrate of 2,3,5-triphenyltetrazolium chloride, which after accepting hydrogen mobilized by dehydrogenases, turned into 2,3,5-triphenylformazane (2,3,5-TFF) with a red color. The amount of 2,3,5-TFF was calculated using a pre-constructed calibration curve [32]. Activity of dehydrogenases was expressed as mg TFF 10 g −1 of dry soil•day −1 .
Activity of catalases (AC) was determined using a titrimetric method [33], based on the amount of undecomposed peroxide which was determined by permanganatometric titration and measuring the rate of decomposition of hydrogen peroxide when interacting with soil. AC was expressed in mL 0.1 N KMnO 4 g −1 of dry soil•h −1 .
The activities of peroxidases (AP) and invertases (AI) were measured photometrically using a LEKI SS2107UV spectrophotometer. The activity of peroxidases was evaluated based on the ability of peroxidases to catalyze the oxidation of hydroquinone in the presence of oxygen peroxide to 1,4-benzoquinone with a yellow color [34]. The amount of 1,4-benzoquinone was calculated using a pre-constructed calibration curve. AP was expressed in mg of 1,4-benzoquinone g −1 of dry soil per 30 min at 30˚C.
The activity of invertases (AI) was determined by a method based on the ability of invertases to catalyze the splitting of sucrose into monosaccharides [21].
Reducing sugars in the filtrate were detected using a 0.2% alkaline solution of ferricyanide, their content was calculated on a standard scale made up of glucose. AI was expressed in mg of glucose•g −1 of dry soil•day −1 . All data on the ac- All the analyses were performed in triplicates. For all the data obtained, average values were calculated, which were compared using statistical indicators of standard deviation and the slightest significant difference. Statistical processing of experimental data was performed using Microsoft Excel 2010 application software package (for Windows). Differences were considered reliable when the probability of error p = 0.05 (95% confidence interval). The relationships between the ecological-geochemical, magnetic and biological properties of soils were evaluated by correlation analysis using the Pearson coefficient.

Results and Discussion
In other to obtain information on the functional and ecological state of the steppe soils of Krasny Kut, we used a comprehensive scientific and methodological approach based on the study of ecological, geochemical, magnetic and biological properties of soils. The results of the ecological-geochemical and petromagnetic analysis of soils in Krasny Kut are presented in Table 1.  [30].
Conducted correlation analysis ( Table 2)  In general, magnetic susceptibility in the city Krasny Kut was evenly distributed without forming continuous anomalous fields. Magnetic susceptibility measured at a high frequency had a similar distribution in the soil cover.
A scale for values of magnetic coefficient reflecting the level of iron content in the soil was proposed by M. V. Reshetnikov et al. [13] [38]: whereby K mag < 1 =     (Table 1) with its substrate when soil particles are enveloped by HCs. HMs partially or completely inactivate enzymes in soil microorganisms, which slows down their synthesis or contributes to blocking of respiratory chains of microorganisms thereby inhibiting their growth [44].
Taking into account the important role of soil dehydrogenases in various biological processes and their sensitivity to pollutants, we determined total dehydrogenase activities in the soil samples of Krasny Kut, which ranged between 2.6 to 9.6 mg TFF 10 g −1 of dry soil•day −1 (Table 3). Minimum values were observed in samples 22 and 24 (2.6 mg TFF 10 g −1 of dry soil•day −1 ), which could indicate the presence of ecotoxicants in the soil sample. In (Table 2) Table 2 showed   (Table 2).
Thus, medium, high and very high levels of dehydrogenase, catalase, peroxidase and invertase activities were observed in the soil samples of Krasny Kut.
According to the results obtained from the conducted enzymatic analysis, the soils of Krasny Kut can be considered to be in a state of ecological balance, although some exceptions exist.
In conditions of moderate technogenic impact of a small industrialized city in a steppe zone, positive correlations were established between the activities of dehydrogenases and catalases in the soil samples and the concentration of HMs: Zn and Pb. It is known that HMs in low concentrations can have a stimulating effect on the activity of soil enzymes [48]. This led to an assumption that the data obtained for HMs and their corresponding concentrations did not have a negative effect on the studied soil enzymes. Significant direct correlations between the activity of enzymes (dehydrogenases, catalases, invertases) in soils and thermomagnetic effect could be an indication of recent hydrocarbon contamination.

Conclusions
As a result of the conducted analysis on the ecological and functional properties