Aquatic systems located in agricultural areas are exposed to complex pesticide mixtures, generally containing ten to twenty pesticides simultaneously. Even though the toxicity of pesticide mixtures can to a large degree be predicted with available mathematical models, the phenomenon known as synergism, defined as the mixture giving a greater biological effect than predicted, poses a challenge for the applicability of these models. Among the different classes of pesticides shown to interact synergistically, azole fungicides and pyrethroid insecticides have gained ecotoxicological concern. The ability of azoles to enhance the toxicity of pyrethroids is proposed to be mainly due to their ability to inhibit cytochrome P450 enzymes playing the major role in detoxification, resulting in higher internal concentration of the pyrethroids.
The objectives of the present study were 1) to investigate the ability of different methods to estimate inhibition strengths toward cytochrome P450 activity of relevant aquatic invertebrates, 2) to test whether measurements of inhibition strength can be used to predict the synergistic potential of azoles and 3) to test whether synergism can also be observed toward sublethal endpoints within environmentally relevant concentrations.
The selected organisms were the insect larva Chironomus riparius, the fresh water crustacean Daphnia magna and the snail Lymnaea stagnalis, due to their employment as test organisms for ecotoxicological studies, while up to 18 azoles were selected based on their use in agriculture and medicine. Inhibition strengths of azole fungicides toward cytochrome P450 activity were investigated by three different methods. Affinities between azoles and cytochrome P450 structures were estimated with a computer based (in silico) model, while inhibitory concentrations (IC50) were measured in vitro on extracted enzymatic material (microsomes), and in vivo with live organisms.
The inhibition strength of the selected azoles toward cytochrome P450 activity depended both on the selected species and on the azole. The ability and applicability of the selected methods to estimate inhibition strength, and the degree by which extrapolations between species were possible, depended strongly on the organism considered. In silico and in vitro assays seem to be promising tools for fast and cheap investigation of inhibition strengths in C. riparius and D. magna. On the other hand, only direct measurements of cytochrome P450 activity in vitro for L. stagnalis can be used in this species. The selected azoles were able to enhance the toxicity of the pyrethroid α-cypermethrin in D. magna by different degrees. However, inhibition strengths
measured in vivo as inhibitory concentrations (IC50) did not seem to predict synergistic potentials measured as the ratio of observed versus predicted toxicities. Nonetheless, measuring inhibition strengths toward cytochrome P450 activity might be used as a screening tool to identify compounds which do not act as synergists through inhibition of cytochrome P450, since the relatively weaker inhibitors did not induce synergism. Regarding the evaluation of synergistic responses on sublethal endpoints, epoxiconazole at 25 μg/L was able to enhance the negative effect of α-cypermethrin up to 3-fold, leading to decreased growth, decreased cytochrome P450 activity, and decreased reproduction of D. magna within the first 14 days of continuous exposure, showing that synergistic interactions can also be observed for sublethal endpoints. However, the synergistic interactions were not observed after 14 days, possibly due to adaptation of the organisms. These results show that time of assessment and exposure duration are important factors to consider also when assessing synergism on sublethal endpoints. Finally, due to the observed complexity of synergistic interactions, other aspects which should be considered for the prediction of synergistic potentials have been presented and discussed.