Estimation of the Enzymatic Activity of Haplic Chernozem under Contamination with Oxides and Nitrates of Ag, Bi, Te and Tl

Abstract

Sustainable agriculture is only possible if the agroecological services of the soil are preserved. Soil contamination with rare elements such as silver (Ag), bismuth (Bi), tellurium (Te), and thallium (Tl) is less studied, but their toxicity is no less high than in other heavy metals. Activity of soil enzymes is of great importance for the healthy functioning of soils, agroecosystem services, and their fertility. It is necessary to assess the ecological state of black soil using the most sensitive and informative indicators of the state of soils—their enzymatic activity. The objective of this research was to evaluate changes in activity of five priority soil enzymes (catalase, dehydrogenases, invertase, phosphatase, and urease) when contaminated with oxides and nitrates of Ag, Bi, Te, and Tl in a laboratory model experiment. The integral toxicity of nitrates and oxides of Ag, Bi, Te, and Tl was assessed by the integrated index of soil enzymatic activity. A comparison of the toxicity of oxides and nitrates of each element, according to the integrated index of soil enzymatic activity, allowed us to establish that Ag oxide is more toxic than Ag nitrate; Bi oxide is equivalent in its toxicity to Bi nitrate; and Tl and Te oxides are less toxic than Tl and Te nitrates. When contaminated with oxides, the most informative indicators are activity of invertase (Ag), urease (Bi, Tl), and phosphatase (Te). When contaminated with nitrates, the most informative indicators are activity of phosphatase (Ag) and invertase (Bi, Tl, and Te). Activity of phosphatase and catalase are the most sensitive to contamination by oxides and nitrates of Ag, Bi, Tl, and Te, and dehydrogenases, invertase, and urease are the least sensitive.

1. Introduction

Sustainable agriculture is only possible if the agro-ecological services of the soil are preserved. More than 90% of human food is obtained from soils [1]. At the same time, the anthropogenic load on soils is growing exponentially and causes economic, environmental, and social damage from reduced soil health, poor crop quality, soil and agricultural product pollution, and increased morbidity and mortality [2,3,4,5,6]. The problems of soil pollution with heavy metals and metalloids have recently become highly relevant. The study of 30 priority chemical elements allowed the establishment of a low degree of knowledge of the ecotoxicity parameters of soil contamination with rare elements (REs): Ag, Bi, Te, and Tl [7]. At the same time, the effects of soil pollution with Ag, Bi, Te, and Tl have been studied to a small extent. However, soil contamination of the Earth’s crust with REs is widespread. Such elements include silver, bismuth, selenium, thallium, indium, cadmium, tellurium, and thallium, which are called chalcophile elements [8,9,10,11].

The ways of self-contamination are also specific for each RE, but they have common sources. The main anthropogenic sources of soil contamination with silver, tellurium, and thallium are the presence of these REs in aerosol impurities of thermal power plants during the combustion of coal and because of the activities of non-ferrous and ferrous metallurgy enterprises [12,13,14,15,16,17,18]. The highest concentrations of thallium in soil were observed near coal mines, including up to 20,000 ppm or 2% of soil mass, which is 100,000 times higher than the average thallium content in soil [19]. In addition, the content of thallium and tellurium in coals is 10–100 times less than other heavy metals and metalloids [14]. Other sources of RE pollution include landfills of solid household waste, production of photographic and electrical materials, use of pesticides, use of sewage sludge as fertilizers, mining, industrial production of glass, ceramics and rubber, and nuclear power [20,21,22,23].

Soil contamination with Ag, Bi, Te, and Tl is very toxic to animals, plants, and humans. By accumulating in the soil in large quantities, silver inhibits the total number of microorganisms and enzymatic activity [24,25,26,27,28,29,30]. The increased content of bismuth in the soil leads to its accumulation in plants [23,31,32,33,34,35]. By transmitting through food chains, bismuth enters the human body, causing numerous pathologies [36,37,38,39,40]. The scale and degree of tellurium contamination of soils and agricultural plants are increasing every year [17,41,42,43,44,45,46]. Soil contamination with thallium creates a high risk of thallium entering agricultural crops and further along food chains [47,48,49].

In this regard, it is relevant to determine the system of biological indicators of soil condition that allow assessing the pollution degree and restoration degree of the soil’s ecological state. According to studies of soil toxicity caused by heavy metal pollution, enzymatic activity is considered one of the most effective and fastest biological indicators, as the first to react to soil pollution compared to other biological indicators [50,51,52]. The aim is to evaluate the change in the enzymatic activity of haplic chernozem when contaminated with oxides and nitrates of Ag, Bi, Te, and Tl.

The novelty of the work lies in the fact that, previously, the state of soils contaminated with rare elements (heavy metals) such as Ag, Bi, Te, Tl was not assessed by changes in the activity of enzymes of the classes of oxidoreductases and hydrolases. The application of this approach to the biomonitoring of heavy metal contaminated soils contributes to sustainable agriculture and control for restoration of agroecosystem services.

