State Enterprise “L.I. Medved’s Research Center of Preventive Toxicology, Food and Chemical Safety, Ministry of Health of Ukraine”, Kyiv, Ukraine
Resume. The study objective was to determine the functional state of infusoria Tetrahymena pyriformis W by parameters of motor activity and energy consumption for movements under acute effects of plant growth regulators — N-oxide pyridine derivatives, their complexes with organic acids and metal salts.
Materials and methods. 2,6-dimethylpyridine-N-oxide, 2-methylpyridine-N-oxide and their complexes with organic acids (succinic, maleic) or metal salts (ZnCl2, ZnI2, CoCl2, MnCl2) — in total, 13 substances, synthesized at the Institute of Bioorganic Chemistry and Petrochemistry of the National Academy of Sciences of Ukraine have been used in the work. Studies have been carried out on the infusoria Tetrahymena pyriformis W in isotoxic doses — at the level of toxic concentrations — LC50, LC16 and inactive concentrations (LC0). Motor activity and energy state of infusoria using an automated laser Doppler spectrometer were evaluated.
Results and conclusion. It was shown that the studied N-oxide pyridine derivatives and their complexes with organic acids or metal salts inhibit the motor activity of the infusoria and increase energy consumption for movements with the greatest effect in the concentrations of the corresponding LC50. 2,6-dimethylpyridine-N-oxide, 2-methylpyridine-N-oxide, and their complexes with succinic acid and metal salts of CoCl2 and ZnCl2 were the most toxic for the viability of infusoria.
Therefore in order to assess the toxicity ofxenobiotics and viability of infusoria Tetrahymena pyriformis W, it is reasonable to recommend parameters of motor activity and energy consumption per unit of the rout that have the highest criterial significance.
Key words: toxicity, plant growth regulators, N-oxide pyridine derivatives, Tetrahymena pyriformis W.
Due to the intensification of the chemization of the national economy, pollution of the environment by various pollutants, which have a high biological activity that may result in changes in the development and functioning of ecosystems and cause significant damage to the environment and human health is noted. Currently, plant growth regulators (PGRs) take the first place among plant protection products. They are widely used to stimulate growth and development of plants, increase their resistance to unfavourable environmental conditions (drought, cold, lodging), infectious and parasitic diseases [1–5]. In the last decade, synthetic PGRs — N-oxide pyridine methyl derivatives (Ivin, Poteityn, Zeastymulin, Agrostymulin, Betastymulin, Tryman-1, Tetran, and others) have been widely used in Ukraine. The range and scope of PGR use is constantly expanding [6], since their application in new technologies contributes to the production of environmentally friendly agricultural products [7–10].
More than 20 new complexes of 2-methylpyridine-N-oxide and 2,6-dimethylpyridine-N-oxide with organic acids or metal salts that have shown high auxin or cytokinin activity and are promising PGRs for different crops have been synthesised at the Institute of Bioorganic Chemistry and Petrochemistry of the National Academy of Sciences of Ukraine. It was shown that they are low or moderately toxic, exhibit membranotropic activity, and intensify protein-synthetic processes in the laboratory animals. Detected effects did not depend on dose and time of action. The dependence curves of the toxic effect of the dose were, in most cases, non-linear — U and bimodal nature [1, 11, 12].
The toxicity of N-oxide pyridine derivatives for environmental objects, in particular, hydrobionts, has not been sufficiently investigated yet. However, it is known that a significant number of pollutants is able to exert a negative influence on various aquatic organisms. For example, it has been shown that high concentrations of the flotation reagent RA-14 (isodecyloxypropylamine acetate), like most surface-active substances (SAS), cause lysis of infusoria, and its low concentrations cause toxic effect [13, 14]. Heavy metals salts have a pronounced toxic effect on cyanobacteria Cynechocystis SP at low doses (1.5 μg/L-1), the toxicity of heavy metals is significantly increased with a decrease in the temperature of the aquatic medium [15]. Toxic effect of heavy metals, some pesticides and PGRs has been also established for Tetrahymena pyriformis infusoria [11, 12, 16–20]. It has been shown that PGRs under acute inflammatory effect on infusoria are moderately or low-toxic substances, and under the chronic effect, infusoria growth-concentration dependence curves have a mono-, bi- or polymorphic form. High comparability of data on acute PGR toxicity for laboratory rats determined experimentally and by the method of express biotesting on Tetrahymena pyriformis W. infusoria has been proved, which gives the possibility of extrapolation of acute toxicity from infusoria to mammals [11, 12].
