УДК 599:574.4+577.15

Екологія. Екологічний моніторинг

O.M. Vasilyuk

Дніпропетровський національний університет ім. Олеся Гончара

Effect of Nickel on Aspartat Aminotransferase activity in Glechoma hederacea L. leaves subject to excretory function of Mammalia

Introduction

We have studied a variety sensitive and simple methods of environmental impact assessment, leading to creation of measures designed to preserve and restore natural biodiversity in areas where diversity has been depleted by man. Thus, the salt stress have been determined (Dzyubak and Vasilyuk, 2009; Vasilyuk and Dzyubak, 2009; Kulik and Vasilyuk, 2009; Shavrukov, 2013), the effect of herbicide and plant growth regulators (Vasilyuk and Kordin, 2005; Vasilyuk and Vinnіchenko, 2006; Vasilyuk and Grycenko, 2008), mechanisms of protection from heavy metals (HM) in plants and resistance against salinity (Varennes A. et al., 1996; Gardea-Torresdey J.L. et al., 2005; Sun et al., 2013; Martín et al., 2013), cold shock and the specificity and nonspecific biochemical reactions to these factors have been established (Hébrard et al., 2013; Naji and Devaraj, 2011). The mechanisms of plant resistance to HM (Perfetto B. et al., 2006; Lefcort et al., 2013) involves both mechanical mechanisms (Vasilyuk and Kulik, 2008, 2009, 2011, 2011) mechanical protection of HM (in the root system) and biochemical metabolisms: plant growth regulators (PGR), phytohormones, vitamins (Clemans, 2001; Bernini et al., 2010), organic compounds (Gallie, D.R., 2013) metabolism under the HM: Cd, Co, Mn, Cr (Vestena et al., 2011; Hameed A. et al., 2011; Hasan et al., 2011; Ruscitti et al., 2011; Millaleo et al., 2013; Becerril et al., 2013). We researched the effect of nickel on plant organisms, nickel being one of the most widespread pollutants in Dnipropetrovs’k region. Nickel belongs to the category of HM on account of its weight, density, toxicity (Alexeev, 1987; Bіlanich, 2008; Clemans, 2001). A reduction of toxicity of Ni ions has been facilitated by such environmental factors as the digging activity of Mammalia (Pakhomov and Vasilyuk, 2011, 2013). Digging activity by Mammalia (Bulakhov and Pakhomov, 2006) affects the physical (hardness, humidity, thermal treatment) and chemical properties of soils (aminoacid composition, soil respiration, microbial and enzymatic activity). Therefore, mammals as biotic factors play a role in ecological restoration of technologically contaminated and biodiversity - depleted areas. A creative environmental role in the creation of ecological buffers against anthropogenic impact on biota, in the process of self-purification of soil, and the restoration of biodiversity in conditions of human impacts on biota is played by mammals. The excretory function of Mammalia both directly and indirectly affected the chemical and biological processes, which was of great practical importance. Using the enzymes of nitrogen metabolism, as a highly sensitive indicator for environmental change, we determined the impact of HM (anthropogenic factors) and excretory function of Mammalia (environmental factor) on plant specimens.

Materials and methods

The experiment was carried out at the International Biosphere Prysamarsky Station (Andriivka village, Novomoskovsk district, Dnipropetrovsk region) in natural floodplain oak forest in a sward area with lime-ash oak and greater stitchwort (Stellaria holostea L.). We studied the excretory function of Mammalia, taking the European mole (Talpa europaea L.) as our example under the condition of artificial Ni soil pollution. As a control we selected an area unaffected by mammals and without Ni pollution. Ni was introduced onto the soil surface in the form of a salt solution Ni(NO₃)₂*6H₂O with concentrations of 0,2; 1,0; 2,0 g/m², which was equivalent to the presence of Ni at 1, 5, 10 times the dose of maximum allowable concentration (MAC). To prevent contamination of the surrounding soil layers by nickel we used isolated soil blocks for our experiment - on the perimeter of each plot vertically placed plates of inert impermeable material were placed into the ground to a depth of 20 cm. A month later we determined the total enzyme activity (nM pyruvic acid/ml*s) of Aspartate Aminotransferase (AST, 2.6.1.1.) by the of method of Polevoy and Maximov (2008), as indicators of the ecological condition of the environment, and the concentration of water-soluble fraction of protein (C, mg/ml) by method of Bradford (1976) in the leaves of Glechoma hederacea L., which dominated in this research area. It was found that AST and ALT (Alanine Aminotransferase (Alt, 2.6.1.2.)), are the part of the system which utilizes the primary product of photosynthesis in the C₄ aspartate plants group – aspartate which is formed in the leaf mesophyll of plants. The reliable difference between the samples in this paper was considered to be at p <0,05. The experiment on excretory function of Mammalia and Ni to restore the biodiversity of anthropogenically polluted areas on the background of exogenous nickel was performed under the following schemes:

- control (the area without pollution of Ni and excretory function of Mammalia), control Ni at a dose 1 МAС, combined effect of excretory function of Sus scrofa and Ni at a dose 1 МAС, combined effect of excretory function of Capreolus capreolus and Ni at a dose 1 МAС, control Ni at a dose 5 МAС, combined effect of Sus scrofa and Ni at a dose 5 МAС, combined effect of Capreolus capreolus and Ni at a dose 5 МAС, control Ni at a dose 10 МAС, combined effect of Sus scrofa and Ni at a dose 10 МAС, combined effect of Capreolus capreolus and Ni at a dose 10 МAС.

Results and discussion

Addition of salts of nickel in the range of concentrations (1 МAС, 5 МAС, 10 МAС) produced a reliable (t/t _{0, 05}=1,94; 3,38; 1,02) decrease in AST activity by 18% - 65% in the Glechoma hederacea L. for all variants of the experiment relative to control (the area without Ni pollution and without of excretory function of Mammalia), which disproves the null hypothesis H₀d=0 (under conditions of criterion of significant differences t/t _{0, 05}≥1, H₀d=0 is discarded). This fact proved the toxic influence of the metal on the growth and development of plants and soils in these concentrations. The concentration of water-soluble protein fraction in the Glechoma hederacea L. leaves under the range of Ni concentrations was inhibited in all variants of the experiment by 31%-64% (t/t _{0, 05}=1,50; 3,14; 3,87), which proved the toxic effect of nickel ions (Table 1).

Table 1

The effect of nickel on total Aspartate Aminotransferase activity and concentration of water-soluble protein fraction in the Glechoma hederacea L. leaves

The variant of the experiment	X± m	Value of experiment relative to control, %	t/t0,05
AST
Контроль	0,88±0,222	100,00	-
Ni 1 МAС	0,53±0,088	60,81	1,94
Ni 5 МAС	0,31±0,051	35,14	3,38
Ni 10 МAС	0,72±0,184	82,43	1,02
Water-soluble protein fraction
Контроль	1,82±0,285	100,00	-
Ni 1 МAС	1,26±0,061	69,40	1,50
Ni 5 МAС	0,67±0,033	36,79	3,14
Ni 10 МAС	1,20±0,127	65,60	3,87

Note: X - average; ± m - confidence interval; t/t_{0, 05} – criterion of significant differences when P <0.05; Н₀d = 0 is rejected under conditions t/t _0,05 ≥ 1;

Adding the nickel salts at a dose of 1 МAС under the influence of excretory function of Sus scrofa L. and Capreolus capreolus L. (combined effect of anthropogenic and natural factors) contributed to a reliable (t/t_0,05=1,78; 1,33) increase in AST activity by 53% and 41% compared with control (сontrol Ni 1 МAС) accordingly, increase in AST activity by 61% (t/t_0,05 =1,45) and 30% (t/t_0,05 =1,03) compared with control (control Ni 5 МAС) accordingly, whereas with an increasing concentration factor (Ni 10 MAC), activity of AST was significantly reduction by 25% (t/t_0,05=1,22) and 42% (t/t_0,05=1,78) compared with control (control Ni 10 МAС) accordingly. Thus, the excretory function of mammals promoted the intensification of nitrate metabolism under the medium and low concentrations of Ni, but under the high concentrations of the metal, the excretory function of Mammalia did not diminish the toxic effect of Ni, which proved the harmful effects of Ni on the process of self-healing in biological systems (table 2).