According to this aim, the objectives of the study included: (1) to evaluate the change in activity of each enzyme of classes of oxidoreductases (catalase and dehydrogenases) and hydrolases (invertase, phosphatase, and urease) when contaminated with oxides and nitrates of Ag, Bi, Te, and Tl; (2) to compare the ecotoxicity of oxides and nitrates of Ag, Bi, Te, and Tl; (3) to determine the most sensitive and informative enzyme by soil contamination by oxides and nitrates of Ag, Bi, Te, and Tl; (4) to measure the highest permissible concentration of Ag, Bi, Te, and Tl in haplic chernozem.

2. Materials and Methods

2.1. Soil Selection

Samples of haplic chernozem were taken from the topsoil of 0–20 cm on the arable land of the Botanical Garden of the Southern Federal University. The Botanical Garden is in the center of Rostov-on-Don (Rostov Region, Russia) and is characterized by a minimal anthropogenic impact on the soil cover. Haplic chernozems are among the most fertile soils in Russia and the world [53]. Studying the effects of pollution with Res on haplic chernozem allows us to predict the consequences of pollution for other fertile soils in the world.

2.2. Experiment Simulation

Ag, Bi, Tl, and Te were introduced in the form of oxides and nitrates (in terms of element): Silver oxide—Ag₂O, CAS No.: 20667-12-3 (Sigma-Aldrich, St. Louis, MO, USA), bismuth(III) oxide—Bi₂O₃ (CAS No.: 1304-76-3, Sigma-Aldrich), thallium(III) oxide—Tl₂O₃ (CAS No.: 1314-32-5, Sigma-Aldrich), tellurium dioxide—TeO₂ (CAS No.:7446-07-3, Sigma-Aldrich), silver nitrate—AgNO₃ (CAS No.: 7761-88-8, Sigma-Aldrich), bismuth(III) nitrate—Bi(NO₃)₃ (CAS No.: 10035-06-0, Sigma-Aldrich), thallium(III) nitrate—Tl(NO₃)₃ (CAS No.: 10102-45-1, Sigma-Aldrich), and tellurium(IV) nitrate basic—Te₂O₃(OH)NO₃ (CAS No.: 64535-94-0, Sigma-Aldrich).

Soil pollution was expressed in approximate permissible concentrations (APC): 0.5, 1, 3, 10, and 30. Oxides of Ag, Bi, Tl, and Te are the most common form of metal compounds found in soil. Oxides are the insoluble form of the compound. Nitrates of Ag, Bi, Tl, and Te are the soluble form of the metal compound in soil also commonly found in soil. The scheme and stages of the experiment are shown in Figure 1. This range of concentrations was chosen to approximate the response of the soil enzyme pool at higher levels of contamination and to calculate the recommended concentrations of metals in each form (oxide and nitrate).

In the prepared soil, dried and freed from roots and other foreign inclusions, sifted through a 2 mm sieve, oxides (in dry form) and nitrate solutions (in terms of an element) of Ag, Bi, Tl, and Te were added. After the introduction of metals, the soil was moistened and incubated for 10 days at 24–25 °C and a soil moisture level of 25–30%. After the end of incubation, the soil was dried and ground through a 1 mm sieve. Soil samples were used to determine the enzymatic activity of soils: activity of catalase, dehydrogenases, phosphatase, invertase, and urease.

2.3. Methods for Assessing Enzymatic Activity

To assess the ecological state of soils after the contamination by Ag, Bi, Tl, and Te, the activity of catalase, dehydrogenases, phosphatase, invertase, and urease was analyzed (

Table 1. Methods for studying the enzymatic activity of soils.

№	List of Determined Enzymes	EC	Methods of Measurement
Class of oxidoreductases
1.	activity of catalase	EC 1.11.1.6.	by the volume of decomposed oxygen during the decomposition of hydrogen peroxide, mL O2/1 g of soil dry/1 min
2.	activity of dehydrogenases	EC 1.1.1	for the reduction of tetrazolium salts to formazan, mg triphenylformazan (TPF)/10 g of soil dry/24 h
Class of hydrolases
3.	activity of invertase (β-fructofuranosidase)	EC 3.2.1.26	by the amount of glucose during the hydrolysis of sucrose, colorimetrically, using Felling’s reagent, mg glucose/10 g of soil dry/24 h
4.	activity of urease (amidohydrolase)	EC 3.5.1.5.	by the amount of ammonia with Nessler’s reagent, with hydrolysis of carbamide, mg NH4+/10 g of soil dry/24 h
5.	activity of phosphatase	EC 3.1.3.1-2	by the amount of inorganic phosphorus during hydrolysis of sodium nitrophenol phosphate, µg p-nitrophenol/1 g of soil dry/1 h

) [54].

The informational value was calculated by the correlation coefficient (R) between the content of Ag, Bi, Tl, and Te in the soil and the value of the indicators of enzymatic activity.

