Embryotoxicity Induced by Triclopyr in Zebrafish (Danio rerio) Early Life Stage

Abstract

Triclopyr, an auxin-like herbicide that is widely employed for managing weeds in food crops and pastures, has been identified in various environmental settings, particularly aquatic ecosystems. Limited understanding of the environmental fate of this herbicide, its potential repercussions for both the environment and human health, and its insufficient monitoring in diverse environmental compartments has caused it to be recognized as an emerging contaminant of concern. In this study, we have investigated how triclopyr affects zebrafish, considering a new alternative methodology. We focused on the endpoints of developmental toxicity, neurotoxicity, and behavior of zebrafish embryos and larvae. We determined that triclopyr has a 96 h median lethal concentration of 87.46 mg/L (341.01 µM). When we exposed zebrafish embryos to sublethal triclopyr concentrations (0.5, 1, 5, 10, and 50 μM) for up to 144 h, we found that 50 µM triclopyr delayed zebrafish egg hatchability. Yolk sac malabsorption was significant at 0.5, 1, 5, and 10 µM triclopyr. In zebrafish larvae, uninflated swim bladder was significant only at 50 µM triclopyr. Furthermore, zebrafish larvae had altered swimming activity after exposure to 10 µM triclopyr for 144 h. In summary, these comprehensive results indicate that even low triclopyr concentrations can elicit adverse effects during early zebrafish development.

Keywords: contaminant of emerging concern; embryotoxicity; new methodology approach; triclopyr; zebrafish.

Figure 1

Determination of triclopyr 96 h median lethal concentration (LC50-96h) in zebrafish embryos. The graph illustrates the mortality rate of zebrafish embryos after exposure to various triclopyr concentrations. LC50-96h was calculated using the sigmoid curve fitting method, with triclopyr concentrations expressed in logarithmic scale.

Figure 2

Hatching rate in zebrafish exposed to triclopyr. Effect of exposure to triclopyr on the hatching rate of zebrafish eggs in relation to exposure time. The proportion of hatched eggs by time and concentration is represented by different blue scales. The bars represent means, and the error bars represent the standard error of the mean (SEM), (n = 60). * One-way ANOVA followed by Dunnett’s test; p ≤ 0.05 for significant differences between exposed and control groups.

Figure 3

Swim bladder development in zebrafish exposed to triclopyr. Graphic representation of swim bladder development rate in zebrafish larvae by time of exposure to triclopyr. The proportion of swim bladder development by time and concentration is represented by different blue scales. The bars represent means, and the error bars represent the standard error of the mean (SEM), (n = 60). * One-way ANOVA followed by Dunnett’s test; p ≤ 0.05 for significant differences between exposed and control groups.

Figure 4

Morphological alterations induced by triclopyr in zebrafish at 96 h post-fertilization (hpf). (A) Larvae length; (B) eye size area; (C) pericardial area; (D) yolk sac area. The bars represent means, and the error bars represent the standard error of the mean (SEM), (n = 45). * One-way ANOVA followed by Dunnett’s test; p ≤ 0.05 for significant differences between exposed and control groups.

Figure 5

Effects of triclopyr on zebrafish larvae development at 96 h post-fertilization (hpf). Representative images of zebrafish larvae exposed to 0.5, 1, 5, 10, or 50 µM triclopyr, demonstrating noticeable yolk sac malabsorption (black arrow) in all treatment groups (magnification: 1.25×).

Figure 6

Total distance moved until 144 h post-fertilization (hpf) by zebrafish larvae exposed to triclopyr. Kinetics of the total distance (mm) moved by larvae during the light/dark stimulus. The lines represent different exposed groups, and the error bars denote the standard error of the mean (SEM).

Figure 7

Total distance traveled until 144 h post-fertilization (hpf) in response to the light/dark stimulus test applied for larvae treated with triclopyr. The X-axis shows the whole experiment time and light/dark transition every 10 min. The bars represent means, and the error bars represent the standard error of the mean (SEM). * Two-way ANOVA multiple comparisons followed by Dunnett’s test, p ≤ 0.05 for significant differences between exposed and control groups.

Figure 8

Swimming speed until 144 h post-fertilization (hpf) in zebrafish larvae exposed to triclopyr. The graph illustrates the kinetics of swimming speed (mm/s) exhibited by larvae during the light/dark stimulus. The lines represent different exposed groups, and the error bars denote the standard error of the mean (SEM).

Figure 9

Acetylcholinesterase activity at 96 h post-fertilization (hpf) in zebrafish larvae exposed to triclopyr. AChE activity was determined in nmol per minute per milligram of protein. The bars represent means, and the error bars represent the standard error of the mean (SEM), (n = 3). * One-way ANOVA followed by Dunnett’s test; p ≤ 0.05 for significant differences between exposed and control groups.