In this regard, it is relevant to study the toxic properties of new PGRs — N-oxide pyridine derivatives, their complexes with organic acids and metal salts for unicellular organisms, in particular, infusoria, and to determine their hazard to the environment and human.
Study objective. Determine the functional state of infusoria Tetrahymena pyriformis W by parameters of motor activity and energy consumption for movements under acute effects of plant growth regulators — N-oxide pyridine derivatives, their complexes with organic acids and metal salts.
Materials and methods. 2,6-dimethylpyridine-N-oxide, 2-methylpyridine-N-oxide and their complexes with organic acids or metal salts (in total, 13 substances), synthesised at the Institute of Bioorganic Chemistry and Petrochemistry of the National Academy of Sciences of Ukraine have been used in the work, they showed high growth-regulating activity and recommended for practical application in agriculture of Ukraine as PGRs on different crops.
The studies have been carried out using Tetrahymena pyriformis W. infusoria. Tetrahymena pyriformis W. infusoria, as in vitro test system, are widely used in toxicology as an alternative test object to study the toxicity of many aquatic pollutants, pesticides, heavy metals, extracts of polymeric materials, preserving additives and disinfectants, organic and inorganic compounds [21, 22]. In general, in most cases, a study on infusoria is performed by the criterion “cell death” to determine the parameters of acute and chronic toxicity and predict their hazard to the environment and human. In addition to the criterion “cell death”, an important characteristic of the functional state of infusoria is their motor activity and energy consumption to maintain cell viability.
Study of motor activity and energy status of Tetrahymena pyriformis W. infusoria was carried out in isotoxic doses: at the level of toxic concentrations — LC50, LC16 and inactive concentrations (LC0) that have been established previously [11].
Motor activity and energy state of infusoria were evaluated using an automated laser Doppler spectrometer (ALDS), which allows recording the following characteristics of the viability of populations of mobile microorganisms:
- Portion of mobile cells (mobility) in the population, %.
- Rate of translation movements of cells, µm/sec.
- Frequency of movements, Hz.
- Median energy consumption of the cell for movement in a viscous medium, cu.
Furthermore, energy consumption for a unit of the route was calculated as follows:
C = A/B, where C is energy consumption per unit of the route per second; A — total energy (cu), B — the rate of movement (µm/sec).
Cultivation of infusoria was performed in digest medium with the following composition: peptones — 20 g, glucose 5 g, NaCl — 1 g, yeast extract — 1 mL, distilled water — 1 L, pH 7.2. Incubation was performed at 22 °С. The study was performed in the stationary phase of growth of Tetrahymena Pyriformis W.
To study the toxic properties of PGRs, the test substance dissolved in the incubation medium, in the volume required for the appropriate final concentration (mg/mL) was introduced into the substance of infusoria cells. Culture without the test substance was used as a control. Incubation of infusoria culture with PGR lasted for 30 min at a room temperature of 20–22 °С [21]. Then measurements were performed on ALDS. All parameters of mobility were automatically registered and averaged. The measurement time was 10 sec. A number of measurements was five. Statistical processing was carried out using generally accepted methods of mathematical statistics: arithmetic mean (M), the error of representativeness (m), Student’s t-test, and the probable difference in the results obtained (P) were calculated [23]. The degree of the severity of the toxic effects of the test substances was determined by changes in the motor activity of cells and energy consumption for movements.
The work was performed at the State Enterprise “Inter-Institutional Scientific and Technical Centre “Agribiotech” of the National Academy of Sciences of Ukraine. We express our sincere gratitude for the advisory support in conducting a study on ALDS to V. V. Vlasenko, Candidate of Biological Sciences.
Results and discussion. Nature of changes in motor activity and energy consumption for movement of Tetrahymena pyriformis W. infusoria under the effects of N-oxide pyridine methyl derivatives and their complexes with organic acids is provided in Table 1.
Table 1. Effect of some N-oxide pyridine methyl derivatives and their complexes with organic acids on the parameters of the viability of Tetrahymena Pyriformis W infusoria population under acute action. Exposure time is 30 min.