The degree of sensitivity of the indicators of enzymatic activity was judged by the degree of decrease in its values in the options with contamination of Ag, Bi, Tl, and Te compared with the control.

2.4. Measurement

The integral index of enzymatic activity (IIEA) of the soil was used to give an integral assessment of the condition of soils after any chemical pollution. For the calculation of the IIEA, the value of each of the above indicators on the control (in unpolluted soil) was taken as 100%. The percentages in other experimental variants (in polluted soil) were expressed as a percentage relative to the control. For the IIEA condition, a maximum value of each index (100%) was chosen from the array data, and in reference to the value of this index, was expressed for other variants of experiments by Equation (1):

$$E_{1 }=\frac{E_{\mathrm{x}}}{E_{\mathrm{cont}}}\times 100\%$$

(1)

where E₁ is the relative score of the enzyme activity; E_x is the actual value of the enzyme activity; and E_ref is the reference value of the enzyme activity.

The relative values of several indicators, activity of catalase, dehydrogenases, invertase, phosphatase, and urease, were summed.

Thereafter, the average assessment point of the studied enzymes was calculated for each variant by Equation (2):

$$E_{av}=\frac{E_1+E_2+\dots +E_{\mathrm{n}}}{\mathrm{N}}$$

(2)

where E_av is the average estimated score of enzyme activity; E₁…E_n is the relative score of the enzyme activity; and N is the amount of enzyme activity.

The integral index of enzymatic activity (IIEA) of the soil is calculated by Equation (3):

$$\mathrm{IIEA}=\frac{E}{E_{\mathrm{ref}}}\times 100\%$$

(3)

where E is the average estimated score of enzyme activity and E_ref is the reference score of enzyme activity.

2.5. Statistical Processing

An analysis of the rate of variation (standard deviation) at p ≤ 0.05 was carried out to determine the reliability of the results. Data were means of triplicate. Statistical data processing was carried out using Statistica 12.0 and the Python 3.6.5 Matplotlib package. The correlation nonparametric Spearman’s coefficient was calculated between the concentration of AgNPs and the average of the biological indicators. These statistical methods make it possible to evaluate the activity of enzymes in comparison with the control, as well as to evaluate the information content and sensitivity of enzymes of different classes after contamination by Ag, Bi, Tl, and Te.

3. Results

3.1. Changes in Activity of Oxidoreductases

Activity of oxidoreductases is an informative and sensitive indicator for soil contamination with heavy metals, petroleum hydrocarbons, and other xenobiotics [4,55,56,57,58,59]. Activity of catalase in haplic chernozem when contaminated with oxides and nitrates of Ag, Bi, Tl, and Te is shown in Figure 2.

With an increase in the concentration from 1, 3, 10, and 30 APC of Ag oxide, a decrease in catalase activity was observed by 10, 26, 42, and 45% relative to the control, respectively. At the lowest concentration of 0.5 APC, no significant differences from the control were observed. However, when contaminated with Ag nitrates, the toxic effect was already detected at 0.5 APC; the inhibition of catalase activity was 21% lower than in the control. A more significant inhibition than with oxide contamination was also found with an increase in the concentration of Ag nitrate from 1 to 30 APC, by 34–50% lower than in the control.

Bi oxide at doses of 0.5, 1, and 3 APC did not affect enzyme activity. At maximum concentrations of Bi 10 and 30 APC, activity inhibition was observed by 37 and 43% relative to the control, respectively. Bi nitrate, as well as Ag nitrate, already had a toxic effect at a dose of 0.5 APC, 27% below the control. With an increase in the dose (from 1 to 30 APC), the inhibition was significant—32–40% relative to the control.

At a minimum concentration of 0.5 APC of Tl and Te oxides, inhibition was 16 and 13%, respectively. With an increase in the concentration from 1 to 30 APC Tl, inhibition was 39–53% relative to the control. Te oxide at concentrations of 1 and 3 APC is less toxic, by inhibiting catalase activity by 19 and 23% relative to the control. When the dose was increased, a similarity with Tl was observed. Tl and Te nitrates had a significant inhibition of activity at 0.5 APC, by 30 and 24%. With an increase in the concentration to 30 UDC, inhibition was 36–75% (Tl nitrate) and 25–75% (Te nitrate).

Activity of dehydrogenases in soil contaminated with Ag oxide at 0.5 APC, compared with catalase, allowed the stimulation of enzyme activity to be 7% higher than in the control, as a manifestation of hormesis (Figure 3). Hormesis is the phenomenon of activity stimulation of a biological indicator by small doses of pollutants in the soil [60].

With an increase in the concentration of Ag oxide to 1.3 and 10 APC, activity inhibition was 7, 13, and 15%, respectively. The maximum inhibition was observed at a concentration of 30 APC—32% lower than in the control. Ag nitrate had a more toxic effect on dehydrogenase activity: already at 0.5 APC, inhibition was 13% lower than the control, and with an increase in concentration from 1 to 30 APC, the change in activity was 18–32% lower than in the control.

