Assessment of Ecological Condition of Haplic Chernozem Calcic Contaminated with Petroleum Hydrocarbons during Application of Bioremediation Agents of Various Natures

Abstract

Petroleum hydrocarbon contamination disrupts ecological and agricultural soil functions. For their restoration, bioremediation agents of various natures are used (nonorganic or organic fertilizers, bacterial preparations, adsorbing agents) featuring different remediation mechanisms (adsorption or biostimulation of petroleum hydrocarbon decomposition). The objective of this research is the assessment of the ecological condition of petroleum hydrocarbon-contaminated Haplic Chernozem Calcic after the application of bioremediation agents of various natures. The influence of glauconite, nitroammophos, sodium humate, the bacterial preparation “Baikal EM-1”, and biochar on the intensity of petroleum hydrocarbon decomposition and the ecological condition of Haplic Chernozem Calcic was analyzed. The ecological condition of Haplic Chernozem Calcic was assessed based on the residual content of petroleum hydrocarbons in soil and the following biological parameters: changes in the number of soil bacteria, activity of catalase and dehydrogenases, soil respiration (CO₂ emission), germinating ability, lengths of roots and shoots, and integrated index of the biological state. The minimum concentrations of residual petroleum hydrocarbons in soil were observed after the use of biochar (44% from initial content) and glauconite (49%). The biological properties of soils were affected in different ways. Soil respiration was stimulated by 3-6-fold after adding nitroammophos. Indices for the intensity of the early growth and germination of radish in soil with glauconite, sodium humate, and biochar were increased by 37–125% (p < 0.01) compared with the reference value. After the application of biochar, sodium humate, and “Baikal EM-1”, the number of soil bacteria was 66–289% higher (p < 0.01) than the reference value. At the same time, the activities of catalase and dehydrogenases were inhibited by up to 35% in variants with bioremediation agents and petroleum hydrocarbons relative to the reference values. The maximum stimulation of the biological activity (as the integrated index of the biological state (IISB)) of Haplic Chernozem Calcic was observed after applying sodium humate and biochar, with 70 and 66% (p < 0.01) increases from the reference value, respectively. Considering the net cost of bioremediation agents, the maximum cost efficiency is achieved with “Baikal EM-1”, sodium humate, and biochar: 110, 527, and 847 USD·10³/ha, respectively. After using Baikal EM-1”, sodium humate, and biochar, the ecological state of Haplic Chernozem Calcic was restored.

1. Introduction

According to data from the Ministry of Energy of Russia, the production of petroleum hydrocarbon products is over 550·10⁶ tons per year [1]. Moreover, petroleum hydrocarbons are exported into neighboring countries of the Russian Federation at rates of over 240·10⁶ tons per year [2]. However, the petroleum hydrocarbon losses that occur during transportation and processing are excessively high. This is very hazardous to the environment. After entering soil, petroleum hydrocarbon products envelope soil particles with dense layers that prevent oxygen access and repel water. As a result, the agrochemical and biochemical properties of the soils change. Petroleum hydrocarbon product contamination of farming lands is the most hazardous to the environment. Petroleum hydrocarbon contamination of soil is often associated with contamination with heavy metals [3,4,5].

The preservation of soils is an important task under various land uses, particularly with respect to contamination [6,7]. Standard methods of removing petroleum hydrocarbons from farming land soil have a number of features. They include mechanical or physical soil cleaning, causing irreparable harm to soil and plant conditions. Restoration of the biological properties of the soil and, thus, the ecological condition and soil capabilities takes a long time. Biological substances can be used to facilitate the petroleum hydrocarbon destruction process by stimulating the activity of native biota. The destruction of hydrocarbons reduces the soil toxicity level and restores its agricultural functions. For the bioremediation and biostimulation of soils by different bacterial preparations with varying natures, nonorganic nitrogen-containing fertilizers such as urea and nitroammophos are used [8,9,10]. However, the use of organic fertilizers and substances can also stimulate native biota and contribute to petroleum hydrocarbon destruction [11,12,13,14,15]. The decomposition process by native microbiota or the creation of favorable conditions for the stimulation of microbiota activity through the application of organic fertilizers is a fundamental function of bio-stimulating organic fertilizers [16,17,18].

