Tebuconazole reduces basal oxidative respiration and promotes anxiolytic responses and hypoactivity in early-staged zebrafish (Danio rerio)

Triazole fungicides are increasingly used in North America to combat mold and fungi, in order to protect vegetables, citrus, ornamental plants and field crops. To determine the biological impacts of tebuconazole in nontarget aquatic organisms, early life stage zebrafish were exposed to 0.1-100 mu M tebuconazole for 120 h (5 dpf). There was a significant increase in mortality over time and at 100 mu M, only 50% of the animals survived 96 h compared to > 95% for all other experimental groups. There was evidence for increased hatching time with 10 mu M tebuconazole compared to the control group (similar to 7 h longer at 50% total hatch) or a lack of hatch observed with 100 mu M. Oxidative respiration and behavior were evaluated to assess whether the fungicide impaired energy-associated processes. Oxygen consumption rates in embryos (exposed from similar to 6 hpf) were determined with exposure to 2.5, 25, 50, 100 pM tebuconazole for 24 h using the XFe24 Extracellular Flux Analyzer. Embryos treated with 100 pM showed a similar to 60% reduction in basal respiration, indicating impaired oxygen consumption and/or changes in resource allocation (e.g. anti-oxidant production, metabolite synthesis). Environmentally-relevant concentrations of tebuconazole did not affect oxidative phosphorylation. As behavior is a sensitive endpoint for toxicity, we measured the dark photokinesis response and conducted a light-dark preference test in 6 dpf larvae following a sub-chronic exposure to 0.1, 1 and 10 mu M tebuconazole beginning with 6 hpf embryos. It was observed in two independent experiments for dark photokinesis that 10 mu M tebuconazole reduced total distance moved (i.e. hypoactivity) in the dark period by similar to 25-35%. In the light-dark preference test, there was an increase for mean time in dark zone (similar to 100% increase in the average time/visits per second) and frequency in dark zone (increase of similar to 35% in average number of visits) with tebuconazole, suggestive of anxiolytic behavior at environmentally-relevant doses. This study demonstrates that exposure to tebuconazole can affect survival, hatch time, oxidative phosphorylation, and behavioral activity of early-staged zebrafish. While survival, hatch time, and mitochondrial bioenergetics were not different than control fish at environmentally-relevant levels of tebuconazole, behavioral responses were detected at concentrations reported in some aquatic environments.