Comparative Assessment of Short- and Long-Term Effects of Triadimenol Fungicide on Danio rerio Erythrocytes Using the Micronucleus and Erythrocyte Nuclear Abnormality Assays

Abstract

Triadimenol is a systemic fungicide widely used in agriculture to manage plant diseases, especially fungal infections. This study aims to evaluate the short-term (24, 48, 72 and 96 h) and long-term (10, 20, and 30 days) genotoxic effects of different concentrations of triadimenol on zebrafish (Danio rerio) erythrocytes using micronucleus (MN) and erythrocyte nuclear abnormal (ENA) assay. Fish were treated with 1.5, 3, and 6 mg/L concentrations of triadimenol for short and long-term periods. After the treatment period, blood was collected with heparin syringe, smears were prepared, the preparations were fixed and stained. For MN assay in short-term treatments, statistically significant MN formation was found at all concentrations of triadimenol for 24 h treatment, at the highest triadimenol concentration for 48 h, at 1.5 and 3 mg/L concentrations for 72 h, and at 3 mg/L concentrations for 96 h, compared to the negative control. In long-term treatments, significant increases in MN formation were observed at all concentrations of triadimenol for 10 and 20 days of treatment compared to the negative control. Mortality occurred at 3 and 6 mg/L concentrations in the 30-day treatment. The most frequently detected abnormalities included echinocytes and binuclear cells. For ENA assay, abnormalities such as echinocytes, binuclear cells, segmented cells, and kidney-shaped nuclei were detected in fish erythrocytes treated with different concentrations of triadimenol. All concentrations of triadimenol caused an increase in the total abnormality level in Danio rerio erythrocytes at all treatment times. These increases were concentration dependent for both short-term and long-term treatments. In conclusion, this study emphasized the potential genotoxic risks of triadimenol fungicide for aquatic organisms in both short-term and long-term treatments and the need for further ecotoxicological evaluation.

Keywords: ecotoxicology; genotoxicity; genotoxicity assays; triadimenol; zebrafish.

Figure 1

Chemical structure of TDML [16].

Figure 2

Total abnormality in D. rerio treated with various concentrations of TDML for short-term (24, 48, 72, and 96 h) and long-term (10, 20, and 30 days) treatment periods. b: p ≤ 0.01; c: p ≤ 0.001.

Figure 3

Graphs showing concentration–response curve slopes and correlation coefficients (R²) of total abnormality in D. rerio erythrocyte cells expose to TDML for 24 (A), 48 (B), 72 (C), and 96 h (D).

Figure 4

Graphs showing concentration–response curve slopes and correlation coefficients (R²) of total abnormality in D. rerio erythrocyte cells expose to TDML for 10 (A), 20 (B), and 30 d (C).

Figure 5

Light micrographs of erythrocyte cells of D. rerio (×400): (a) normal nucleus; (b) micronucleus; (c) binucleated cell; (d) polymorphic nucleus; (e) kidney-shaped nucleus; (f) segmented nucleus; (g,h) blebbed nucleus; (i) notched nucleus; (j) echinocytes.

Figure 6

Scanning electron micrographs of erythrocyte cells of D. rerio (×10,000): (a) normal; (b) kidney-shaped nucleus; (c) notched nucleus; (d) polymorphic nucleus; (e) segmented nucleus; (f) echinocyte.