To study the consequences of pollution of ordinary chernozem by antibiotics, the following antibiotics were chosen: bactericidal antibiotics (benzylpenicillin, ampicillin, streptomycin), bacteriostatic antibiotics (oxytetracycline, tylosin, pharmasin), antibiotics used as growth stimulators in animal industry (tromexin, aliseryl), fungicidal antibiotics (nystatin). Full information on antibiotics is in the database on medicines http://drugreg.ru/Bases/.
The object of the study was ordinary chernozem of the South European facies (Botanical Garden, Southern Federal University), carbonate, powerful, slightly humic, heavy clay on yellow-brown and loessial loam (Table 1). The investigated type of soils was chosen in connection with the fact that chernozems constituted the majority of the soil cover of the south of Russia and were of particular importance in the country's food supply. The humus horizon thickness of the used chernozem was about 80 cm, the granulometric composition was heavy loamy, the reaction of environment was 7.7, the humus content was 4.1%, the total nitrogen (according to Kjeldahl) was 0.25%, total phosphorus (according to Ginzburg et al.) – 0.18, labile phosphorus (according to Machigin) – 28.8, total potassium (according to Berzelius) – 2.06% (Valkov et al., 2008).
Experimental test procedure
Laboratory model research
The soil for laboratory model research was selected from the arable layer (0-25 cm). Air-dry soil samples were treated with solutions of medical antibiotic – benzylpenicillin, veterinary antibiotic – pharmasin - and its mixture with fungicidal antibiotic nystatin in a wide range of concentrations of 1-1000 mg/kg of soil. Biological properties were studied on the 10th, 60th, and 120th days of incubation. The concentrations used were selected from literature data according to the residual amounts of antibiotics found in the environment (Christian et al., 2003; Grote et al., 2004; Thiele-Bruhn et al., 2004; Kay et al., 2005; Sarmah et al., 2006; Sun et al., 2014). Then the effect of medical antibiotics was studied – ampicillin, streptomycin, and veterinary antibiotics – tylosin, tromexin, aliseryl in a concentration of 500 mg/kg of soil, using the results of previous reconnaissance studies (Akimenko et al., 2014, 2015). Biological indicators were examined on the 3rd, 30th and 90th day of incubation. All soil samples were incubated at a temperature of 20-25 °C and optimum moistening (60% of the field moisture capacity). The control was soil, not polluted by antibiotics.
Field model research
In the field model experiments, the plots were laid according to the generally accepted method of field experimentation (Field Methods..., 1967). We used a plot of 1 m2. Repeatability was threefold. To study the dynamics of changes in biological properties, the soil samples were collected on the 3rd, 30th and 60th days after administering antibiotics. Antibiotics (oxytetracycline, tylosin) were introduced into the soil in the form of solutions in a concentration of 500 mg/kg of soil. Control areas were not treated with antibiotics. Within the framework of the field modeling, the influence of antibiotics on the growth and development of peas was studied, and the "Aksaiskii usatyi 7" variety was selected from Cirrosut vulgatum. Antibiotics were introduced into the soil by watering, after seed germination on the 10th day. The control was the plots sown with peas, without antibiotics.
Laboratory and analytical methods of research
Laboratory and analytical studies were conducted at the Department of Ecology and Nature Management of the Southern Federal University, using methods, which were common in ecology, biology, and soil science (Galstyan, 1978; Khaziev, 1991; Kazeev, & Kolesnikov, 2012). A comprehensive study of the microbiological properties of ordinary chernozem included determining the total number of bacteria by the method of luminescence microscopy with acridine orange staining, as well as the number of viable microorganisms of various ecological and trophic groups by the method of seeding the corresponding dilutions into solid nutrient medium. The enzymatic activity of the soil was judged by the activity of enzymes of the class of oxidases and hydrolases. The catalase and dehydrogenase activity was determined by the methods of A.Sh. Galstyan (1978), invertase activity – by the modified colorimetric method of F.Kh. Khaziev (1991), the activity of phosphatase – by the method of A.Sh. Galstyan and E.A. Arutyunyan (1966), the activity of peroxidase and polyphenol oxidase – by the method of L.A. Karyagina and N.A. Mikhailova (1986). The medium reaction (pH) was determined by the potentiometric method in a soil suspension of 1:2.5. The peas' indicators were studied by the following morphometric parameters and components of the crop structure: total phytomass (g), total number of pods (pcs.), average length of pods (mm), total weight of pods (g), weight of pods without peas (g), the average number of peas in pods (pcs.), the total weight of peas (g), the weight of 100 peas (g) and yield (dt/ha).
The biological properties of soil were analytically determined in 3-fold repetition for studying the microbiological properties of soils and in 4-9-fold repetition for studying the biochemical properties of soils.
To determine the general regularities of the influence of antibiotic pollution on the biological condition of chernozem, an integral indicator of the biological state (IIBS) of the soil was used. IIBS was determined by the most informative biological indicators (Kazeev, & Kolesnikov, 2012). From microbiological ones, the number of ammonifiers, amylolitic enzymes and micromycetes was taken into account, from biochemical ones – the activity of enzymes of the class of oxidases (catalase, dehydrogenase) and hydrolases (invertase, phosphatase) was used. To calculate the IIBS of the soil, the value of each of the selected indicators was taken as 100% (in the control and unpolluted soil sample) and in relation to the control, the values of other indicators (in polluted soil) were reflected in percentage terms. Then the average value of the selected indicators for each option was determined. This technique of calculation allows combining the relative values of various indicators, the absolute values of which cannot be summed up, since they have different units of measurement.
Statistical processing of research results
Since the biological properties of soils vary widely (Kazeev, & Kolesnikov, 2012), the biological values obtained in the work were subjected to statistical variation analysis, dispersion and correlation analysis. Statistical processing of data was conducted using statistical package Statistica 10.0. The following main indicators of the variation statistics were calculated: average (M±m), the coefficient of variation (CV), standard deviation (s). The sensitivity of the studied indicator was evaluated according to the degree of its decrease depending on the concentration of the antibiotic, and the information content – according to the degree of value correlation of the indicator and the concentration of the antibiotic.