Effects of long-term exposure to two fungicides, pyrimethanil and tebuconazole, on survival and life history traits of Italian tree frog (Hyla intermedia)

• Sublethal effects of tebuconazole and pyrimethanil on Hyla intermedia.
• Long-term effects of fungicides realistic concentrations on amphibian life history traits.
• Evaluation of developmental parameters, deformity and metamorphosis traits.
• Fungicides induce reduced survival and increase of deformities in Hyla intermedia.
• Reduced metamorphic success and delayed time to metamorphosis.

Over the last few years, the hazards associated with the extensive use of fungicides have become an issue of great concern but, at present, the effects of these substances on amphibians remain poorly understood. The goal of the present study was to assess the effects of two commonly used fungicides, tebuconazole and pyrimethanil, on Italian Tree Frog (Hyla intermedia), a species frequently found in agricultural areas. Tadpoles were exposed to fungicides from developmental Gosner stage 25 (GS 25) to completion of metamorphosis (GS 46) and the whole exposure period lasted 78 days. For both tested fungicides we used two concentrations (5 and 50 μg/L) that are comparable to those detected in surface waters, near agricultural fields.A variety of sublethal effects—on growth, development, behavior, and physiology—may be used for evaluating alterations induced by pollutants in amphibians. We estimated whether pyrimethanil and tebuconazole exposure impacted on H. intermedia life history traits. For this purpose, survival, growth, development, initiation of metamorphosis, success and size at metamorphosis, time to metamorphosis, and frequency of morphological abnormalities were evaluated. We showed, for all considered endpoints, that the exposure to tebuconazole exerts more harmful effects on H. intermedia than does exposure to pyrimethanil.Before the onset of metamorphic climax we showed, for both fungicides, that the low concentrations (5 μg/L) induced significantly greater effects than the higher ones (50 μg/L) on survival and deformity incidence. During the metamorphic climax, a complete reversal of this nonlinear trend takes place, and the percentage of animals initiating metamorphosis was reduced in fungicide-exposed groups in a concentration-dependent manner. Furthermore, a strong correlation emerged between fungicide exposure and the incidence of morphological abnormalities such as tail malformations, scoliosis, edema, mouth and limb deformities.Exposure to tested fungicides also caused a reduction in developmental rates just prior to the onset of metamorphic climax, which translated to a significant delay in timing of metamorphosis. We detected a drastic decrease in the success at metamorphosis in all exposed groups, compared to control group (86.25%). In fact, the percentage of survived larvae to GS 46, in the high and low concentrations, respectively, was only 22.5% and 36.25% in tebuconazole-exposed groups and 43.75% (50 μg/L) and 56.25% (5 μg/L) in pyrimethanil-exposed groups.Our findings underscore the hazardous properties of these two fungicides for non-target species in the context of ecotoxicological risk assessment. No published studies have addressed the long-term effects of tebuconazole and pyrimethanil on amphibians. To date, this is one of only a few studies documenting the effects of fungicide exposure over the whole larval development.