Bi nitrate and oxide had almost the same effect on activity of dehydrogenases in haplic chernozem. The difference between the effects of different forms of chemical compounds was observed only at a dose of 0.5 and 1 APC: Bi oxide inhibited the dehydrogenase activity by 9 and 16%, and nitrate by 19 and 21%, relative to the control. With an increase in the concentration from 1 to 30 UDC, inhibition was the same for both oxide and nitrate, decreased by an increase in concentration by 20–30% than in the control.

Tl oxide inhibited enzyme activity at 0.5 and 1 APC by 14 and 24%, and nitrate by 21 and 27%, respectively. With an increase in the concentration from 1 to 20 APC, activity inhibition in haplic chernozem when contaminated with Tl oxide decreased by 24–39% relative to the control, and nitrate by 27–65%.

Tellurium affected activity of dehydrogenases similarly to Te. In small doses (0.5 APC) of Te oxide, a 9% decrease in activity was observed, and ofTe nitrate, F 24%. With an increase in the concentration from 1 to 30 APC, a significant inhibition of activity was observed with the addition of Te oxide by 14–28% and of Te nitrate by 27–65%.

Activity of oxidoreductases was quite sensitive to the introduction of oxides and nitrates of Ag, Bi, Tl, and Te. According to the data obtained above and the assessment of the correlation with the dose of each element, the series of informativeness of each biological indicator in relation to metal oxides and nitrates were compiled.

The close correlation of activity of oxidoreductases in haplic chernozem with the dose of the introduced oxide and nitrate of Ag, Bi, Tl, and Te was estimated by the correlation coefficients (r) and presented as series 4 and 5 (catalase), and series 6 and 7 (dehydrogenases):

Activity of catalase:

Tl (−0.65) < Ag (−0.81) < Bi (−0.88) = Te (−0.88)

(4)

Bi (−0.51) < Ag (−0.67) <Te (−0.76) (2) < Tl (−0.81)

(5)

Activity of dehydrogenases:

Bi (−0.67) < Tl (−0.73) < Te (−0.82) < Ag (−0.89)

(6)

Te (−0.62) < Bi (−0.64) < Tl (−0.70) < Ag (−0.72)

(7)

According to these series, Bi and Te oxides and Tl nitrate have the greatest toxic effect on catalase activity; nitrate and Ag oxide have the greatest toxic effect on dehydrogenase activity.

3.2. Changes in Hydrolase Activity

Hydrolases are soil enzymes involved in carbon, nitrogen, and phosphorus cycles [61]. Phosphatase activity during contamination with oxides and nitrates of Ag, Bi, Tl, and Te is shown in Figure 4. Ag oxide at a dose of 0.5 and 1 APC inhibited phosphatase activity by 26 and 38% relative to the control. With an increase in concentration, this trend persisted: from 3 to 30 APC, the decrease in enzyme activity was the same for all doses and was 40–42% lower than the control.

Ag nitrate had a less toxic effect on activity of phosphatase in haplic chernozem. At 0.5, 1, and 3 APC of Ag nitrate, no significant decrease in activity relative to the control was observed. A decrease in enzyme activity was observed at 10 and 30 APC of Ag nitrate by 12 and 20% below the control.

Bi oxide at a minimum concentration (0.5 APC) had no effect on activity of the enzyme. With an increase in the concentration from 1 to 30 APC of Bi oxide, activity inhibition was observed by 12–32% below the control. Bismuth nitrate in 0.5 APC also did not significantly affect enzyme activity. At the same time, when the concentration of 1 APC was reached, activity inhibition was 38%, and with an increase in the concentration of Bi nitrate from 3 to 30 APC, 44–58% below the control.

Tl and Te oxides at doses of 0.5, 1, 3, and 10 APC had no significant effect on phosphatase activity. Only at a concentration of 30 APC, activity inhibition was observed by 13 and 12%, respectively, for Tl and Te oxides.

Invertase activity under the influence of small doses of Ag oxide (0.5, 1 APC) did not significantly differ from the control (Figure 5). With an increase in concentration to 3, 10, and 30 APC, activity inhibition was observed by 11, 24, and 45% relative to the control.

Ag nitrate at low doses (0.5 and 1 APC), as well as Ag oxide, did not affect invertase activity. With an increase in the concentration of Ag nitrate to 3, 10, and 30 APC, activity inhibition was 12, 13, and 24% relative to the control.

Bi oxide did not significantly affect invertase activity at a dose of 0.5 UDC. At a dose of 1 to 30 APC, the decrease in activity was 9–20% relative to the control. Bi nitrate did not significantly affect invertase activity at low doses of 0.5, 1, and 3 APC. At the same time, Bi nitrate at concentrations of 10 and 30 APC inhibited enzyme activity by 28 and 72% relative to the control.

Tl oxide already inhibited invertase activity by 13 and 15% at 0.5 and 1 APC. With an increase in the concentration of Tl oxide from 3 to 30 APC, inhibition was 17–22%. In contrast, Tl nitrate at low doses of 0.5, 1, and 3 UDC had no significant effect on invertase activity. Only at 10 and 30 APC was a decrease in enzyme activity observed by 22 and 60% relative to the control, respectively.

