Subterranean but vulnerable: Sub-lethal effects of substrate contamination by a glyphosate-based herbicide on the physiological condition and behavior of a fossorial reptile

Abstract Ecosystems worldwide face increasing levels of environmental pollutants, including agrochemicals which are widely used in modern agriculture to improve crop productivity and food production. Among them, glyphosate-based herbicides (GBHs) are globally the most widely used. However, although the potential toxicity of GHHs has been evaluated in a myriad of vertebrate species, its impact on fossorial reptiles remains unexplored. We evaluated physiological and behavioral responses of the fossorial amphisbaenian reptile Blanus cinereus after exposure to substrates treated with a commercial GBH Roundup Ultra Plus® (RU-UP). Individuals were kept for 21 days in clean or contaminated coconut fiber substrates treated with a high RU-UP application solution (7.2 g a.e. L−1) to assess effects on body condition, levels of fecal corticosterone metabolites (FCM), skin inflammatory response (phytohemagglutinin), and antipredator behavior. After 21 days, body condition did not change, but exposed individuals showed higher FCM levels and reduced inflammatory responsiveness. They also increased antipredator behaviors (coiling, shaking) and fled by burrowing faster, indicating behavioral alterations likely linked to an increase in the stress response. A subsequent avoidance experiment using clean, low (3.6 g a.e. L−1) and high (7.2 g a.e. L−1) RU-UP application solutions revealed that individuals failed to avoid RU-UP-treated substrates and showed a preference for low-dose RU-UP concentrations (3.6 g a.e. L−1), potentially increasing vulnerability to RU-UP exposure, which may affect their health status. To our knowledge, these findings provide the first evidence of GBH effects in a fossorial reptile and highlight the need to combine behavioral and physiological endpoints in ecotoxicological research.