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Amphibians have experienced an accentuated population decline in the whole world due to many factors, one of them being anthropogenic contamination. The present study aimed to assess the potential effect of copper, as a worldwide and reference contaminant, on the immediate decline of exposed population due to avoidance and mortality responses in tadpoles of three species of amphibians across climatic zones: a South American species, Leptodactylus latrans, a North American species, Lithobates catesbeianus, and a European species, Pelophylax perezi. A non-forced exposure system with a copper gradient along seven compartments through which organisms could freely move was used to assess the ability of tadpoles to detect and avoid copper contamination. All species were able to avoid copper at a concentration as low as 100 μg L(-1). At the lowest (sublethal) concentrations (up to 200 μg L(-1)) avoidance played an exclusive role for the population decline, whereas at the highest concentrations (> 450 μg L(-1)) mortality was the response determining population decline. The median concentrations causing exposed population immediate decline were 93, 106 and 180 μg L(-1) for Le. latrans, Li. catesbeianus and P. perezi, respectively. Contaminants might, therefore, act as environmental disruptors both by generating low-quality habitats and by triggering avoidance of tadpoles, which could be an important response contributing to dispersion patterns, susceptibility to future stressors and decline of amphibian populations (together with mortality).

Several authors have suggested that nitrogen-based fertilizers may be contributing to the global amphibian decline. We have studied the impact of sodium nitrite on early aquatic stages of Epidalea calamita, Pelophylax perezi and Hyla meridionalis larvae from Doñana National Park (coastal wetland) and P. perezi from Gredos Mountain (high mountain ponds), exposed during 10 to 16 days. After 8 days of exposure all P. perezi larvae from Doñana presented 100% mortality at 5 mg l(-1)N-NO2(-) while E. calamita larvae mortality rates were significantly lower at that concentration after 15 days. However, for H. meridionalis at day 15 no deaths were registered at 5 mg l(-1)N-NO2(-) and at 20 mg l(-1)N-NO2(-) presented intermediate mortality rates. In Doñana the 10 d LC50 of older H. meridionalis larvae was between 20 and 30 mg l(-1)N-NO2(-) whilst for P. perezi it was below 5 mg l(-1)N-NO2(-). These results indicate inter-specific variation of the sensitivity of larval amphibians to nitrite. Gredos Mountain P. perezi larvae exposed since the egg stage were highly sensitive to nitrite, with a 16 d LC50 below 0.5 mg l(-1)N-NO2(-). The same species in Doñana had a 15 d LC50 between 5 and mg l(-1)N-NO2(-). These results suggest that there is also intra-specific variation in sensitivity of amphibian larvae to nitrite: mountain amphibian populations appear to be more sensitive to polluted environments than coastal populations. Geographic and genetic variation and evolutionary adaptation of tolerance may also be the keys to variation amongst populations of the same species.

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Tadpoles of two amphibian species, the neotropical anuran Leptodactylus latrans and the North American bullfrog Lithobates catesbeianus, were used in experiments to assess their preferred spatial distribution along habitat gradients and, thus, to what extent contamination by the fungicide pyrimethanil could trigger active spatial avoidance. The tadpoles were tested in a non-confined multi-compartment static system with a pyrimethanil contamination gradient through which organisms could move freely. Two samples, with and without (reference) pyrimethanil contamination, taken from outdoor mesocosms, were assayed. Tadpoles showed to be able to detect and move to the most favorable environment by preferring compartments containing reference mesocosm water. Pyrimethanil concentrations from 0.2 to 1.4 mg L(-1) were below lethal levels, but acted as habitat disturber since spatial avoidance was triggered. Avoiders of L. latrans reached almost 50 % at 1.4 mg L(-1). The present data reinforces the hypothesis regarding the risk of plant protection products to act, not only as toxicants, but also as habitat disturber, potentially leading to avoidance-driven population decline of amphibians.

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a b s t r a c tReactive nitrogen compounds such as nitrite (NO -) are highly toxic to aquatic animals and are partly responsible for the global decline of amphibians. On some fish and Caudata amphibian species low levels of sodium chloride significantly reduce the toxicity of nitrite. However, the nitrite-salinity interaction has not been properly studied in anuran amphibians. To verify if chloride (Cl -) attenuates NO -toxicity, eggs and larvae of three anuran species were subjected to a series of NO -solutions combined with three salt concentrations (0, 0.4 and 2 or 0, 0.052 and 0.2 g L -1 NaCl). One of the species tested originated from two different populations inhabiting highly contrasted nutrient richness environments: lowland Doñana Natural Park and Sierra de Gredos Mountain. In general, the presence of Cl -increased survival and growth of lowland Pelophylax perezi and activity of mountain P. perezi larvae exposed to NO -, thus attenuating the toxicity of NO -to developing amphibians. Mountain amphibian populations appeared to be much more sensitive to the concentrations of NO -and Cl -used in this experiment than coastal conspecifics, suggesting possible adaptation of populations to local conditions. Nitrogen pollution in coastal wetlands poses a serious threat to aquatic organisms, causing direct toxicity or indirect effects via ecosystem eutrophication. The presence of low to medium levels of salinity that would be common in coastal wetlands may attenuate the direct effects of increasing concentrations of nitrogenous compounds in water bodies. Furthermore, treating cultures of endangered anurans with small amounts of NaCl may provide an additional protective measure.

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