Multiple Assays on Non-Target Organisms to Determine the Risk of Acute Environmental Toxicity in Tebuconazole-Based Fungicides Widely Used in the Black Sea Coastal Area

Abstract

The widespread use of Tebuconazole-based fungicides in phytosanitary treatments on a wide range of crops, on the one hand, and the lack of official reports on the amount of fungicide residues in nearby water basins, on the other hand, may lead to uncontrolled and hazardous contamination of water sources used by the resident population, and to serious effects on the environment and public health. Our study explores the acute toxicological risk of this fungicide on various organisms, from bacteria and yeast to fish, using a battery of tests (standardized Toxkit microbiotests and acute semi-static tests). By investigating the interaction between Tebuconazole and bacteria and yeast organisms, we observed that Gram-negative bacteria displayed a strong tolerance for Tebuconazole, while Gram-positive bacteria and yeasts proved to be very sensitive. The fish experiment was conducted on Chelon auratus juveniles exposed to five concentrations of the fungicide Tebustar EW (Tebuconazole, 250 g/L as active substance). After 96 h of exposure, the LC₅₀ for C. auratus was 1.13 mg/L. In the case of the Toxkit microbiotests' application, the following results were recorded: Spirodela polyrhiza EC₅₀ = 2.204 mg/L (after 72 h exposure), Thamnocephalus platyurus EC₅₀ = 0.115 mg/L (after 24 h), and Daphnia magna EC₅₀ = 2.37 mg/L (after 24-48 h). With the exception of bacteria and yeast, the same response pattern was observed for all non-target species tested; the response range expressed by concentrations causing growth inhibition or mortality was small, ranging between very close values that are quite low, thereby demonstrating the high toxicity of Tebuconazole-based fungicides to the environment.

Keywords: Gram-positive and Gram-negative bacteria; Tebuconazole; Toxkit microbiotests; acute semi static; acute toxicity; environmental risk; fungicides; pelagic fish.

Figure 1

Frond area of Spirodela polyrhiza plants after 72 h of exposure to Tebuconazole. ECx (x = 5, 10, 15, 20 and 50) values are represented.

Figure 2

Number of immobilized D. magna neonates after 48 h exposure to Tebuconazole.

Figure 3

Number of immobilized T. platyurus individuals after 24 h of exposure to Tebuconazole.

Figure 4

Mortality of C. auratus after 96 h exposure to Tebuconazole (LC₅₀ = 1.13 mg/L).

Figure 5

Time-kill in E. coli ATCC 25922 at different concentrations of Tebuconazole (Ec4 = 15.625; Ec5 = 7.81 mg/mL; Ec6 = 3.90 mg/mL; Ec8 = 0.97 mg/mL; Ec9 = 0.48 mg/mL; Ec10 = 0.24 mg/mL).

Figure 6

Time-kill in P. fluorescens at different concentrations of Tebuconazole (Psa1 = 125 mg/mL; Psa2 = 62.5 mg/mL; Psa3 = 31.25 mg/mL).

Figure 7

Time-kill in P. aeruginosa ATCC 27853 at different concentrations of Tebuconazole (Psa1 = 125 mg/mL; Psa2 = 62.5 mg/mL; Psa3 = 31.25 mg/mL).

Figure 8

Time-kill in Bacillus sp. at different concentrations of Tebuconazole (BS1 = 0.25 mg/mL; BS2 0.025 mg/mL; BS3 = 0.0125 mg/mL).

Figure 9

Time-kill in Candida albicans ATCC 10231 at different concentrations of Tebuconazole (CA1 = 0.25 mg/mL; CA2 = 0.05 mg/mL; CA3 = 0.025 mg/mL).