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A study of acute toxicity of newly synthesized compound on adult hydrobiont Danio rerio

Materials and methods. Determination of acute toxicity LC50 of the test compound was performed in vivo on a model of the aquatic organism Zebrafish (Danio rerio) according to the instruction OECD No 203 (Fish, Acute Toxicity Test) for testing chemical compounds (acute toxicity test on fish from 10.12.2009). It was used fish 2 months of age, 11.8 ± 0.1 mm long, and weighing 2.6 ± 0.2 g in the experiment. The concentration of the test compound ranged from 5.0 mg/l to 100.0 mg/l. Test water-soluble compounds were dissolved in distilled water. Each mini-aquarium with a certain dose of the compound contained at least 7 individuals of Danio rerio. During the experiments, the fish were kept on a diet for a test 96 hours and their mortality was checked every 24, 48, 72 and 96 hours. Statistical analysis of the results was performed using the program Statistica 6.

The study of newly synthesized compounds for acute to xicity is a necessary component for further determination of biological activity and effective dose of the future drug [5][6][7][8][9].
Not only mice or rats can be used to determine acute toxicity, but it can also be an aquatic organism -Zebrafish (Danio rerio) [10,11].

Materials and methods
Determination of acute toxicity LC 50 of the test compound was performed in vivo on a model of the aquatic organism Zebrafish (Danio rerio) according to the instruction OECD № 203 (Fish, Acute Toxicity Test) for testing chemical compounds (acute toxicity test on fish from 10.12.2009).
It was used fish 2 months of age, 11.8 ± 0.1 mm long, and weighing 2.6 ± 0.2 g in the experiment.
The concentration of the test compound ranged from 5.0 mg/l to 100.0 mg/l. Adults Danio rerio were stored in ventilated aquariums with carbonfiltered tap water (pH = 7.3 ± 0.3; 95 % Cl) at a temperature of 26.5 ºC. This oxygenenriched water was used for experiments. Prior to the experiments, the fish were acclimatized, with a mortality rate of no more than 1 in 500 individuals. Test watersoluble compounds were dissolved in distilled water. Each miniaquarium with a certain dose of the compound contained at least 7 individuals of Danio rerio. During the experiments, the fish were kept on a diet for a test 96 hours and their mortality was checked every 24, 48, 72, and 96 hours.
Statistical analysis of the results was performed using the program Statistica 6.
The degree of toxicity of the tested compounds was determined according to the classification of D. R. Passino [11].
The research was conducted in accordance with the national "General Ethical Principles of Animal Experimentation" approved by the First National Congress on Bioethics and the "Bioethical Expertise of Preclinical and Other Animal Research". These principles have been developed in accordance with the basic principles of bioethics and bioethical expertise in the interests of human protection and the entire biological diversity of the world. They are in line with the provisions of the "European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes".
Fish are considered dead if there are no visible movements (for example, gill movements) and if touching the tail stalk does not cause a reaction. Dead fish were removed by observation and mortality was recorded.

Discussion
Every 24, 48, 72, and 96 hours, mortality of the Zebrafish was entered in the Table 1.
After examining the mortality of animals at appropriate intervals, it is possible to draw conclusions about the relationship between "mortality -a period of time". Table 1 shows that the highest fish mortality occurs on the last day of observation (96 hours). This can be explained by the accumulation of the compound in the body of animals (Fig. 1).
After 48 hours, acute poisoning occurs, as evidenced by the mortality of animals at high concentrations.
According to the obtained data, graphs of the dependence of the concentration on the fish mortality percentage were constructed (Fig. 2, 3, 4) According to the acute toxicity of LC50 (96 hours) of sodium 2((4amino5(thiophen2ylmethyl)4H1,2,4tri-azole3yl)thio)acetate, which according to the classification of D. R. Passino and coauthors allowed it to be classified as a moderately toxic compound.
At low concentrations (5-10 mg/l) of the test substanceexophthalmos was observed (swelling in the orbital fossae, which leads to convexity of one or both eyes). In some cases, there was an increase in spontaneous activity.
Clinical signs in the middle range of concentrations (18-65 mg/l) were marked by the appearance of petechiae (spots the size of the head). Hematomas (area of blood) due to intradermal or submucosal bleeding in fish, as well as excessive mucus production, was observed in some individuals.  The effect of high concentrations (100 mg/l) on the aquatic organism Zebrafish (Danio rerio) caused abdominal edema due to fluid accumulation.

Conclusions
The acute toxicity LC 50 in vivo of sodium 2((4amino5 (thiophen2ylmethyl)4H1,2,4triazole3yl)thio)acetate on the Zebrafish (Danio rerio) aquatic model was investigated. The research was conducted in accordance with the national "General Ethical Principles of Animal Experimentation" approved by the First National Congress on Bioethics and the "Bioethical Expertise of Preclinical and Other Animal Research".
The highest fish mortality occurred on the last day of observation (96 hours).
The acute toxicity LC 50 (96 hours) of the test substance was 4.5364 mg/l. It can be concluded that the compound belongs to the class of moderately toxic substances.

Funding
The research is carried out within the SRW of Zaporizhzhia State Medical University "Synthesis, modification and study of the properties of 1,2,4-triazole derivatives to create an antimicrobial drug" state registration number 0120U101649.