Note. * – Р ≤ 0.05
As Table 1 shows, the effect of 2,6-dimethylpyridine-N-oxide with the increase in the active concentration resulted in the increased proportion of mobile infusoria, and at the highest test concentrations 34 mg/mL (at the level LC50), it was approximately three-fold higher than in the control. Frequency and rate of infusoria movements in the lowest of test concentrations of substance slightly (by 11 %) increased. With the increase in the active concentration, these parameters decreased with the greatest effect (by 51.4 %) in the concentration corresponding to LC50. Total energy consumption on the motor activity of infusoria was the highest when exposed to 2,6-dimethylpyridine-N-oxide at a concentration of 9 mg/mL (LC0) due to an increase in the rate of movement. In relation to the control of energy consumption per unit of route increased by 25 % to 91.7 %, depending on the active concentration.
The same trend was maintained for the 2-methylpyridine-N-oxide and 2,6-dimethylpyridine-N-oxide complex with succinic acid. In case of exposure to 2-methylpyridine-N-oxide, the number of mobile cells increased with an increase in the active concentration and reached a maximum (2.06 times) in the concentration at the level of LC50 and, under the effect of 2,6-dimethylpyridine-N-oxide complex with succinic acid at a maximum concentration, the number of mobile cells increased only 0.55-fold. The frequency and rate of movements at LC50 level decreased by 26.6 % with 2-methylpyridine-N-oxide, by 42.2 %, with 2,6-dimethylpyridine-N-oxide complex with succinic acid. Total energy consumption on the movement was high in all test concentrations of 2-methylpyridine-N-oxide and increased by 44.6 % to 64 %, while energy consumption per unit of the route was higher by 35.4 % to 125 % than in the control. For 2,6-dimethylpyridine-N-oxide complex with succinic acid, the total energy consumption of the movement was less pronounced than that of 2,6-dimethylpyridine-N-oxide, and the energy consumption per unit of route increased by 25 % to 47.7 % compared to the control, however at a maximum concentration they were lower by 44 % compared with exposure to 2,6-dimethylpyridine-N-oxide.
Under the effect of complexes of 2,6-dimethylpyridine-di-N-oxide with succinic acid and 2,6-dimethylpyridine-N-oxide with maleic acid, the portion of mobile infusoria in 2 lower concentrations (at the levels of LC0 and LC16) decreased slightly and amounted from 14.1 % to 22.7 %, the frequency and rate of movements virtually did not change (in the case of 2,6-dimethylpyridine-di-N-oxide complex with succinic acid) or increased by 22 % to 24 % (in the case of 2,6-dimethylpyridine-N-oxide complex with maleic acid); total energy consumption on the movement was slightly different from the control. At a maximum concentration (at the LD50 level), as well as under the effect of other test substances mentioned above, there was an increase in the number of mobile infusoria under the effect of the complexes of 2,6-dimethylpyridine-di-N-oxide with succinic acid by 26.14 %, 2,6-dimethylpyridine-N-oxide with maleic acid — by 42.15 %, the frequency and rate of movements under the action of these substances decreased by 22.4 % and 37.9 %, respectively. Total energy consumption in the first case increased by 76.43 %, while in the second, it decreased by 9.4 %, however energy consumption of cells per unit of route, as compared to the control, was higher by 130 % under the action of 2,6-dimethylpyridine-di-N-oxide complex with succinic acid and by 47.6 % under the action of the 2,6-dimethylpyridine-N-oxide complex with maleic acid.
