Effects of glyphosate-based herbicides on survival, development, growth and sex ratios of wood frogs (Lithobates sylvaticus) tadpoles: I, Chronic laboratory exposures to VisionMax®

The purpose of this study was to determine if chronic exposure to the glyphosate-based herbicideVisionMax®affects the survival, development, growth, sex ratios and expression of specific genesinvolved in metamorphosis of wood frog tadpoles (Lithobates sylvaticus). We hypothesized that expo-sure to this herbicide will affect developmental rates by disrupting hormone pathways, sex ratios and/orgonadal morphology. Tadpoles were chronically exposed in the laboratory from Gosner developmen-tal stage 25 to 42 to four different concentrations of VisionMax® (ranging from 0.021 to 2.9 mg acidequivalents/L). Chronic exposures to VisionMax® had direct effects on the metamorphosis of L. sylvaticustadpoles by decreasing development rates, however, there was a decrease in survival only in the group exposed to the highest dose of VisionMax® (2.9 mg a.e./L; from approximately 96% in the control groupto 77% in the treatment group). There was a decrease in the number of tadpoles reaching metamor-phic climax, from 78% in the control group to 42% in the VisionMax®(2.9 mg a.e./L) group, and a 7-daydelay to reach metamorphic climax in the same treatment group. No effects of exposure on sex ratiosor gonadal morphology were detected in tadpoles exposed to any of the concentrations of VisionMax®tested. Gene expression analyses in brain and tail tissues demonstrated that exposure to VisionMax® alters the expression of key genes involved in development. Results showed significant interaction (two-way ANOVA, P < 0.05) between developmental Gosner stage and treatment in brain corticotropin-releasingfactor, deiodinase type II (dio2) and glucocorticotiroid receptor (grII) and tail dio2 and grII. This demon-strates that mRNA levels may be differently affected by treatment depending on the developmental stageat which they are assessed. At the same time there was a clear dose–response effect for VisionMax®toincrease thyroid hormone receptor ˇ in tadpole brain (F(2,69)= 3.475, P = 0.037) and tail (F(2,69)= 27.569,P < 0.001), regardless of developmental stage. Interestingly, delays in development (or survival) wereonly observed in the group exposed to 2.9 mg a.e./L of VisionMax®, suggesting that tadpoles need to be exposed to a “threshold” concentration of glyphosate-based herbicide to exhibit phenotypic observable effects. We suggest that the up regulation of genes that trigger metamorphosis following VisionMax® herbicide exposure might result from a compensatory response for the delays in development observed. Further studies are needed to determine if disruption of expression of these key genes leads to long-termeffects when metamorphs reach adult stages