Altered development, oxidative stress and DNA damage in Leptodactylus chaquensis (Anura: Leptodactylidae) larvae exposed to poultry litter.

Poultry litter (PL), which is usually used as organic fertilizer, is a source of nutrients, metals, veterinary pharmaceuticals and bacterial pathogens, which, through runoff, may end up in the nearest aquatic ecosystems. In this study, Leptodactylus chaquensis at different development stages (eggs, larval stages 28 and 31 here referred to as stages I, II and III respectively) were exposed to PL test sediments as follows: 6.25% (T1), 12.5% (T2); 25% (T3); 50% (T4); 75% (T5); 100% PL (T6) and to dechlorinated water as control. Larval survival, development endpoints (growth rate -GR-, development rate -DR-, abnormalities), antioxidant enzyme activities (Catalase -CAT- and Glutathione-S-Transferase -GST-), and genotoxic effect (DNA damage index by the Comet assay) were analyzed at different times. In stage I, no egg eclosion was observed in treatments T3-T6, and 50% of embryo mortality was recorded after 24h of exposure to T2. In stages II and III, mortality in treatments T3-T6 reached 100% between 24 and 48h. In the three development stages evaluated, the DR and GR were higher in controls than in PL treatments (T1, T2), except for those T1-treated larvae of stage II. Larvae of stage I showed five types of morphological abnormalities, being diamond body shape and lateral displacement of the intestine the most prevalent in T1, whereas larvae of stages II and III presented lower prevalence of abnormalities. In stage I, CAT activity was similar to that of control (p>0.05), whereas it was higher in T1- and T2- treated larvae of stages II and III than controls (p<0.05). In stages I and III, GST activity was similar to that of controls (p>0.05), whereas it was inhibited in T1-treated larvae of stage II (p<0.05). T1- and T2-treated larvae of stages II and III caused higher DNA damage respect to controls (p<0.05), varying from medium to severe damage (comet types II, III and IV). These results showed that PL treatments altered development and growth and induced oxidative stress and DNA damage, resulting ecotoxic for L. chaquensis larvae.

Effect of diet on carboxylesterase activity of tadpoles (Rhinella arenarum) exposed to chlorpyrifos.

An outdoor microcosm was performed with tadpoles (Rhinella arenarum) exposed to 125µgL-1 chlorpyrifos and fed two types of food, i.e., lettuce (Lactuca sativa) and a formulated commercial pellet. Acetylcholinesterase (AChE) and carboxylesterase (CbE) activities were measured in liver and intestine after 10 days of pesticide exposure. Non-exposed tadpoles fed lettuce had an intestinal AChE activity almost two-fold higher than that of pellet-fed tadpoles. No significant differences were observed, however, in liver AChE activity between diets. Likewise, intestinal CbE activity - measured using two substrates, i.e. 1-naphthyl acetate (1-NA) and 4-nitrophenyl valerate (4-NPV) - was higher in tadpoles fed lettuce than in those fed pellets. However, the diet-dependent response of liver CbE activity was opposite to that in the intestine. Chlorpyrifos caused a significant inhibition of both esterase activities, which was tissue- and diet-specific. The highest inhibition degree was found in the intestinal AChE and CbE activities of lettuce-fed tadpoles (42-78% of controls) compared with pellet-fed tadpoles (<60%). Although chlorpyrifos significantly inhibited liver CbE activity of the group fed lettuce, this effect was not observed in the group fed pellets. In general, intestinal CbE activity was more sensitive to chlorpyrifos inhibition than AChE activity. This finding, together with the high levels of basal CbE activity found in the intestine, may be understood as a detoxification system able to reduce intestinal OP uptake. Moreover, the results of this study suggest that diet is a determinant factor in toxicity testing with tadpoles to assess OP toxicity, because it modulates levels of this potential detoxifying enzyme activity.

Toxicity of the fungicide trifloxystrobin on tadpoles and its effect on fish-tadpole interaction.

Contamination of aquatic systems is a major environmental stress that can interfere with predator-prey interactions, altering prey or predator behavior differentially. We determined toxicity parameters of the fungicide trifloxystrobin (TFS) and examined its effects on predation rate, using a fish predator (Synbranchus marmoratus) and four anuran tadpole species as prey (Rhinella arenarum, Physalaemus santafecinus, Leptodactylus latrans, and Elachistocleis bicolor). TFS was not equally toxic to the four tadpole species, E. bicolor being the most sensitive species, followed by P. santafecinus, R. arenarum, and L. latrans. Predation rates were evaluated using different treatments that combined predator and prey exposed or not to this fungicide. TFS would alter the outcome of eel-tadpole interaction by reducing prey movements; thus, prey detection would decrease and therefore tadpole survival would increase. In addition, eels preyed selectively upon non-exposed tadpoles avoiding the exposed ones almost all throughout the period evaluated. Predation rate differed among prey species; such differences were not due to TFS exposure, but to interspecific differences in behavior. The mechanism that would explain TFS-induced reduction in predation rates remains unclear; however, what is clear is that sublethal TFS concentrations have the potential to alter prey behavior, thereby indirectly altering predator-prey interactions. In addition, we consider that predator-prey relationships are measurable responses of toxicant exposure and provide ecological insight into how contaminants modify predator-prey interactions.

Apoptotic cell death in the central nervous system of Bufo arenarum tadpoles induced by cypermethrin.

Tadpoles of the toad Bufo arenarum treated with cypermethrin (CY) at concentrations above 39 mug CY/L showed dose-dependent apoptotic cell death in immature cells of the central nervous system as demonstrated by morphometric analysis, the TUNEL method, and DNA fragmentation assay. Light-and electron-microscopic studies showed structural alterations in the intermediate and marginal layers of the brain. Immature cerebral tissue showed cellular shrinkage, nuclear fragmentation and increase of intercellular spaces. In this study we demonstrated high toxicity of CY to larval stages of Bufo arenarum. Our results show that doses lower than those used in routine insecticide applications can cause massive apoptosis in the immature cells of the central nervous system. These results coincide with our previous studies in Physalaemus biligonigerus, confirming the severe toxic effects of CY to the central nervous system of anuran species from Argentina. This may increase the mortality index in wild animals and contribute to the loss of biodiversity in our agroecosystems. We postulate that CY induces apoptosis in central nervous system cells of Bufo arenarum tadpoles by specific neurotoxic mechanisms.

Paraquat tolerance and alteration of internal gill structure of Scinax nasica tadpoles (Anura: Hylidae).

Paraquat acute tolerance, and histological and ultrastructural response analysis of gills were evaluated in Scinax nasica tadpoles at stages 25-26. The treated tadpoles were able to survive with a mortality rate similar to the controls in media containing 6.48 to 10.80 mg PQ/L. At 18.00 mg PQ/L survival rate was about 85%; at 30.00 and 50.00 mg PQ/L an incremented mortality rate was detected. Their internal gills appear to have a critical role in adaptation to exposure to PQ. The LC-50 values found helped us conclude that increasing time of exposure to PQ produces a decrease of tadpole survival rate. With light microscopy, the apical ramification gill tufts exhibited a dose response increase of the total volume. At the ultrastructural level, an increase in the intercellular spaces and alteration in gill epithelium organization were the most notable changes. Furthermore, the capillaries showed enlarged endothelial cells, irregular nuclei, and numerous cytoplasmatic projections.