title: What causes the difference in synergistic potentials of propiconazole and prochloraz toward pyrethroids in Daphnia magna?
abstract: 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 (1.4μM) and prochloraz (1.7μM) for 18h resulted in internal concentrations of 22.7 and 53.5μmolkg(-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±0.002μM) compared to propiconazole (IC50: 4.9±0.06μM). 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 12h following transfer to azole-free medium, which correlated with elimination of the measured internal prochloraz concentration (DT95≈13h). 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.
authors.affiliation: Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg C, Denmark. Electronic address: Dalhoff@plen.ku.dk.
authors.forename: Kristoffer
authors.initials: K
authors.lastname: Dalhoff
authors.affiliation: Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg C, Denmark.
authors.forename: Michele
authors.initials: M
authors.lastname: Gottardi
authors.affiliation: Department of Analytical Biosciences, Institute of Pharmacy, University of Copenhagen, Universitetsparken 2, 2100, København Ø, Denmark.
authors.forename: Andreas
authors.initials: A
authors.lastname: Kretschmann
authors.affiliation: Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg C, Denmark.
authors.forename: Nina
authors.initials: N
authors.lastname: Cedergreen
mesh_terms: D000818:Animals; D003374:Coumarins / Q000378:metabolism; D003621:Daphnia / Q000187:drug effects*; D004357:Drug Synergism; D005659:Fungicides, Industrial / Q000633:toxicity; D007093:Imidazoles / Q000633:toxicity*; D020128:Inhibitory Concentration 50; D011722:Pyrethrins / Q000633:toxicity*; D014230:Triazoles / Q000633:toxicity*; D014468:Umbelliferones / Q000737:chemistry; D014874:Water Pollutants, Chemical / Q000633:toxicity
chemical_list: D003374:Coumarins; D005659:Fungicides, Industrial; D007093:Imidazoles; D011722:Pyrethrins; D014230:Triazoles; D014468:Umbelliferones; D014874:Water Pollutants, Chemical; C045950:propiconazole; C017299:7-ethoxycoumarin; C031477:7-hydroxycoumarin; C045362:prochloraz
keywords: Azole fungicides; Daphnia magna; Synergism; Toxicodynamics; Toxicokinetics