Tellurium oxide at 0.5 APC had no significant effect on soil enzyme activity. By starting with doses of 1, 3, 10, and 30 APC, the activity decrease was 8, 13, 17, and 23% relative to the control. Tellurium nitrate had no effect on invertase activity at low doses of 0.5, 1, and 3 APC. When the concentration was increased to 10 and 30 APC, activity inhibition was 54 and 77% relative to the control.

Influence of contamination with oxides and nitrates of Ag, Bi, Tl, and Te urease activity in haplic chernozem is shown in Figure 6. Silver oxide inhibited urease activity from a low dose of 0.5 APC to a maximum dose of 30 APC in this study—11–28% lower than in the control. At the same time, Ag nitrate was less toxic judging by the activity of urease in haplic chernozem. At a low dose of Ag nitrate (0.5 APC), the effect of hormesis was observed, with stimulation by 10% relative to the control. At 1, 3, and 10 APC, there were no significant differences from the control.

Only at a maximum concentration of Ag nitrate—30 APC—was the decrease in activity 11% relative to the control.

Bi oxide at a dose of 0.5 and 1 APC had no effect on urease activity. Only when the dose was increased to 3, 10, and 30 UDC, a decrease in enzyme activity was observed by 12.23 and 32% relative to the control. Bi nitrate, as well as Ag nitrate, at a dose of 0.5 APC stimulated urease activity up to 13% relative to the control. At 1 APC of Bi nitrate, no significant differences from the control were found. At a Bi nitrate dose of 3, 10, and 30 APC, inhibition was 11, 13, and 16% relative to the control.

Tl oxide had no significant effect on urease activity at doses of 0.5, 1, 3, and 10 APC. Activity inhibition by 17% was observed only at 30 APC. Tl nitrate had no effect at doses of 0.5, 1, and 3 APC. Only at concentrations of 10 and 30 APC, a decrease in activity was observed by 12 and 18% relative to the control.

Te oxide at low doses caused stimulation of urease activity by 14 and 12% relative to the control. At 3, 10, and 30 APC, no significant differences from the control were observed. Tellurium nitrate at 0.5 and 1 APC stimulated urease activity by 6 and 3%. At 3 UDC, no significant differences from the control were found. Urease activity inhibition was detected at concentrations of Te nitrate 10 and 30 APC—12 and 16%, respectively.

The close correlation of activity of hydrolases in haplic chernozem with the dose of the introduced oxide and nitrate of Ag, Bi, Tl, and Te was estimated by the correlation coefficients (r) and presented as series 8 and 9 (phosphatase), series 10 and 11 (invertase), and series 12 and 13 (urease).

Activity of phosphatase:

Ag (−0.45) < Bi (−0.67) < Tl (−0.73) < Te (−0.82)

(8)

Bi (−0.68) < Te (−0.73) (2) < Tl (−0.79) <Ag (−0.95)

(9)

Activity of invertase:

Tl (−0.62) < Bi (−0.80 < Te (−0.84) < Ag (−0.98)

(10)

Ag (−0.90) < Te (−0.93) < Tl (−1.00) = Bi (−1.00)

(11)

Activity of urease:

Te (−0.62) < Ag (−0.82) < Bi (−0.90) < Tl (−0.95)

(12)

Bi (−0.71) < Ag (−0.79) < Tl (−0.89) = Te (−0.89)

(13)

According to these series, the greatest toxic effect on phosphatase activity is exerted by Te oxide and Ag nitrate; on invertase activity, Ag oxide and Tl and Bi nitrates; and on ureases, Tl oxide and Tl and Te nitrates.

3.3. Changes in the Integral Index of Soil Enzymatic Activity (IIEA)

According to the obtained indicators of enzymatic activity, an integrated index of soil enzymatic activity (IIEA) was calculated (Figure 7). At Ag concentrations from 0.5 to 1 APC, no differences in oxides and nitrates in their toxicity (in enzymatic activity) were revealed. However, with an increase in the concentration from 3 to 30 APC, the difference between the toxicity of oxide and nitrate was 7, 6 and 11% for 3, 10, and 30 APC, respectively. Bi nitrate was more toxic than oxide starting from a dose of 1 APC by 8%. With an increase in concentration, greater toxicity was found for nitrate contamination—9 and 13% for 3 and 30 APC, respectively. At 10 APC, Bi nitrate and oxide have the same toxicity.

Tl nitrate toxicity compared to its oxide was detected already with 0.5 APC—8%; with an increase in concentration, an increase in the toxicity of nitrate was observed by 9, 15, 22, and 32% for 1, 3, 10, and 30 APC, respectively. By analogy with Tl, Te nitrate toxicity was already detected with 0.5 APC—16%; with an increase in concentration, an increase in nitrate toxicity was observed by 17, 23, 41, and 42% for 1, 3, 10, and 30 APC, respectively.