The use of bacterial fertilizers or bioaugmentation can induce the oxidation of petroleum hydrocarbons in the soil. Microorganisms use petroleum hydrocarbons for their life activities by decomposing them into simple products: carbon gas and water, which are partially converted into organic substances in the soil and cell biomass [19,20,21,22,23,24]. After 30 weeks, the efficiency of microbial preparations in combination with mineral and organic fertilizers for petroleum hydrocarbon decomposition in soil had a favorable impact on the activity of dehydrogenase and the abundance and composition of microbial communities in soils [25,26]. The bioremediation process is performed with the use of different groups of microorganisms by means of microbial preparations or bioaugmentation: Gram-negative oxidase-negative cocci bacteria (Acinetobacter), Gram-positive corynebacteria (Rhodococcus, Bravobacterium, Arthrobacter, Micrococcus), Gram-positive spore-forming bacteria (Bacillus), and Gram-negative oxidase-positive bacteria (Flavobacterium, Chromobacterium) [27]. In addition to bacteria, petroleum hydrocarbon-destroying agents include micromycetes of the following genera: Phoma, Penicillium, Aspergillus, Fusarium, and Candida [28]. The use of microbial preparations contributes to petroleum hydrocarbon destruction for the restoration of biological properties [29,30,31]. The petroleum hydrocarbon decomposition process in soil with native or applied microorganisms depends on its agrochemical and agrophysical properties, including the structure, moisture content, and fertilizer element content (nitrogen, phosphorus, and potassium) [32,33]. When the soil is contaminated with petroleum hydrocarbons, changes in its water permeability and oxygen inhibit the activity of soil enzymes, alter microbiological indices, and reduce plant growth and development [13,34,35,36,37].

Upon bioremediation agent application, it is necessary to analyze the restoration of agricultural soil functions. Such an assessment can be performed using biological indices that have been proven to be sensitive to petroleum hydrocarbon contamination [10,11,35,36,38,39,40,41]. It has been previously shown that during soil petroleum hydrocarbon contamination, changes in CO₂ emission, inhibition or stimulation of enzyme activity, and changes in phytotoxic indices can be used as indicators of the restoration of the agricultural functions of the soil [42,43].

The objective of this work is to assess the ecological condition of Haplic Chernozem Calcic contaminated with petroleum hydrocarbons after the application of bioremediation agents of various natures.

According to the objective, the research tasks include the following: (1) assessing the residual content of petroleum hydrocarbons in Haplic Chernozem Calcic after applying bioremediation agents of various natures; (2) analyzing the biological indices of the soil ecological condition; (3) calculating and assessing the cost efficiency of bioremediation agent use; and (4) assessing the ecological condition of Haplic Chernozem Calcic after bioremediation.

2. Materials and Methods

The research object is Haplic Chernozem Calcic (according to IUSS Working Group, WRB). The soil was sampled in the upper layer (top layer of soil: 0–10 cm) of arable land in the territory of the Botanical Garden of the Southern Federal University (SFU). Soil samples were taken in the territory of the Botanical Garden, far from industrial enterprises and other anthropogenic sources (Figure 1). This area is protected by the specialists of the Botanical Garden of SFU and is an experimental site for the cultivation of agricultural crops.

Currently, the Botanical Garden of SFU occupies an area of 160.5 hectares with a varied conformation, microclimate, soil, vegetation, and fauna. Most of its territory is located in the floodplain of the river Temernik. The highest point of the Botanical Garden of SFU is fixed at 84.5 m. The site is not subject to any anthropogenic impacts. In Rostov-on-Don, the natural conformation has largely not been preserved, and the territory of the Botanical Garden of SFU is a unique natural ecosystem that includes the poorly transformed conformation of the Botanical Garden of SFU [44]. The experimental plots of the Botanical Garden of SFU are located at a sufficient distance from emission sources in the forest thicket. In the soils of these botanical gardens, an increase in the content of organic carbon and an increase in pH values (pH > 7) are observed [45]. Increases in the degree of saturation with metabolic bases and the content of plant nutrients (phosphorus, potassium) are observed. Haplic Chernozem Calcic in the south of Russia is the most widespread soil in Russia. Considering the high anthropogenic load on these soils, their restoration and preservation are especially relevant.

For the restoration of petroleum hydrocarbon-contaminated Haplic Chernozem Calcic functions through petroleum hydrocarbon degradation, a wide range of bioremediation agents was used: a natural mineral adsorbent (glauconite), a nonorganic mineral substance (nitroammophos), a biological biota stimulant (sodium humate), microbiological fertilizer (“Baikal EM-1”), and organic carbon-bearing fertilizer (biochar). These ameliorants differ in their aggregate composition (liquid, solid state) and in their physical and biological properties (adsorption and stimulation of native biota).

Glauconite is a complex potassium-containing aqueous aluminium silicate mineral from the hydromica group of the layered silicate subclass with an unstable and complex composition. The absorbing agent is produced by Glauconite LLC in the Chelyabinsk Region (Kunashaksky District) for the physical and chemical cleaning of soil and waste waters (

Table 1. Glauconite’s specifications [46].