A portion of mobile infusoria increased by 35.4 % and 28.6 %, with the rate and frequency of movements increased by 20 % and 46 %, respectively, under the effect of the 2-methylpyridine-N-oxide complex with succinic acid in 2 lower concentrations (at levels of LC0 and LC16). Under the effect of the 2-methylpyridine-di-N-oxide complex with succinic acid — the portion of mobile infusoria decreased by 12.2 % and 14.5 % according to the active concentrations; frequency and rate of movements in relation to the control virtually did not change. At a maximum concentration (at LD50 level), the number of mobile cells decreased significantly less than under the action of 2-methylpyridine-N-oxide complex with succinic acid than that of 2-methylpyridine-di-N-oxide with succinic acid and amounted to 10.2 % and 47.1 % compared to the control; the frequency and rate of movements roughly decreased to the same extent (by 32 % and 29 %, respectively). Total energy consumption on the movement of cells under the effect of the 2-methylpyridine-di-N-oxide complex with succinic acid at all tested concentrations virtually did not differ from the control, and 2-methylpyridine-N-oxide complex with succinic acid they significantly increased. Detected effect did not depend on the active concentration and it was 174 to 45.7 % higher compared to the control. At the same time, energy consumption of cells per unit of the route of both of these substances had a concentration dependence and increased in comparison with the control under the effect of the maximum concentration of 2-methylpyridine-N-oxide complex with succinic acid by 113 %, and by 90 % for 2- methylpyridine-di-N-oxide complex with succinic acid.
As can be seen from the provided data, increasing of concentration for 2,6-dimethylpyridine-N-oxide, 2-methylpyridine-N-oxide, 2,6-dimethylpyridine-N-oxide complex with succinic acid cause an increase in the portion of mobile infusoria, a decrease in frequency and rate of movement, total energy consumption on movement, with an increase in the energy consumption of cells per unit of a route. Increase in the portion of mobile cells can be linked to the chemotactic cellular response to the increased content of the chemical factor in the digesting medium. Under the action of substances in lower concentrations, change in the frequency and rate of infusoria is not significant compared to the control, however, there is an increase in the energy consumption of cells per unit of a route, indicating the strain of the adaptive capacity of infusoria. Under the effect of substances at a maximum test concentration, along with the decrease in the frequency and rate of movements, energy consumption per unit of route significantly increased, which indicates an increase in the toxic effect and deeper changes in the viability of infusoria.
Toxic effect 2,6-dimethylpyridine-di-N-oxide complexes with succinic acid and 2,6-dimethylpyridine-N-oxide with maleic acid, 2-methylpyridine-N-oxide with succinic acid and 2-methylpyridine-di-N-oxide with succinic acid on infusoria in 2 concentrations is not pronounced, as evidenced by the slightly divergent directional changes in the studied parameters and increase in energy consumption on movement. At the level of median lethal concentrations for 2,6-dimethylpyridine-di-N-oxide complexes with succinic acid and 2,6-dimethylpyridine-N-oxide with maleic acid, as well as for the other compounds mentioned above, an increase in the number of mobile infusoria is typical; for 2-methylpyridine-N-oxide complexes with succinic acid and 2-methylpyridine-di-N-oxide with succinic acid, as with lower concentrations, there is a more pronounced decrease in the portion of mobile cells, decrease in the frequency and rate of movements, and increase in energy consumption by cells per unit of route is observed that indicates an increase in the toxic effects of these substances.
Nature of changes of studied parameters of the viability of Tetrahymena pyriformis W. under the effects of N-oxide pyridine methyl derivatives and their complexes with metal salts is provided in Table 2.
Table 2. Effect of some N-oxide pyridine methyl derivatives with metal salts on the parameters of the viability of Tetrahymena Pyriformis W infusoria population under acute action. Exposure time is 30 min.
Note. * – Р ≤ 0.05
As Table 2 shows, 2,6-dimethylpyridine-di-N-oxide complex with ZnCl2 and 2-methylpyridine-di-N-oxide complex with ZnCl2 at the lowest concentration increased the portion of mobile cells, with the increase in the concentration of active ingredients, the number of mobile infusoria decreased and at a maximum concentration, reduction of this parameter was 57.3 % and 59.1 %, respectively. Frequency and rate of movements, total energy consumption of cells on movements was reduced in most cases due to the effect of 2,6-dimethylpyridine-di-N-oxide with ZnCl2 than due to 2-methylpyridine-di-N-oxide with ZnCl2. At a maximum concentration (corresponding to LC50), the reduction of these parameters compared to the control for 2,6-dimethylpyridine-di-N-oxide with ZnCl2 was 77.4; 77.4 and 59.6 %, for 2-methylpyridine-di-N-oxide with ZnCl2 — 48.1; 48.1 and 1.4 %. The energy consumption of cells per unit of route under the effect of these complexes with ZnCl2 increased by 74 % and 90 %, respectively.
