Essential Oils Produce Developmental Toxicity in Zebrafish Embryos and Cause Behavior Changes in Zebrafish Larvae

Abstract

Essential oils have gained significant popularity in various industries due to their biological properties, but their potential toxic effects on living organisms have been poorly investigated. This study aimed to evaluate the effects of lemongrass, thyme, and oregano essential oils on zebrafish embryos and larvae as animal models. Embryos were exposed to different concentrations of essential oils, and various endpoints were assessed, including epiboly, mortality (LC50), morphometry, and behavioral changes. All three essential oils reduced epiboly, affecting embryonic development. LC50 values were calculated for lemongrass (3.7 µg/mL), thyme (14.4 µg/mL), and oregano (5.3 µg/mL) oils. Larvae exposed to these oils displayed morphological defects, including growth reduction, spinal deformation, pericardial edema, eye size reduction, and reduced swim-bladder inflation. Morphometric analysis confirmed reduced larval length at higher oil concentrations. Essential-oil exposure altered zebrafish larval swimming behavior, with lemongrass oil reducing dark-cycle activity and oregano oil increasing light-cycle activity, suggesting neurodevelopmental toxicity. These findings illustrate the adverse effects of these oils on zebrafish embryos and larvae and reveal essential-oil toxicity, indicating careful use should be considered, particularly during pregnancy.

Keywords: embryogenesis; essential oils; toxicology; zebrafish.

Figure 1

Schematic representation of tests performed for toxicity evaluation of plant essential oils in embryo and larvae zebrafish.

Figure 2

Epiboly percentages at 8 hpf in zebrafish embryos exposed to (A) lemongrass, (B) thyme, and (C) oregano essential oils. Test F by one-way ANOVA: lemongrass EO (F(5, 77) = 48.83, p < 0.05); thyme EO (F(5, 76) = 48.97, p < 0.05); and oregano EO (F(5, 72) = 29.95, p < 0.05). * Statistically different from the control group (one-way ANOVA test, p < 0.05).

Figure 3

Representative zebrafish embryo epiboly and typical teratogenic effects observed in zebrafish embryos exposed to lemongrass, thyme, and oregano essential oils. Legend: (A) Control group epiboly at 8 hpf. (B) Thyme 25 µg/mL group epiboly at 8 hpf (scale bar 500 μm). (C,D) Fish affected by thyme essential-oil (20 µg/mL) exposure. Arrows and abbreviations indicate the main teratogenic effects observed as spine deformation (SD), small eyes (EY), pericardial edema (PE), swim-bladder inflation (SB), and growth retardation (GR). (E,F) Control group in lateral and dorsal views, respectively (scale bar 1 mm).

Figure 4

Survival curve and the mortality linear regression of zebrafish embryos after exposure to lemongrass (LGEO), thyme (TEO), and oregano (OREO) essential oils (A, B, and C, respectively). The linear regression and relationship of probit essential-oil concentrations were used to calculate LC50 values in larvae at 6 days post-fertilization.

Figure 5

Zebrafish larvae behavior in excitatory dark–light locomotor test. (A–F) Bars show the average of small (between 4 and 8 mm/s) or large (>8 mm/s) activity movement for each dark or light period (10 min). (G–L) Average swimming behavior (2 min) in each complete dark–light cycle (20 min). Behavior test was analyzed by one-way ANOVA in light period (lemongrass EO small activity (F(2, 15) = 2.273, p = 0.14), large activity (F(2, 15) = 3.943, p < 0.05); thyme EO small activity (F(2, 15) = 3.463, p = 0.06), large activity (F(2, 15) = 0.5043, p = 0.61); oregano EO small activity (F(2, 15) = 1.984, p = 0.17), large activity (F(2, 15) = 4.964, p < 0.05)) and dark period (lemongrass EO small activity (F(2, 15) = 13.10, p < 0.05), large activity (F(2, 15) = 12.77, p < 0.05); thyme EO small activity (F(2, 15) = 2.829, p = 0.09), large activity (F(2, 15) = 2.547, p = 0.11); oregano EO small activity (F(2, 15) = 2.925, p = 0.08), large activity (F(2, 15) = 8.370, p < 0.05)). When the time variable was considered, two-way ANOVA test was realized in small activity: lemongrass EO small activity (F(2, 69) = 9.015, p < 0.05) and large activity (F(2, 69) = 9.924, p < 0.05); thyme EO small activity (F(2, 69) = 1.436, p = 0.24) and large activity (F(2, 69) = 0.456, p = 0.64); oregano EO small activity (F(2, 69) = 1.951, p = 0.15) and large activity (F(2, 69) = 2.746, p = 0.07). * Represents statistically significant (p < 0.05) difference by control group. ^# Represents statistically significant (p < 0.05) difference by another essential-oil group.