No.	Characteristic	Unit	Value
1.	Bulk density	kg/m3	1300–1400
2.	Specific surface	m2·g	180–200
3.	Intergranular porosity	%	60–72
4.	Petroleum product capacity	mg/g	200–220
5.	Phenol capacity	mg/g	1000
	Distribution ratio:
6.	by Sr90	mL/g	214
7.	by Cs137	mL/g	2.6·103
8.	Fractional composition	mm	0.1–0.6

).

Nitroammophos (NH₄H₂PO₄ + NH₄NO₃) is a mineral fertilizer with a nitrogen content of up to 15% (Figure 2). When applied to the soil, nitroammophos quickly dissolves and dissociates mainly into ammonia ions, nitrate ions, and ions of orthophosphoric acid (H₂PO₄).

Nitrate ions (NO₃⁻) are absorbed by plants and microorganisms only within the vegetation period for biological purposes. In the autumn and winter period, nitrate ions migrate with descending water flows from the root-containing layer, which results in significant nitrogen losses, especially in areas with excessive moistening in soils with simple particle-size distributions. Ammonia ions (NH⁴⁺) are less mobile in the soil. Ammonia ions are quickly bound with a soil-cover complex, thereby becoming exchangeable ions.

Sodium humate is a biological stimulant of soil biota made of peat and brown coal, wastes obtained from the production of paper and alcohol by organic methods (

Table 2. Characteristics of sodium humate by the content of salts of humic acids and micro- and macroelements, % (V/V) [47].

Characteristics	Value
Humic acid salts	84.0–86.0
Silicon, Si water-soluble.	4.0–5.0
Carbon, C	46.0–49.0
Oxygen, O	17.0–19.0
Hydrogen, H	3.0–4.0
Nitrogen, N	0.8–1.0
Phosphorus, P	0.5–0.7
Potassium, K	6.0–8.0
Sulfur, S	0.75
Calcium, Ca	1.0–2.0
Magnesium, Mg	0.15
Sodium, Na	3.0–5.0
Silicon, Si	9.0–10.0
Iron, Fe	0.4–0.5
Manganese, Mn	0.12
Molybdenum, Mo	0.02
Cobalt, Co	0.02
Zinc, Zn	0.30
Boron, B	0.30
Copper, Cu	0.20

). Sodium humate is a waste product of Californian worms. Sodium humate is one of the best stimulating agents for the growth of vegetable and fruit crops.

The microbiological fertilizer “Baikal EM-1” contains 60 beneficial microorganisms: as shown in Figure 3, the composition of the main microorganisms of “Baikal EM-1” includes strains of bacteria (Paenibacillus pabuli, Azotobacter vinelandii, Lactobacillus casei, Clostridium limosum, Cronobacter sakazakii, Rhodotorulla mucilaginosa, Cryptococcu), fusion yeasts (Saccharomyces, Candida lipolitica, Candida norvegensis, Candida guilliermondii), and fungi (Aspergillus, Penicillium, Actinomycetales).

The biochar was pure birch (Betulaceae) wood charcoal (A grade), GOST 7657-84, with a carbon content of at least 85%. The product is made by wood pyrolysis (800 °C) in retort units without oxygen access (Figure 4).

The product has high carbon content (93–99%) and lacks harmful or toxic impurities. The amount of carbon in 1 ton of biochar is equivalent to the content of 3 tons of carbon in carbon dioxide. When using biochar, plants capture carbon from the air and bind it in the soil. In relation to petroleum hydrocarbons, biochar serves as a sorbent and stimulator of the native soil biota.

After sampling, the prepared Haplic Chernozem Calcic was dried, cleaned from plant roots, and screened through a metallic sieve with a mesh diameter of 2 mm. Samples of air-dried soil (200 g) were placed in vegetative pots; triple biological replicates were performed. Petroleum hydrocarbons at a concentration of 5% of soil mass were applied to moistened soil (up to 60% of soil mass).

For contamination modeling, petroleum hydrocarbons provided by Novoshakhtinsk Refinery were used. This petroleum hydrocarbon is a light petroleum hydrocarbon (density 0.818 g/m³) with a sulfur mass fraction of 0.43%, mechanical impurity mass fraction of 0.0028%, water mass fraction of 0.03%, and chloride salt concentration of 40.1 mg/dm³. Measurements in API degrees were taken to determine the relative petroleum hydrocarbon density (relative to water density at the same temperature).

Thereafter, bioremediation agents were applied to the soil according to the experimental scheme (

Table 3. Scheme of an experiment on the bioremediation of petroleum hydrocarbon-contaminated Chernozem.

