Recommended rates of azoxystrobin and tebuconazole seem to be environmentally safe but ineffective against target fungi

Abstract

The use of fungicides in agriculture has been playing a role in the enhancement of agricultural yields through the control of pathogens causing serious diseases in crops. Still, adverse environmental and human health effects resulting from its application have been reported. In this study, the possibility of readjusting the formulation of a commercial product combining azoxystrobin and tebuconazole (active ingredients - AIs; Custodia®) towards environmentally safer alternative(s) was investigated. Specifically, the sensitivity of non-target aquatic communities to each AI was first evaluated by applying the Species Sensitivity Distributions (SSDs) approach. Then, mixtures of these AIs were tested in a non-target organism (Raphidocelis subcapitata) denoting sensitivity to both AIs as assessed from SSDs. The resulting data supported the design of the last stage of this study, where mixtures of those AIs at equivalent vs. alternative ratios and rates as in the commercial formulation were tested against two target fungal species: Pyrenophora teres CBS 123929 and Rhynchosporium secalis CBS 110524. The comparison between the sensitivity of non-target aquatic species and the corresponding efficacy towards target fungi revealed that currently applied mixture and rates of these AIs are generally environmentally safe (antagonistic interaction; concentrations below the EC₁ for R. subcapitata and generally below the HC₅ for aquatic non-target communities), but ineffective against target organisms (maximum levels of inhibition of 70 and 50% in P. teres CBS 123929 and R. secalis CBS 110524, respectively). Results additionally suggest a potentiation of the effects of the AIs by the other formulants added to the commercial product at tested rates. Overall, this study corroborates that commercial products can be optimized during design stages based on a systematic ecotoxicological testing for ingredient interactions and actual efficacy against targets. This could be a valuable pathway to reduce environmental contamination during transition to a more sustainable agricultural production.

Keywords: Azoxystrobin; Mixture toxicity; Pyrenophora teres; Rynchosporium secalis; Tebuconazole.

Fig. 1

Mixture test design applied for assessing the efficacy of equivalent and alternative application rates of azoxystrobin (A) and tebuconazole (T) towards the target fungi Pyrenophora teres CBS 123929 and Rynchosporium secalis CBS 110524. ECx/HCx and a commercial application rate of these fungicides were included to relate effectiveness (target fungi) with environmental safety considering a non-target aquatic representative (Raphidocelis subcapitata) and aquatic communities as a whole (HCx estimated in the SSDs approach), respectively. Predicted environmental concentrations (PECs) equal to the estimated ECx were converted to application rates (AR; g ha⁻¹)

Fig. 2

Species Sensitivity Distribution curves (SSDs) for azoxystrobin and tebuconazole built with EC₅₀ values (Table S4) estimated from short-term toxicity assays with aquatic organisms. The central lines represent the SSD model fitted to the data regarding each fungicide (R² = 0.960 for azoxystrobin; R² = 0.976 for tebuconazole) and the dotted lines represent corresponding 95% confidence intervals

Fig. 3

Response of Raphidocelis subcapitata measured following 96 h of exposure to single and mixture treatments of tebuconazole (T) and azoxystrobin (A). The bars graph (a) shows the average yield (n = 3; error bars represent the standard error) and significant differences among treatments are assigned using low case letters (Tukey test, p < 0.05). The isobologram (b) illustrates the mixture response surface as predicted by the IA model with an antagonistic type of deviation, using a TU dimensionless strength scaling (TU is the concentration in the mixture normalized to the single-chemical EC₅₀) and a grey-scale gradient indicative of the level of the effect (the lighter the colors, the lower the yield). The scatter plot (c) shows the regression between experimental data and data predicted by the IA – Antagonism model, disclosing the respective equation and coefficient

Fig. 4

Growth response of the target fungi Pyrenophora teres CBS 123929 (a) and Rynchosporium secalis CBS 110524 (b) to single and mixture treatments of azoxystrobin (A) and tebuconazole (T), or to Custodia® diluted to the same concentration of active ingredients tested in one of the mixture treatments. The growth response is presented in terms of average mean diameter ± standard error (n = 3) of the fungi mycelium at 7, 14 and 21 days of exposure. Low case letters are used to assign significant differences between conditions within each assessed timepoint (post hoc Tukey test, p < 0.05). Lines were added only for visualization purposes