The toxicity series of oxides and nitrates were calculated according to IIEA data (series 14 and 15).

Toxicity of oxide of Ag, Bi, Tl, and Te, according to the IIEA presented as series 14:

Tl (88) < Bi (82) = Te (82) < Ag (78)

(14)

Toxicity of nitrate of Ag, Bi, Tl, and Te, according to the IIEA presented as series 15:

Ag (83) < Bi (75) < Tl (65) < Te (60)

(15)

For Bi, the difference in toxicity between oxides and nitrates was not revealed, while for Ag, the oxide is more toxic, especially at a high concentration of 30 APC, and for Tl and Te, the nitrate (15–42%) relative to the control. A comparison of the toxicity in oxides and nitrates of each element allowed us to establish that Ag oxide is more toxic than Ag nitrate; Bi oxide is equivalent in its toxicity to Bi nitrate; and Tl and Te oxides are less toxic than Tl and Te nitrates.

4. Discussion

4.1. Informative Value of Biological Indicators

The informative value of each enzyme was evaluated by correlation coefficients with the content of each heavy metal (

Table 2. Correlation coefficients (r) between the content of Ag, Bi, Tl, and Te in soil and enzymatic activity (α = 0.05).

Biological Indicators	Ag		Bi		Tl		Te
	Oxide	Nitrate	Oxide	Nitrate	Oxide	Nitrate	Oxide	Nitrate
Catalase activity	−0.81 *	−0.67 *	−0.89 *	−0.51	−0.65	−0.81 *	−0.88 *	−0.76 *
Dehydrogenase activity	−0.89	−0.70 *	−0.67 *	−0.64	−0.73 *	−0.70 *	−0.82 *	−0.62
Phosphatase activity	−0.45	−0.95 *	−0.71 *	−0.68	−0.83 *	−0.79	−0.96 **	−0.73 *
Invertase activity	−0.98 *	−0.90 **	−0.80	−1.00 **	−0.62	−0.99 **	−0.84 *	−0.93 **
Urease activity	−0.82 *	−0.79 *	−0.90	−0.71 *	−0.95 **	−0.89 *	−0.60	−0.89 **
IIEA	−0.85 *	−0.82 *	−0.84 *	−0.86 *	−0.80 *	−0.87 *	−0.89 *	−0.83 *

Note: * p < 0.05, ** p < 0.01.

). This indicator allows the assessment of the closeness of the relationship of changes in enzyme activity with an increase in the concentration of Ag, Bi, Tl, and Te [62]. The higher the value of the correlation coefficient (r) and the closer to (→−1.00), the more informative the enzyme is.

The series of biological indicators according to the degree of informative value in the case of contamination with oxides of Ag, Bi, Tl, and Te are presented below.

Ag oxide: invertase (−0.98) > dehydrogenases (−0.89) > urease (−0.82) > catalase (−0.82) > phosphatase (−0.45)

Bi oxide: urease (−0.90) > catalase (−0.89) > invertase (−0.80) > phosphatase (−0.71) > dehydrogenases (−0.67)

Tl oxide: urease (−0.95) > phosphatase (−0.83) > dehydrogenases (−0.73) > catalase (−0.65) > invertase (−0.62)

Te oxide: phosphatase (−0.96) > catalase (−0.88) > invertase (−0.84) > dehydrogenases (−0.82) > urease (−0.60)

When contaminated with oxides of Ag, Bi, Tl, and Te, the most informative indicators are activity of invertase (Ag), urease (Bi, Tl), and phosphatase (Te), and the least are phosphatase (Ag), dehydrogenase (Bi), invertase (Tl), and urease (Te).

The series of biological indicators according to the degree of informative value in the case of nitrate contamination are presented below.

Ag nitrate: phosphatase (−0.95) > invertase (−0.90) > urease (−0.79) > dehydrogenases (−0.72) > catalase (−0.67)

Nitrate Bi: invertase (−1.00) > urease (−0.71) > phosphatase (−0.68) > dehydrogenases (−0.64) > catalase (−0.51)

Tl nitrate: invertase (−0.99) > urease (−0.89) > catalase (−0.81) > phosphatase (−0.79) > dehydrogenases (−0.70)

Te nitrate: invertase (−0.93) > urease (−0.89) > catalase (−0.76) > phosphatase (−0.73) > dehydrogenases (−0.62)

When contaminated with Ag, Bi, Tl, and Te nitrates, the most informative indicators are activity of phosphatase (Ag) and invertase (Bi, Tl, and Te), and the least informative are activity of catalase (Ag, Bi) and dehydrogenases (Tl, Te).

4.2. Sensitivity of Biological Indicators

The enzymatic activity of soils during soil contamination with heavy metals, along with microbiological properties, is a sensitive biological indicator [63]. The degree of indicator sensitivity was judged by the degree of decrease in its values in the options with contamination compared to the control.

Table 3. The degree of reduction of biological indicators in terms of sensitivity to pollution Ag, Bi, Tl, and Te, % of the control.