No.	Type of Bioremediator
1	Control
2	C + glauconite
3	C + nitroammophos
4	C + sodium humate
5	C + Baikal EM-1
6	C + biochar
7	petroleum hydrocarbons (5% of soil)—PHC
8	PHC + glauconite
9	PHC + nitroammophos
10	PHC + sodium humate
11	PHC + “Baikal EM-1”
12	PHC + biochar

, Figure 5). As described in Table 3, in summary, the experimental scheme used a control sample as a reference, taking into account two scenarios for the application of bioremediation agents: (i) the control sample (control) was not contaminated (No. 1–6 in Table 3) and (ii) the control sample was contaminated with petroleum hydrocarbons (PHCs) at a concentration of 5% of the soil (No. 7–12 in Table 3). A model experiment on soil pollution with petroleum hydrocarbons and bioremediation with substances of various natures was carried out at controlled humidity and temperature: soil moisture—60%; temperature of air—25–26 °C. Bioremediation agents differ in the methods of their application to the soil.

Dry substances (glauconite, nitroammophos, and biochar) were applied by mixing them with moistened soil and adding petroleum hydrocarbons. Other bioremediation agents with various natures (“Baikal EM-1” and sodium humate) were applied to the soil in the form of a solution, followed by petroleum hydrocarbon addition.

Upon completion of the bioremediation agent exposition period for petroleum hydrocarbon-contaminated soil, the residual content of petroleum hydrocarbon and a number of biological indices characterizing the ecological condition of the soil after bioremediation were determined.

The residual petroleum hydrocarbon content was determined by infrared spectrometry with the use of carbon tetrachloride as an extracting agent [48].

For the assessment of the ecological condition, soil phytotoxicity (germinating ability, lengths of roots and shoots), soil respiration (CO₂ emission), and the activity of soil enzymes (catalase and dehydrogenases) were determined. For the assessment of the toxicity level due to the applied bioremediation agents, the soil conditions without petroleum hydrocarbons but with similar doses of bioremediation agents were compared with those of petroleum hydrocarbon-contaminated soil.

Soil phytotoxicity was assessed using standard procedures to evaluate the plant sprout and root lengths. The phytotesting of contaminated soil was performed using the garden radish (Raphanus sativus var. radicula Pers) cultivar “Rubin”.

With the increasing time of exposition to petroleum hydrocarbon decomposition, the contents of CO₂ and CH₄ increase in the soil air. When adding bioremediation agents, the decomposition speed increases. The petroleum hydrocarbon decomposition intensity was assessed by the gas-analyzer TESTO-535 with an error of ±50 ppm, and the respiration of soil was recalculated by Equation (1), expressed in mg C/kg of soil [49]:

$$C=\frac{Dc-D_0}{dt}\times \frac{V}{S}\times 1000\times 60$$

(1)

where Dc is the emission of carbon dioxide in the air above the contaminated soil, ppm; D₀ is the emission of carbon dioxide in the air, ppm; dt is the time period within which measurements are performed, sec.; V is the chamber volume, m³; and S is the chamber cross-sectional area, m².

The strength of enzymes of the oxidoreductase class (catalase, dehydrogenases) was assessed as per A.Sh. Galstyan, F.Kh. Khasiev in a modification of K.Sh. Kazeev and S.I. Kolesnikov [50,51,52]. Enzyme activity based on the recommendation of A.Sh. Galstyan (1978) was analyzed at the natural pH of soil.

The activity of catalase (H₂O₂: H₂O₂—oxidoreductase, EC number 1.11. 1.6.) was determined via the gasometric method as per A.Sh. Galstyan (1978), in which the amount of decomposed peroxide during the reaction with the soil is determined by measuring the volume of segregated oxygen that displaces water from a burette [50]. Enzyme activity was expressed in mL O₂ per 1 g of soil within 1 min.

Dehydrogenase activity (substrata: NADP—oxidoreductase, EC number 1.1.1) was determined as per A.Sh. Galstyan (1978) by measuring the restoration of tetrazolium chloride in triphenylformazan (TPF) [50]. Enzyme activity was expressed in mg TFP per 10 g of soil within 24 h.

The number of soil bacteria was determined by luminescence microscopy according to the number of soil bacteria stained with acridine orange dye (Zvyagintsev, 1991) [53]. The result was expressed as 10⁹ bacteria per 1 g of soil (Equation (2)):

$$M=\frac{4\times A\times H\times {10}^{10}}{p}$$

(2)

where M is the number of cells per 1 g of soil; A is the average number of cells within 1 visual field; H is the dilution index; and P is the area of the visual field, µm².

For the assessment of soil conditions using soil biological activity indices, the integral index of the biological state (IIBS) of Haplic Chernozem Calcic was determined [54]. The IIBS of Haplic Chernozem Calcic was calculated in this research using 6 indices of the soil biological activity: activity of catalase, activity of dehydrogenases, germinating ability, lengths of root and sprouts, and soil respiration.

