What causes the difference in synergistic potentials of propiconazole and prochloraz toward pyrethroids in Daphnia magna?

• Toxicity of azole fungicides is driven by toxicokinetics (hydrophobicity).
• The synergistic potential of azole fungicides is based on their toxicodynamic properties.
• The inhibition potential of prochloraz toward in vivo cytochrome P450 (ECOD) activity is approximately 450 times stronger than the inhibition potential of propiconazole.

Azole fungicides (imidazoles and triazoles) are known to function synergistically with several compounds, especially with pyrethroid insecticides, most likely by inhibiting cytochrome P450. Different azole fungicides have been shown to differ in their synergistic potentials usually with the imidazoles being stronger synergists than the triazoles. This study investigated whether the toxicokinetic and toxicodynamic (TKTD) properties of the imidazole prochloraz and triazole propiconazole can explain their different synergistic potential toward the freshwater macroinvertebrate Daphnia magna. Pulse exposure to external concentrations of propiconazole (1.4 μM) and prochloraz (1.7 μM) for 18 h resulted in internal concentrations of 22.7 and 53.5 μmol kg−1 w.w. for propiconazole and prochloraz, respectively. This 2-fold difference in bioaccumulation corresponded very well with the observed 2.7-fold lower external EC50-estimate (7 days) for prochloraz compared to propiconazole. The estimated IC50 for the in vivo inhibition of cytochrome P450 (ECOD) activity, however, measured as transformation of 7-ethoxycoumarin into 7-hydroxycoumarin, was almost 500-fold higher for prochloraz (IC50: 0.011 ± 0.002 μM) compared to propiconazole (IC50: 4.9 ± 0.06 μM). When indirectly measuring the binding strength of the two azoles, daphnids exposed to propiconazole recovered roughly 80% of their ECOD activity compared to the control shortly after being moved to azole-free medium, indicating that propiconazole causes reversible inhibition of cytochrome P450. In contrast, the ECOD-activity remained inhibited in the prochloraz-exposed daphnids for 12 h following transfer to azole-free medium, which correlated with elimination of the measured internal prochloraz concentration (DT95≈ 13 h).These results indicate that lethal toxicity of the azole fungicides is mainly driven by toxicokinetics through their hydrophobicities resulting in different internal concentrations. Their synergistic potential toward pyrethroid toxicity, on the other hand, is mainly governed by their toxicodynamic effects measured as the differences in IC50-values toward in vivo cytochrome P450 (ECOD) activity together with the proposed binding strength measured indirectly through the recovery of ECOD activity as a function of internal azole concentrations.