Biological Indicators	Ag		Bi		Tl		Te
	Oxide	Nitrate	Oxide	Nitrate	Oxide	Nitrate	Oxide	Nitrate
Catalase activity	74	62	81	65	60	51	70	50
Dehydrogenase activity	88	77	81	76	73	54	82	47
Phosphatase activity	63	91	80	62	98	47	97	33
Invertase activity	82	88	87	77	83	83	87	74
Urease activity	82	99	85	96	98	91	103	96
IIEA	78	83	83	75	82	65	88	60

shows the values of the enzyme sensitivity assessment.

According to the results of this study, when contaminated with oxides of Ag, Bi, Tl, and Te, the sensitivity of enzymes is presented in the form of series.

Ag oxide: dehydrogenases (88) < invertase (82) = urease (82) < catalase (74) < phosphatase (63)

Bi oxide: invertase (87) < urease (85) < dehydrogenases (81) = catalase (81) < phosphatase (80)

Tl oxide: phosphatase (98) = urease (98) < invertase (83) < dehydrogenases (82) = catalase (60)

Te oxide: urease (103) < phosphatase (97) < invertase (87) < dehydrogenases (73) = catalase (70)

Phosphatase (Ag, Bi) and catalase (Tl, Te) have the highest sensitivity to oxide contamination, while dehydrogenases (Ag), invertase (Bi), phosphatase (Tl), and urease (Te) have the lowest sensitivity.

The sensitivity of enzymatic activity in the case of contamination with nitrates of Ag, Bi, Tl, and Te is presented in the form of series:

Ag nitrate: urease (99) < phosphatase (91) < invertase (88) < dehydrogenases (77) < catalase (62)

Bi oxide: urease (96) < invertase (77) < dehydrogenases (76) < catalase (65) < phosphatase (62)

Tl oxide: urease (91) < invertase (83) < dehydrogenases (54) < catalase (51) < phosphatase (47)

Te oxide: urease (96) < invertase (76) < catalase (50) < dehydrogenases (47) < phosphatase (33)

The greatest sensitivity to nitrate contamination was found in catalase (Ag) and phosphatase (Bi, Tl, Te), and the least in urease (Ag, Bi, Tl, and Te). The high sensitivity of catalase, along with phosphatase, was found when contaminated with nitrates of Bi and Tl.

The high sensitivity of catalase in the case of soil contamination with oxides and nitrates of Ag, Bi, Tl, and Te is associated with the active course of biochemical processes in the soil, in particular, the carbon cycle [64,65]. Catalase is a stable enzyme in the soil and for the enzyme activity to be inhibited, it is necessary that the toxicant affects the microbiological component of the soil.

Phosphatase is a sensitive enzyme in the case of soil contamination with a wide range of heavy metals [66,67,68]. Phosphatase, along with dehydrogenases and microbial biomass, is considered a marker of soil contamination with heavy metals [69,70]. Such indicators can reflect the direction and degree of biochemical reactions in the soil and serve as potential biological indicators for the diagnosis of soil health [52].

The most informative enzymes in the case of contamination with Ag, Bi, Tl, and Te include the enzymes of the hydrolase class, and the most sensitive, the enzymes of the oxidoreductase class. In this regard, all biological indicators used in the work have confirmed their compliance with the requirements for indicators used for monitoring, diagnostics, and normalization of chemical contamination of soils [4].

A comparison of the greater sensitivity of silver oxides and nitrates made it possible to reproduce the previously presented small studies assessing the toxicity of various chemical forms of Ag [71,72,73]. Quite a lot of studies have been conducted to assess the toxicity of Ag nitrate, and the toxicity of Ag oxide has been studied to a lesser extent [74,75].

Bismuth, compared to Ag, Tl, and Te, was equally toxic both in the form of oxide and in the form of nitrate. Previously, an analysis of the ecotoxicity of Bi chemical compounds made it possible to establish a greater toxicity of nitrate than oxide [58,76]. This is because nitrate has good solubility and greater mobility in the soil solution of Bi3+ cations, and the water-insoluble bismuth oxide showed a slightly less negative effect.

The toxicity of Tl and Te in haplic chernozem was previously assessed only in the form of oxides [57,77]. Even at a minimum concentration in the soil (less than 1 ppm), Tl oxide has a toxic effect on living organisms and soils [14,48,78]. The high informative value of enzymes of the oxidoreductase class (catalases and dehydrogenases) and the sensitivity of microbiological parameters when contaminated with Te oxide were established [57]. The toxicity of Te, as well as Tl, is assessed as high already at a minimum concentration in soil, water, and other environmental objects [17,45,79].

Based on the results of the study, regression equations were constructed that characterize ecosystem and agroecosystem services of contaminated soil. With their help, the concentrations of Ag, Bi, Tl, and Te leading to the violation of certain ecological functions of soils were calculated (

Table 4. Scheme of ecological regulation of the content of Ag, Bi, Tl, and Te in ordinary chernozem according to the degree of violation of ecological functions and optimal methods of soil sanitation.