Effect of the 2,6-dimethylpyridine-di-N-oxide complex with ZnI2 was slightly less pronounced in comparison with 2,6-dimethylpyridine-di-N-oxide complex with ZnCl2. In all test concentrations of this substance, the portion of mobile cells was higher than in the control by 48.2–106.3 %, however, the frequency and rate of movement decreased, and at a concentration of 2.76 mg/mL (LC50) they were lower than in the control by 47.4 %. At the same time, total energy consumption on the movement in lower concentrations tended to increase, and at a maximum concentration, they decreased by 18.56 %. With an increase in the active concentration of the substance, energy consumption of cells per unit of route increased, and at a maximum concentration, they were 56 % higher than in the control.
2,6-dimethylpyridine-di-N-oxide complex with CoCl2 and 2-methylpyridine-N-oxide complex with CoCl2 increased the portion of mobile cells in all test concentrations: the first substance — by 17.87–71.5 %, the second — by 2.87–42.58 %, the degree of significance of changes did not depend on the active concentration. With increasing concentrations of these substances, the frequency and rate of movements, total energy consumption on movement decreased, and the energy consumption of cells per unit of route increased, and at a maximum concentration corresponding to LC50 they were: for 2,6-dimethylpyridine-di-N-oxide complex with CoCl2 — 63.4; 63.4; 48.2, and 42.55 %, respectively, for 2-methylpyridine-N-oxide complex with CoCl2 — 64.7; 64.7; 51.0, and 40.0 %, respectively. By these parameters, it can be considered that both complexes with CoCl2 according to the studied criteria have the same toxic effect on infusoria.
Under the acute effect on the infusoria of 2-methylpyridine-N-oxide complex with MnCl2 in all test concentrations, the portion of mobile cells was higher than in the control by 21.58–47.30 %, the frequency and rate of movements of infusoria, total energy consumption on movement decreased depending on the active concentration and were most pronounced at a maximum concentration (at the level of LC50) and were by 47.13; 47.13, and 21.12 % lower than in the control, respectively. With an increase in the active concentration of the substance, energy consumption of cells per unit of route increased, and at a maximum concentration, they were 50 % higher than in the control.
As it can be seen from the above data, with the increase of the active concentration, 2,6-dimethylpyridine-di-N-oxide complex ZnCl2 and 2-methylpyridine-di-N-oxide complex with ZnCl2 reduced the portion of mobile infusoria, and 2,6-dimethylpyridine-di-N-oxide complex with ZnI2, 2,6-dimethylpyridine-di-N-oxide complex with CoCl2, 2-methylpyridine-N-oxide complex with CoCl2 and 2-methylpyridine-N-oxide complex with MgCl2 increased the portion of mobile infusoria without a clear concentration dependence. For all studied complexes of N-oxide pyridine methyl derivatives with metal salts, decrease in the frequency and rate of movements, total energy consumption on movement are typical, and significance of the changes depended on the active concentration. The energy consumption of cells per unit of route increased with increasing concentration. The most toxic effect on the viability of infusoria was induced by the 2-methylpyridine-di-N-oxide complex with ZnCl2, 2,6-dimethylpyridine-di-N-oxide complex with ZnCl2, and 2,6-dimethylpyridine-di-N-oxide complex with CoCl2.
Thus, based on the data obtained, the most informative in assessing the toxic effects of the substances studied are parameters of the reduction of motor activity and an increase of energy consumption per unit of a route, which may be one of the criteria for toxicity of xenobiotics for infusoria and assessment of their viability.
Conclusion
1. Studied N-oxide pyridine methyl derivatives and their complexes with organic acids (succinic, maleic) and metal salts (ZnCl2, ZnI2, CoCl2, MnCl2) inhibit the mobile activity of infusoria and increase energy consumption on movement with the greatest effect at concentrations corresponding to LC50.
2. 2,6-dimethylpyridine-N-oxide, 2-methylpyridine-N-oxide, and their complexes with succinic acid and metal salts of CoCl2 and ZnCl2 were the most toxic for the viability of infusoria.
3. In order to assess the toxicity of xenobiotics and viability of infusoria Tetrahymena pyriformis W., it is reasonable to recommend parameters of motor activity and energy consumption per unit of the route that have the highest criterial significance.
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Надійшла до редакції 20.06.2018 р.