Evaluating the Impact of Individual and Combined Toxicity of Imidacloprid, Cycloxaprid, and Tebuconazole on Daphnia magna

Abstract

The risks posed by chemicals in the environment are typically assessed on a substance-by-substance basis, often neglecting the effects of mixtures. This may lead to an underestimation of the actual risk. In our study, we investigated the effects of three commonly used pesticides-imidacloprid (IMI), cycloxaprid (CYC), and tebuconazole (TBZ)-both individually and in combination, using various biomarkers to assess their impact on daphnia. Our findings indicated that the order of toxicity, from highest to lowest, was TBZ, IMI, and CYC, as determined by acute toxicity as well as reproduction. The effects of the ITmix (IMI and TBZ) and CTmix (CYC and TBZ) combinations on immobilization and reproduction were evaluated by MIXTOX, revealing a higher risk of immobilization at low concentrations for ITmix. The effect on reproduction differed depending on the ration of pesticides in the mixture, with synergism observed, which may be caused mainly by IMI. However, CTmix showed antagonism for acute toxicity, with the effect on reproduction depending upon the composition of the mixture. The response surface also exhibited a switch between antagonism and synergism. Additionally, the pesticides extended the body length and inhibited the development period. The activities of superoxide dismutase (SOD) and catalase (CAT) content was also significantly induced at different dosage points in both the single and combination groups, indicating changes in the metabolic capabilities of detoxifying enzymes and target site sensitivity. These findings highlight the need for more attention to be focused on the effects of pesticide mixtures.

Keywords: MIXTOX; combined toxicity; ecotoxicity; pesticide mixture; risk assessment.

Figure 1

Test design of single and combined acute and reproduction test. (A) Acute toxicity under combined exposure of IMI and TBZ; (B) Acute toxicity under combined exposure of CYC and TBZ; (C) Chronic toxicity under combined exposure of IMI and TBZ; (D) Chronic toxicity under combined exposure of CYC and TBZ; TBZ, Tebuconazole; IMI, Imidacloprid; CYC, Cycloxaprid.

Figure 2

Response surface curve representing inhibition percentage of reproduction (A,C) and body length (B,D) of Daphnia magna exposed to pesticide mixtures. Points M and N represent the effects of the highest concentrations of tebuconazole and neonicotinoid (imidacloprid and cycloxaprid) tested alone. Color levels represent changes in percentage of inhibition. TBZ, Tebuconazole; IMI, Imidacloprid; CYC, Cycloxaprid.

Figure 3

Growth parameters effect by mixture of IMI and TBZ: (A) first birth time; (B) first spawning number; (C) number of litters per daphnia; (D) total number of brood per daphnia; (E) body length. L: low concentration NOEC; M: medium concentration EC₂₀; H: high concentration EC₅₀. In the figure, “a–i” indicate significant differences among 15 treatments and control analyzed by one-way ANOVA; “**” indicate the significant differences between the exposure groups and the control, and the results were analyzed by two-way ANOVA (**: p < 0.01), The difference in test item exposure (e.g., mixture in difference concentration as a whole group compared with control). TBZ, Tebuconazole; IMI, Imidacloprid; ITmix, mixture of TBZ and IMI.

Figure 4

Growth parameters effect by mixture of CYC and TBZ: (A) first birth time; (B) first spawning number; (C) number of litters per daphnia; (D) total number of brood per daphnia; (E) body length. L: low concentration NOEC; M: medium concentration EC₂₀; H: high concentration EC₅₀. In the figure, “a–i” indicate significant differences among 15 treatments and control analyzed by one-way ANOVA; “**” indicate the significant differences between the exposure groups and the control, and the results were analyzed by two-way ANOVA (**: p < 0.01), the difference in test item exposure (e.g., mixture in difference concentration as a whole group compared with control). TBZ, Tebuconazole; CYC, Cycloxaprid; CTmix, mixture of TBZ and CYC.

Figure 5

Growth parameters effect by mixture of ITmix and CTmix: (A) first birth time; (B) first spawning number; (C) number of litters per daphnia; (D) total number of brood per daphnia; (E) body length. L: low concentration NOEC; M: medium concentration EC₂₀; H: high concentration EC₅₀. In the figure, “a–i” indicate significant differences among 18 treatments and control analyzed by one-way ANOVA; “**” indicate the significant differences between the exposure groups and the control, and the results were analyzed by two-way ANOVA (**: p < 0.01), The difference in test item exposure (e.g., mixture in difference concentration as a whole group compared with control). ITmix, mixture of TBZ and IMI; CTmix, mixture of TBZ and CYC.

Figure 6

Enzyme content of daphnia exposed to single and combined treatments of IMI, CYC, and TBZ with different treatment (A); SOD content treated with single exposure (B); SOD content treated with ITmix (C); SOD content treated with CTmix (D); CAT content treated with single exposure (E); CAT content treated with ITmix (F); CAT content treated with CTmix. The value of each column represents the mean ± standard deviation (n = 10). “* and **” indicate the significant differences between the exposure groups and the control, and the results were analyzed by two-way ANOVA (*: p < 0.05; **: p < 0.01). ITmix, mixture of TBZ and IMI; CTmix, mixture of TBZ and CYC; L: low concentration NOEC, M: medium concentration EC₂₀; H: high concentration EC₅₀.