L.I. Medved's Research Center of Preventive Toxigology, Food and Chemical Safety Ministry of Health of Ukraine, Kyiv, Ukraine
Abstract. The aim of this work was to determine the character and type of combined action of plant growth regulators —Ivin and Poteytin with pesticides of different chemical groups.
Methods. Toxicological —acute toxicity (LD50), clinical signs, the type of combined action.
Results. The study combined effect plant growth regulators — Ivin (2,6-dimethyl pyridine N-oxide) and Poteytin (complex of 2,6-dimethyl pyridine N-oxide with succinic acid) with some pesticides in izotoxic doses corresponding 1 LD50 or 1/2 LD50 (in 1:1) showed that both plant growth regulators reduce acute oral toxicity of pesticides and severity of clinical signs of toxicity in rats. The character of the intoxication in rats under the combined effect was due to the influence of the individual pesticide. For the combined action of most investigated pesticides with Ivin is characteristic antagonism, but for Ivin with lambda-cyhalothrin — potentiation of toxicity. For Poteytin combinations with studied pesticides also preferred antagonism, with exception for Poteytin with lambda-cyhalothrin or flutriafol —potentiation of toxicity and with imidacloprid or cypermethrin —summation of toxicity.
Conclusions. Plant growth regulators Ivin and Poteytin in most cases reduce the toxicity of studied pesticides and severity of clinical signs of intoxication. Antagonism was establish for the combined action of Ivin with 2,4-D-2-ethylhexyl, tebukonazole, difenoconazole, flutriafol, cypermethrin, thiamethoxam, chlorpyrifos, imidacloprid and potentiation of toxicity for the combined action Ivin with lambda-cyhalothrin. Also, antagonism was found at the combined action of Poteytin with 2,4-D-2-ethylhexyl, tebukonazole, difenoconazole, thiamethoxam and chlorpyrifos, summation — for Poteytin with imidacloprid and cypermethrin, and potentiation of toxicity — for Poteytin with flutriafol and lambda-cyhalothrin.
Key words: plant growth regulators, pesticides, acute toxicity, rat, type combined action.
Pesticides and plant growth regulators (PGRs) are widely used in Ukraine to protect agricultural crops from pests and diseases. Pesticides are the main chemical protection of plants from weeds (herbicides), harmful insects and mites (insecto-acaricides), fungi and fungal diseases of plants (fungicides), etc. [1]. PGRs is used to stimulate the growth and development of plants, increase their resistance to adverse environmental conditions, pathogenic fungi, pests, reducing the toxicity of heavy metals to plants and their accumulation in plant products [2-7]. In recent years, it has been shown that domestic PGRs, in particular, based on pyridine N-oxide derivatives (Ivin, Poteitin), and natural growth biostimulants (Emistim, Biolan, Regoplant, Stimpo, etc.) can be used to reduce the rate of pesticide consumption without reducing their efficacy for their intended purposes [2, 8-10]. Both pesticides and PGRs are biologically active substances. The combined effect of pesticides and PGRs on the body has not been studied.
Previously, it was shown that methyl derivatives of pyridine N-oxide (Ivin, Poteitin, Triman-1, etc.) are moderately toxic substances, Ivin at subtoxic dosages increases the content of endogenous NO, stabilizes hepatocyte membranes, reduces peroxide oxidation of lipids, intensifies protein-synthetic processes in the body of animals [11-16], which may affect the toxicity and nature of toxic action of pesticides.
Proceeding from this, the important task of toxicology is the study of the effects of pesticides and PGRs with their co-occurrence in the body, the definition of the nature and type of the combined effect and, if necessary, the development of preventive measures of their harmful effects on the body.
Purpose of the work. Determine the nature and type of the combined effect of plant growth regulators — Ivin and Poteitin with pesticides of various chemical groups.
Materials and methods of the study.
This work uses PGRs —2,6-dimethyl N-pyridine (Ivin) and its complex with amber acid (Poteitin), pesticides from various chemical groups most widely used in the agriculture of Ukraine: Phenoxyalcanecarboxylic acid derivatives are 2,4-D ethylhexyl ether (2,4-D EHE); triazole— tebuconazole, difenoconazole, flutriafol; synthetic pyrethroids— lambda-cyhalothrin, cypermethrin; neonicotinoids – imidacloprid; thiamethoxam; organophosphorus compounds— chlorpyrifos.