Table 2

The effect of combined excretory function of Mammalia and Ni on total Aspartate Aminotransferase activity in Glechoma hederacea L. leaves

	The variant of the experiment	X± m	Value of experiment relative to control, %	t/t0,05
Control: Ni 1МРС		0,53±0,081	100,00	-
Sus scrofa L. + Ni 1МРС		0,82±0,083	153,37	1,78
Capreolus capreolus L. + Ni 1МРС		0,75±0,088	140,05	1,33
Control: Ni 5МРС		0,31±0,051	100,00	-
Sus scrofa L. + Ni 5МРС		0,50±0,085	161,51	1,45
Capreolus capreolus L. + Ni 5 МРС		0,40±0,051	130,80	1,03
Control: Ni МРС		0,73±0,102	100,00	-
Sus scrofa L. + Ni 10 МРС		0,56±0,051	75,81	1,22
Capreolus capreolus L. + Ni 10МРС		0,43±0,084	58,06	1,78

Note: see Table 1

It was observed that the combined effect of excretory function of Sus scrofa L., Capreolus capreolus L and nickel reliably (t/t_0,05=4,64; t/t_0,05=2,00) increased the concentration of water-soluble protein fractions by 123% and 48% compared with controls (Ni 1 МAС) accordingly, by 32% and 8% (t/t_0,05=2,75; t/t_0,05=1,03) compared with controls (Ni 5 МAС) accordingly. The excretory function of Sus scrofa L. and Capreolus capreolus L under the maximum concentration of nickel (Ni 10 МAС) did not provide a reduction in metal toxicity, there was a reliable (t/t_0,05=1,58; t/t_0,05=1,11) reduction in the concentration of water-soluble proteins fraction by 21% and 10% compared to the control (control Ni 10 МAС). The excretory function of mammals under conditions of nickel salts at the maximum dose did not provide a reduction of metal toxicity due to the inability of biological systems to adapt and restore functional activity of metabolic processes at this concentration. The representatives of Mammalia reduced anthropogenic pressure, produced a reduction of toxicity of metal components in biosystems and the environment under the low and medium doses of nickel (Table 3).

Table 3

The effect of combined excretory function of Mammalia and Ni on the concentration of water-soluble protein fraction in Glechoma hederacea L. leaves

Варіанти досліду	X± m	М, %	t/t0,05
Control: Ni 1МРС	1,26±0,061	100,00	-
Sus scrofa L. + Ni 1МРС	2,83±0,257	223,81	4,64
Capreolus capreolus L. + Ni 1МРС	1,87±0,229	148,03	2,00
Control: Ni 5МРС	0,67±0,033	100,00	-
Sus scrofa L. + Ni 5МРС	0,89±0,052	132,35	2,75
Capreolus capreolus L. + Ni 5 МРС	0,72±0,026	108,12	1,03
Control: Ni МРС	1,20±0,080	100,00	-
Sus scrofa L. + Ni 10 МРС	0,94±0,101	78,23	1,58
Capreolus capreolus L. + Ni 10МРС	1,08±0,026	90,02	1,11

Примітка: див. таблицю 1

Conclusions

Thus, we discovered the fact of the reduction in total activity of AST and concentrations of water-soluble protein fraction relative to control (the area without pollution of Ni and without excretory function of Mammalia) in leaves of Glechoma hederacea L., which dominated in the area under study, which was subject to considerably toxic concentrations of Ni, at doses of 1 MAC, 5 MAC, 10 MAC. The combined effect of the excretory function of Mammalia under the low and medium doses of nickel contributed to the increased activity of the enzyme and under the Ni at maximum concentration was reliably reduced compared with the control in the corresponding concentration factor. The concentration of water soluble protean fraction under the combined effect of the excretory function of Mammalia and Ni at maximum concentration was reliably reduced, because it was difficult for the system to operate the mechanisms of recovery and normalization function, while at low-and medium metal concentration the processes of protein metabolism increased. Thus, the use of individual elements of zoocoenosis in the system of nature conservation and improvement of the transformed ecosystems in the Steppe Dnieper region had positive results.

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