For the IIBS of Haplic Chernozem Calcic, the maximum value of each index (100%) was chosen from the array of data, and this value was used as a reference for this index, which was expressed for other variants of experiments by Equation (3):

$$B_1=\frac{B_x}{B_{max}}\times 100\%$$

(3)

where B₁ is the relative score of the indicator, B_x is the actual value of the indicator, and B_max is the maximum value of the indicator.

Then, the relative values of several IIBSs of Haplic Chernozem Calcic, such as the activity of catalase, activity of dehydrogenases, germinating ability, length of roots, length of sprouts, and soil respiration, were summed. Thereafter, the average value of the studied indices was calculated for each variant by Equation (4):

$$B=\frac{B_1+B_2+\dots +B_n}{N}$$

(4)

where B is the average estimated score of indicators, B₁…B_n is the relative score of the indicator, and N is the number of indicators.

The IIBS was calculated by Equation (5):

$$IIBS=\frac{B}{B_{max}}\times 100\%$$

(5)

where B is the average estimated score of all indicators, and B_max is the maximum estimated score of all indicators.

For the calculation of the contamination value of each index, non-contaminated soil is taken as 100% as the reference, and the value of the same index for the contaminated soil is expressed as a percent.

The efficiency of using each bioremediation agent was assessed as per

Table 4. Consumption (C_BR, kg/ha) and cost (C, USD 10³/kg) of bioremediation agents.

No.	Type of Bioremediator	Appearance	C 1	CBR 2
1.	Glauconite	Gray fine powder	3.7 3	16,600
2.	Nitroammophos	Gray-white solid opaque crystals	5.2	300
3.	Sodium humate	Dark brown viscous paste	4.5	200
4.	“Baikal EM-1”	Dark brown liquid	7.5	2
5.	Biochar	Black coal coarse powder	7.5	11

Note: ¹ C—cost of bioremediator, USD 10³/kg; ² C_BR—bioremediator consumption, kg/ha; ³ according to the exchange rate as of 17 September 2020, 1 USD = 75 rubles.

.

The cost efficiency of bioremediation agent use was assessed by Equation (6), in USD 10³/ha:

$$E=C_{BR}\times C\times D_{PCH}$$

(6)

where C is the cost of the bioremediator, USD 10³/kg; C_BR is the bioremediator consumption, kg/ha; and D_PCH is the fraction of residual petroleum hydrocarbon content.

Statistical processing of the data obtained was carried out using the software package of STATISTICA 12.0. Statistics (mean values, dispersion) were determined, and the reliability of different samples was established by using dispersion analysis (Student t-test).

3. Results and Discussion

3.1. Residual Petroleum Hydrocarbon Content in Petroleum Hydrocarbon-Contaminated Soil after Application of Bioremediation Agents

The petroleum hydrocarbon content remaining in the soil after incubation with bioremediation agents and changes in the soil ecological condition was assessed. The petroleum hydrocarbon content after incubation with bioremediation agents changed in different degrees (Figure 6). The addition of glauconite to soil contaminated with petroleum hydrocarbons reduced the petroleum hydrocarbon content by 6%. The sample with biochar resulted in the maximum reduction of petroleum hydrocarbon content, which was 16% lower than in the petroleum hydrocarbon-contaminated variant.

This effect is conditioned by the physical structure of the bioremediation agent. The composition, porosity, and surface area of biochar are similar to those of activated carbon, but it has wider range of initial raw materials [55]. Over the last several years, biochar has been actively used as an organic fertilizer for the restoration of the fertility and agricultural functions of soils. The use of biochar has a stimulating effect on microbiological activity. The preparation based on the brown coal “Gumikom” is rather efficient in the bioremediation of petroleum hydrocarbon-contaminated soils [56].

The petroleum hydrocarbon decomposition efficiency and economic feasibility of the researched bioremediation agents are in the following order:

biochar (57) > glauconite (51) > sodium humate (50) = nitroammophos (50) > “Baikal EM-1” (49).

Glauconite application led to the maximum petroleum hydrocarbon adsorption (51%) from the soil, and after biochar application, the maximum ameliorative effect was obtained, with 57% petroleum hydrocarbon decomposition relative to the initial content.

3.2. Change in the Number of Soil Bacteria of Non-Contaminated and Petroleum Hydrocarbon-Contaminated Haplic Chernozem Calcic after Application of Bioremediation Agents

In pure soil without petroleum hydrocarbons, after glauconite and biochar were applied, the number of soil bacteria increased by 49 and 243% (p < 0.01) from the reference value, respectively (Figure 7). The final calculation of the number of soil bacteria was performed using Equation (2). Other bioremediation agents either did not influence the number of bacteria, such as nitroammophos and sodium humate, or reduced the number (32% decrease relative to the reference value), which occurred with “Baikal EM-1”.