The biologic model for the determination of acute oral toxicity of these substances with isolated and combined effects was Wistar Han rats (SPF) from the nursery of small animals of the State Enterprise ‘Academician L. I. Medved Scientific Centre for Preventive Toxicology, Food and Chemical Safety of the Ministry of Health of Ukraine’. Experimental animals were quarantined for 5 days. Animals were kept indoors at a temperature of 22 ± 3 оС, air relative humidity — 40–60 %. The diet of animals consisted of concentrated granular formulated feed manufactured by Altromin (Germany). Animals received water disinfected with ultraviolet irradiation and filtering through reverse osmosis from 0.5-litre glass bottles through metal tips (without limitation).
The study was conducted in accordance with the GLP (Good Laboratory Practice) principles set out in Directive 2004/10/EC [17]. Acute oral toxicity after isolated intake of the substances studied was studied in Wistar Han rats (males) using the OECD 425 method [18]. Solutions of the xenobiotics studied were prepared ex tempore on distilled water, and for pesticides insoluble in water, they were pre-diluted with an emulsifier OP-10 (GOST 8433-81). An administration technique was followed when the substances were injected into the stomach taking into account data on the amount of fluid that can be administered to animals, depending on the route of administration, their type and body weight [19].
The main criterion for acute toxicity was the dose causing the death of 50 % of animals (LD50), which was determined by the Statistical Program (AOT 425 Stat Program) [20]. During 14 days, a clinical examination of animals was performed, clinical signs of intoxication and their duration were recorded.
The combined effect of PGRs with pesticides of various chemical groups was studied after single oral administration. Wistar Han rat males received orally Ivin or Poteitin once and after 30 minutes active pesticide substances at isotoxic doses corresponding to 1LD50 or 1/2 LD50 (in the ratio of 1:1). For the studied combinations of substances, the LD50 value was determined using the 2-point method [21], the type of combined effect was calculated using Finney D.J. formula. [22].
Study results and their discussion.
It has been established that the clinical signs of intoxication in rats after single concomitant oral administration of pesticides with PGRs Ivin or Poteitin were characteristic for the effect of a specific pesticide, but in most cases, they were less pronounced than with the isolated action of pesticides.
Thus, under the effect of both PGRs with 2,4-D EHE at the doses of the corresponding 1 LD50, no severe clinical signs of intoxication (ataxia, rigidity of the tail and paresis of the hind limbs), characteristic of 2,4-D EHE, were not observed, the duration of intoxication was reduced approximately to 2 times (9 days for 2,4-D EGE, 3 days in combination with Ivin and 4 days in combination with Poteitin). Mortality of rats was observed only in the combination of Ivin with 2,4-D EHE.
Under the combined effect of the organophosphorus pesticide chlorpyrifos with PGRs at the doses of the corresponding 1 LD50, the severe cholinergic symptoms of intoxication were not detected, indicating a decrease in the anticholinesterase activity of the organophosphorus substance, but the general toxicological symptoms of intoxication (decreased activity, tousled fur, sanious discharge from the nose) were observed 1–2 days longer than the effects of chlorpyrifos. Mortality of rats with a combined effect of chlorpyrifos with Ivin or Poteitin was similar.
Under the effects of triazole compounds at the doses of the corresponding 1 LD50 (tebuconazole, flutriafol, difenoconazole), clinical signs of intoxication were characterized by decreased activity, tousled fur, salivation, sanious discharge from the nose and continued respectively 8, 9 and 7 days. Under the combined effect with PGRs at the doses of the corresponding 1 LD50, the clinical signs of intoxication were similar. The duration of intoxication under the combined effect of Ivin with tebuconazole was 2 days, with flutriafol — 7 and difenoconazole — 9 days; Poteitin with tebuconazole — 8 days, with flutriafol — 8 and difenoconazole — 10 days. Therefore, only under the combined effect of PGRs with difenoconazole, the duration of intoxication increased by –-2 days, Poteitin with flutriafol — decreased by 1 day. Higher mortality rates in rats were observed in combinations of tebuconazole with Ivin than Poteitin, flutriofol and difenoconazole with Poteitin than Ivin.
