Amphibians are frequently found exploring subterranean environments. Even though in Europe there are already some reports of amphibians present in subterranean habitats, in Portugal there is a major lack of knowledge of these ecosystems, namely in the karst region. Considering the relevance of amphibians and their current conservational status, it is important to assess their presence in every potential habitat. In order to provide further knowledge over this subject a preliminary survey was carried out in two Portuguese karst massifs, Estremenho and Sicó. A total of 24 caves were surveyed and as result three species (Tarentola mauritanica; Salamandra salamandra and Pleorodeles waltl) were found in Estremenho karst massif, and two species (Triturus marmoratus and Bufo bufo) were observed in Sicó karst massif. Furthermore, evidences were found that P. waltl uses caves for reproduction, which had only been reported once in Spain. This allowed perceiving that herpetofauna species occupy caves of Portuguese karst massifs and are also using them as reproduction sites.

Amphibians in Portugal are commonly found in karst ecosystems. Nonetheless, these ecosystems are threatened by activities such as agricultural and industrial activities and mining, which contaminate both surface water and groundwater. Due to the high permeability of the karst systems, the existence of water on the surface is scarce, with sinkhole ponds and springs representing essential water sources for amphibians, especially for their reproduction. Furthermore, water from springs may reflect the effects of surface contamination on groundwater. Considering the relevance of these water sources, specially to amphibians, which are considered highly sensitive to a widerange of contaminants, our study aimed at assessing the suitability of water from springs and sinkholes from two Portuguese karst massifs, for the development of the early lifestages of Hyla arborea. To achieve this, eggs from H. arborea were exposed to water from seven springs and four sinkholes, from the Estremenho and Sicó karst massifs, anda control (FETAX). The exposure was carried out with five replicates per condition, each with 20 tadpoles, and ended once the 25th Gosner stage was reached. The parameters evaluated were growth, antioxidant enzyme activity (Glutathione STransferases, Glutathione Peroxidase Se-dependent, total-Glutathione Peroxidase and Glutathione Reductase) and lipid peroxidation (LPO). Also, water samples were screened for the presence of six commonly used pesticides (Terbuthylazine, Tebuconazole, Deltamethrin, Dimethomorph, Chlorpyrifos and Glyphosate). The results revealed the presence of glyphosate in three of the sampled water bodies. Concurrently, in one of the sites presenting detectable glyphosate concentrations, tadpoles presented smaller body lengths, and mortality was also observed. In the remaining sites, despite the absence of the screened pesticides, there were some alterations of the biochemical parameters assessed, namely LPO. These results raised some concerns regarding the impact of human activities in surface and groundwater in the Sicó and Estremenho karst massifs.