In variants with petroleum hydrocarbons, after the application of bioremediation agents, the changes were more significant. After the application of glauconite, sodium humate, “Baikal EM-1”, and biochar, the number of soil bacteria was 43, 289, 89, and 66% (p < 0.01) higher than the reference value, respectively. After sodium humate application to the petroleum hydrocarbon-contaminated soil, the bacteria number was 3.8 times higher than the reference value (p < 0.01) and 7 times higher (p < 0.01) than that in contaminated soil; this is the result of the stimulation of native biota. In comparison, with the application of microorganism strains by means of “Baikal EM-1”, the stimulation was 89% (p < 0.01) relative to the reference value and 136% (p < 0.01) compared with the petroleum hydrocarbon-contaminated Haplic Chernozem Calcic without bioremediation agents. The stimulation of native microbiota was also detected after the application of biochar, with a 66% increase (p < 0.01) from the reference value.

3.3. Change in the Enzyme Activity of Non-Contaminated and Petroleum Hydrocarbon-Contaminated Haplic Chernozem Calcic after Application of Bioremediation Agents

The soil enzyme activity was assessed by the activity of catalase and dehydrogenase. Enzymes of this class are used to monitor the chemical contamination of soils in the south of Russia [10,35,41]. The maximum activity of dehydrogenases in the control soil without the application of petroleum hydrocarbons was detected in the samples with glauconite: 50% (p < 0.01) over the reference value (Figure 8).

Other bioremediation agents either did not influence the enzyme activity (sodium humate and “Baikal EM-1”) or inhibited the activity (nitroammophos) by 35% (p < 0.01). In the petroleum hydrocarbon-contaminated Haplic Chernozem Calcic, significant differences in the activity of dehydrogenases from the reference value were not detected: in the petroleum hydrocarbon-contaminated Haplic Chernozem Calcic without bioremediation agents and with “Baikal EM-1”, dehydrogenases were found to be stimulated by 19 and 17% (p < 0.01) compared with the reference value, respectively.

The activity of the other representative of the oxidoreductase class—catalase—was changed in the same way (Figure 9).

All bioremediation agents in the pure soil had an inhibiting influence on the activity of catalase (by 13–15%, p < 0.01), especially nitroammophos, for which catalase activity was 53% (p < 0.01) lower than the reference value. In the petroleum hydrocarbon-contaminated soil, the catalase activity was inhibited to 27–36% (p < 0.01) of the reference value after the application of bioremediation agents. The oxidoreductase activity of the soils after bioremediation agent application underwent different changes: the activity of dehydrogenases remained almost the same as that in the petroleum hydrocarbon-contaminated variant, and the catalase activity was inhibited by 26–36% (p < 0.01) of the reference value.

3.4. Change in Soil Respiration of Non-Contaminated And Petroleum Hydrocarbon-Contaminated Haplic Chernozem Calcic after Application of Bioremediation Agents

As a result of natural transformation processes and degradation, petroleum hydrocarbons in the soil slowly decompose. When adding bioremediation agents for petroleum hydrocarbon degradation, the speed of decomposition increases. This results in carbon dioxide and water vapor formation. The biochemical condition of the soils is assessed not only by the activity of soil enzymes but also by the products that characterize petroleum hydrocarbon decomposition: carbon dioxide and water vapor. For the correct assessment of petroleum hydrocarbon decomposition into simple decomposition products (carbon dioxide and water vapor), non-contaminated and petroleum hydrocarbon-contaminated soil samples were analyzed (Figure 6). The final calculation of soil respiration was performed using Equation (1).

Figure 10 shows the change in soil respiration when applying “Baikal EM-1”. Soil respiration was 8 times higher than the reference value 14 days after the experiment commenced. The soil is conditioned by the 60 strains of microorganisms in the preparation, which change the content and concentration of the carbon dioxide in the soil air for their life activities. Upon the completion of the 30-day period, soil respiration with different bioremediation agents did not differ from the reference value.

From the analysis of data on petroleum hydrocarbon-contaminated soil according to CO₂ emission intensity, the following results were obtained. Seven days after the experiment commenced, with the addition of glauconite, nitroammophos, and biochar, soil respiration was increased by 29, 115, and 24% (p < 0.01) compared with the reference value in the previous period.

Within the remaining exposition period, soil respiration was increased by 10–232% (p < 0.01) compared with the reference value after adding nitroammophos, which is connected to not only petroleum hydrocarbon decomposition but also the decomposition of the applied substance to CO₂ and water. Nitroammophos compensates for nitrogen loss in petroleum hydrocarbon-contaminated soils and thus contributes to the optimization of native biota conditions in the soil.