Metal	Rare Metal’s Content in Soil, mg/kg
Ag	<0.197	0.197–0.395	0.395–3.206	>3.206
Bi	<0.513	0.513–0.968	0.968–6.522	>6.522
Tl	<0.376	0.376–0.601	0.601–2.454	>2.454
Te	<0.999	0.999–1.475	1.475–4.753	>4.753
Decrease in soil IIEA	<5%	5–10%	10–25%	>25%
Disturbed ecological functions of the soil	–	Informational	Chemical, physico-chemical, biochemical, holistic	Physical
Degree of soil pollution	Not polluted	Weakly polluted	Medium polluted	Heavily polluted
Soil sanitation method	Not required	Phytoremediation, leaching	Chemical melioration	Removal of contaminated soil layer

).

To calculate the environmentally safe concentration of each metal in the soil, its most toxic form was used: for Ag—oxide, and for Bi, Tl, and Te—nitrate. The breakdown of the ecological functions of the soil occurs in a certain order, and it is advisable to use the soil as an indicator of the violation of ecosystem and agro-ecosystem services according to IPFA [4]. With a decrease in IPFA by less than 5%, soil functions are not disturbed. A decrease in the values of IPBS by 5–10% diagnoses a violation of information functions: by 10–25%—biochemical, physico-chemical, chemical and holistic functions; more than 25%—physical functions. As can be seen from Table 4, if the Ag content does not exceed 0.197 mg/kg, then the soil is functioning normally. If the concentration of Ag is from 0.197 to 0.395 mg/kg, there is a violation of the informational ecological functions of the soil; from 0.395 to 3.206 mg/kg, in addition to information, chemical, physico-chemical, biochemical, and integral functions are violated; more than 3.206 mg/kg, there is also a disruption of the physical functions of the soil. Obviously, it is impossible to allow violations of the chemical, physicochemical, biochemical, and, most importantly, integral functions of the soil, which ensure soil fertility. Therefore, an Ag concentration of 0.395 mg/kg should be considered the maximum allowable concentration (MAC) of Ag. The most effective methods of rehabilitation of ordinary chernozem with Ag, Bi, Tl, and Te are presented. The higher the concentration of HMs in the soil, the more “radical” the rehabilitation method should be. If the content of Ag does not exceed 0.197 mg/kg and there is no violation of its ecological functions, then sanitation of the soil in this case is not required. If the concentration of Ag is from 0.197 to 0.395 mg/kg, then to reduce its concentration to 0.197 mg/kg or less, phytomelioration is sufficient to remove Ag from the soil. If the concentration of Ag reaches 0.395 to 3.206 mg/kg, then chemical reclamation is required, for example, the use of zeolites to bind Ag and prevent its entry into the food chain. If the Ag content exceeds 3.206 mg/kg, reclamation measures are less effective than the removal of the top layer of contaminated soil.

According to the degree of toxicity of the content of elements in the soil, heavy metals are arranged in the following order (from the lowest to the highest permissible concentration), mg/kg:

Ag (0.396) < Tl (0.601) < Bi (0.968) < Te (1.475)

Thus, the IIEA can be used to assess the degree of disruption of agro-ecosystem services. A similar approach has been tested in several studies [4], including silver [56] and bismuth [58]. The application of this approach contributes to the sustainable development of agricultural land. The use of the results of the study contributes to the transition to highly productive and environmentally friendly agriculture, obtaining competitive, environmentally friendly agricultural products, ensuring food and environmental security in the world.

5. Conclusions

According to the results of this study, it was found that when the chernozem was contaminated with Ag, Bi, Tl, and Te by five enzymes (invertase, urease, phosphatase, dehydrogenases, catalase), the maximum decrease in activity was observed with the introduction of Tl and Te. A comparison of the ecotoxicity of oxides and nitrates of each element allowed us to establish that Ag oxide is more toxic than Ag nitrate; Bi oxide is equivalent in its toxicity to Bi nitrate; and Tl and Te oxides are less toxic than Tl and Te nitrates. The most informative enzymes in the case of contamination with Ag, Bi, Tl, and Te include the activity of phosphatase, invertase, and urease (hydrolase class), and the most sensitive of enzymes, activity of catalase (oxidoreductase class) and phosphatase (hydrolase class). The highest permissible concentrations of contamination in haplic chernozem by Ag, Bi, Tl, and Te differ by several times, which emphasizes the toxicity of each element, mg/kg: Ag (0.396 mg/kg) < Tl (0.601 mg/kg) < Bi (0.968 mg/kg) < Te (1.475 mg/kg). The results of this study confirm the expediency of using activity of soil enzymes in biomonitoring and diagnostics of the ecosystem and agroecosystem services of soils with contamination of Ag, Bi, Tl, and Te. The application of this approach to biomonitoring heavy metal-contaminated soils contributes to sustainable agriculture and control for restoration of agroecosystem services.