With concomitant administration of PGRs with synthetic pyrethroids at the doses corresponding to 1 LD50 (lambda-cyhalothrin and cypermethrin), intoxication was characterized by the nervous system dysfunction (difficult breathing, tremor, paresis of hind limbs, convulsions) and was more severe in combination with lambda-cyhalothrin than cypermethrin. Duration of intoxication of lambda-cyhalothride was 5 days, cypermethrin — 2 days; with the concomitant administration of lambda-cyhalothrin or cypermethrin with Ivin — 1 day, with Poteitin, respectively, 5 and 3 days. The mortality of rats with the combination of lambda-cyhalothrin or cypermethrin with Ivin was higher than with Poteitin.
For neonicotinoids — imidacloprid and thiamethoxam at the doses corresponding to 1 LD50 intoxication were characterized mainly by decreased activity, kyphosis, in the case of imidacloprid with tremor, thiamethoxam — salivation. The duration of intoxication under the effect of imidacloprid was 7 days, thiamethoxam — 1 day. With the concomitant administration of PGRs at the doses corresponding to 1 LD50 the nature of intoxication was similar, the duration of intoxication of the combination of Ivin or Poteitin with imidacloprid was 14 days, Ivin with thiametoxam — 2 days, Poteitin with thiametoxam — 1 day. Higher mortality rates of rats was observed in combinations of imidacloprid with Poteitin than Ivin, and thiamethoxam with Ivin than with Poteitin.
As can be seen from Table 1, antagonism was observed in combinations of Ivin with the pesticides studied (2,4-D EHE, chlorpyrifos, tebuconazole, difenoconazole, flutriafol, cypermethrin, thiamethoxam, imidocloprid). The combined effect ratio ranged from 0.43 to 0.83. The potentiation of toxicity was detected in a combination of Ivin and lambda-cyhalothrin.
Table 1. Acute combined effect of Ivin and some pesticides for male rats at isotoxic doses
Notes: 1. A — Ivin,
2. В — corresponding active substance of pesticides,
3. * — the numerator is the number of dead animals, the denominator is the total number of animals
In combinations of Poteitin with pesticides (Table 2) (2,4-D EHE, chlorpyrifos, tebuconazole, difenoconazole, thiamethoxam) antagonism was observed. The combined effect ratio ranged from 0.31 to 0.73. Under the combined effect of Poteitin with imidacloprid, Poteitin with cypermethrin, summation was detected, Poteitin with lambda-cyhalothrin, Poteitin with flutriafol – potentiation of toxicity was observed.
Table 2. Acute combined effect of Poteitin and some pesticides for male rats at isotoxic doses
Notes: 1. A — Poteitin,
2. В — corresponding active substance of pesticides,
3. * — the numerator is the number of dead animals, the denominator is the total number of animals
Thus, for most of the pesticides studied with Ivin or Poteitin, antagonism was detected, indicating a decrease in the acute toxicity of pesticides when co-administered with PGRs. Considering that Ivin and Poteitin are stabilizers of biological membranes [13 - 15], the reduction of toxicity of the pesticides studied may be due to the hepatoprotective effect of the PGRs studied.
Potentiation of toxicity was detected in 2 cases - lambda-cypermethrin and flutriafol. It is possible that the increased toxicity of lambda-cyhalothrin, which has pronounced neurotoxic properties, with Ivin or Poteitin, and flutrifol, containing 2 fluorine atoms in its structure, with Poteitin may be associated with a change in their toxicinetics in an animal body requiring further researches.
Conclusions.
1. The plant growth regulators Ivin and Poteitin in the vast majority of cases reduce the toxicity of the tested pesticides and severity of their clinical signs of intoxication.
2. Under the combined effect of Ivin with 2,4-D acidethylhexyl ether, tebuconazole, difenoconazole, flutriafol, cypermethrin, thiamethoxam, chlorpyrifos, imidacloprid, antagonism has been established, with lambda-cyhalothrin — potentiation of toxicity.
3. Under the combined effect of Poteitin with 2,4-D acid ethylhexyl ether, tebuconazole, difenoconazole, thiamethoxam and chlorpyrifos, antagonism has been established; with imidacloprid and cypermethrin — summation; flutriafol and lambda-cyhalothrin — potentiation of toxicity.
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