3.5. Change in the Intensity of Initial Growth and Development of Radish Seeds in Non-Contaminated and Petroleum Hydrocarbon-Contaminated Haplic Chernozem Calcic after Application of Bioremediation Agents

For the assessment of the soil toxicity level after the application of bioremediation agents, a plant sensitive to phytotesting, Raphanus sativus var. radicula Pers cultivar “Rubin”, was used. The use of radish produces a quick response to changes in petroleum hydrocarbon-contaminated Haplic Chernozem Calcic toxicity within a short period, particularly during the use of nonorganic bioremediation agents [10,39]. Toxicity was assessed by indices for the germinating ability of seeds (Figure 11) and its morphological indices: lengths of shoots (Figure 12A) and roots (Figure 12B).

In non-contaminated soil, the germinating ability of radish seeds did not change after the application of glauconite, nitroammophos, and biochar. However, the application of sodium humate and “Baikal EM-1” to the non-contaminated soil in dosages intended for remediation reduced the germinating ability by 40 and 38% (p < 0.01) of the reference value. In petroleum hydrocarbon-contaminated soil, after the application of nitroammophos, sodium humate, and “Baikal - EM1”, the germinating ability was stimulated by 23, 44 and 16% (p < 0.01) compared with the reference value from petroleum hydrocarbon contamination.

Morphological indices of radish after cultivation in non-contaminated and petroleum hydrocarbon-contaminated soil are shown in Figure 12A,B.

In pure soil that was not contaminated with petroleum hydrocarbons, the lengths of sprouts in the soil with glauconite, nitroammophos, and biochar were 50, 41 and 55% higher (p < 0.01) than the reference value. Maximum sprout growth (72% higher than the reference value) was detected for the application of sodium humate. This effect is caused by the stimulation of soil microbiota, causing the acceleration of plant growth. In petroleum hydrocarbon-contaminated soil, the lengths of sprouts were 3 times lower than the reference value. When adding glauconite, sodium humate, and biochar, sprout lengths were increased by 11, 68, and 66% (p < 0.01) in comparison with those from petroleum hydrocarbon-contaminated soil.

Radish root lengths changed in the same way. The application of nitroammophos, “Baikal EM-1”, and sodium humate caused root lengths to increase by 32, 49, and 60% (p < 0.01) from the reference value. Maximum stimulation of the root length was 121 and 138% (p < 0.01) relative to the reference value and was detected after the application of glauconite and biochar. In petroleum hydrocarbon-contaminated soil, the root length was reduced by 5 times; after adding glauconite, nitroammophos, and “Baikal EM-1”, roots were 37, 22, and 28% (p < 0.01) longer than the reference value. Applying glauconite and biochar produced the maximum increases in root length, which were 119 and 125% (p < 0.01) higher than the reference value from the petroleum hydrocarbon-contaminated variant.

In the presence of petroleum hydrocarbon contamination, glauconite and biochar had a more favorable influence on the intensity of radish seed sprouting. Due to its chemical nature, glauconite decontaminated petroleum hydrocarbons in its crystal latitude and contributed to the optimal growth and development of plants. Radish growth stimulation in the soil with biochar is conditioned by the availability of organic and mineral elements.

3.6. Change in the Integral Index of the Soil Biological State for Non-Contaminated and Petroleum Hydrocarbon-Contaminated Haplic Chernozem Calcic after Application of Bioremediation Agents

Biochar as a fertilizer not only ensures moisture and nutrient elements (carbon, nitrogen, phosphorus, potassium) for the soil but also contributes to the decomposition of organic compounds and the sorption of heavy metals [57,58]. Soil quality was assessed by calculating the integral index of the biological state of Haplic Chernozem Calcic using Equations (3)–(5). The integral index of the soil biological state (IIBS) was calculated using the average values of phytotoxicity (root and sprout lengths), enzyme activity (activity of catalase and dehydrogenases), number of soil bacteria, and soil respiration (emission CO₂) over a period of 30 days (Figure 13).

According to Figure 13, the studied bioremediation agents according to the change in ecological properties of the soil IIBS are in the following order:

sodium humate (145) > biochar (106) > “Baikal EM-1” (91) > glauconite (77) > nitroammophos (64).

Restoration of soil biological properties after petroleum hydrocarbon contamination is a long-term process. The degree of restoration was assessed by measuring the increase or stimulation of the following soil ecological indices: intensity of initial growth and development of radish seeds, release of carbon dioxide, and change in the soil enzyme activity in comparison with the reference values. The application of sodium humate and biochar resulted in 145 and 106% (p < 0.01) restoration of the ecological condition compared with the reference value. After applying the bacterial preparation “Baikal EM-1”, soil properties were restored by 91% (p < 0.01) compared with the reference value. The other bioremediation agents (glauconite and nitroammophos) did not have a more favorable influence on the soil ecological properties relative to the reference value. However, compared with the petroleum hydrocarbon-contaminated soil in variants with glauconite, stimulation of 14% (p < 0.01) relative to the reference value was detected.

3.7. Assessment of the Cost Efficiency for Bioremediation Agent Use in Petroleum Hydrocarbon-Contaminated Haplic Chernozem Calcic

Considering the range of soil ecological condition restoration based on the integral index of the soil biological condition, the economic feasibility of using bioremediation agents was assessed. Based on the data on the efficiency of petroleum hydrocarbon decomposition, the economic feasibility of bioremediation agent use was calculated (

Table 5. Economic efficiency (E) of using various bioremediators of different natures, USD·10³/ha.

No.	Type of Bioremediators	DPCH	C	CBR	E
1.	Glauconite	0.51	16,600	3.75	31,996
2.	Nitroammophos	0.50	200	5.25	527
3.	Sodium humate	0.50	200	4.50	446
4.	“Baikal EM-1"	0.49	30	7.50	110
5.	Biochar	0.57	200	7.50	847

Note: D_PCH—fraction of residual petroleum hydrocarbons content (without units of measure); C—cost of bioremediator, USD 10³/kg; C_BR—bioremediator consumption, kg/ha; E—economic efficiency, USD·10³/ha.

). The cost efficiency of the bioremediators was calculated in USD per 1 ha of arable land.

Moreover, the efficiency of petroleum hydrocarbon decomposition, cost, and use of bioremediation agents in terms of agricultural application were chosen as assessment criteria. According to Equation (6), the most efficient bioremediation agent for the cleaning and restoration of petroleum hydrocarbon-contaminated soils is biochar. When applying this bioremediation agent, in addition to efficient petroleum hydrocarbon decomposition (57%), the economic feasibility of its use in the case of Haplic Chernozem Calcic after petroleum hydrocarbon contamination is 847 USD·10³/ha. The use of “Baikal EM-1”, sodium humate, and nitroammophos is only economically sound, but it is not efficient in the case of soil remediation. The use of glauconite is reasonable only at low levels of soil contamination, as this absorbing agent does not decompose petroleum hydrocarbons but fixes it in its crystal latitude.

The bioremediation agents can be classified by cost efficiency in the following order (in increasing order of cost, in USD ·10³/ha):

“Baikal EM-1” (110) > nitroammophos (446) > sodium humate (527) > biochar (847) > glauconite (31,996).

Thus, for Haplic Chernozem Calcic contaminated with petroleum hydrocarbons, the most efficient bioremediator is biochar, as it is economically sound and ecologically advisable. Biochar stimulates native biota in soils to decompose petroleum hydrocarbon as a concentrated carbon source. The use of glauconite is economically inadvisable but efficient in relation to the petroleum hydrocarbon concentration in the soil. However, due to the fact that petroleum hydrocarbons are not decomposed and remain in the soil thanks to the active surface of glauconite bars, its use within the wide scope of restoration of farming lands is economically inadvisable.

4. Conclusions

A model experiment on the influence of a wide range of bioremediation agents of different natures on the condition of Haplic Chernozem Calcic contaminated with petroleum hydrocarbon shows the following results. Petroleum hydrocarbons have an inhibiting influence on all indices of the soil biological state, causing 30–80% inhibition in comparison with the initial contents of petroleum hydrocarbons.

The minimum concentrations of residual petroleum hydrocarbons in soil were observed after the use of biochar (44% of the initial content) and glauconite (49%). The biological properties of soils were affected in different ways. After adding nitroammophos, soil respiration was stimulated by 3-6-fold. Indices for the intensity of the early growth and germination of radish in the soil with glauconite, sodium humate, and biochar were increased by 37–125% (p < 0.01) from the reference value.

After the application of biochar, sodium humate, and “Baikal EM-1”, the number of soil bacteria was 66–289% (p < 0.01) higher than the reference value. At the same time, catalase and dehydrogenases were observed to be inhibited by up to 35% (p < 0.01) in variants with bioremediation agents and petroleum hydrocarbons relative to the reference values. The maximum stimulation of the biological activity (IIBS) of Haplic Chernozem Calcic was observed after applying sodium humate and biochar, with 70 and 66% increases from the reference value, respectively.

Considering the net cost of bioremediation agents, the maximum cost efficiency was found for “Baikal EM-1”, sodium humate, and biochar, with values of 110, 527, and 847 USD·10³/ha, respectively. After using “Baikal EM-1”, sodium humate, and biochar, the ecological state of Haplic Chernozem Calcic was restored.