Lethal and sublethal effects of the emerging contaminant oxytetracycline on the embryo-larval development of Rhinella arenarum.

The antibiotic oxytetracycline (OTC) is commonly used in animal production and can enter aquatic ecosystems, causing adverse effects on non-target species. The aim of this work was to evaluate the lethal and sublethal effects of OTC on the embryonic and larval period of Rhinella arenarum, through standardized bioassays and oxidative stress (catalase-CAT-, superoxide dismutase-SOD-, glutathione S-transferase-GST-, reduced glutathione-GSH- and lipid peroxidation-TBARS-), neurotoxicity (acetylcholinesterase-AChE- and butyrylcholinesterase-BChE-) and genotoxicity (micronuclei test) biomarkers. Mortality was time and stage dependent, being the embryos (504 h-LC50 = 64.04 mg/L) more sensitive than the larvae (504 h-LC50 = 97.74 mg/L). Alterations in the oxidative stress biomarkers were observed mainly in larvae: CAT, SOD and GST decreased and GSH increased significantly. In embryos, only GST decreased significantly. Also, OTC increased the AChE and BChE activities but did not increase the micronuclei frequency. This study shows evidence that the presence of OTC in the environment may have negative effects on amphibians.

Morphological and histological abnormalities of the neotropical toad, Rhinella arenarum (Anura: Bufonidae) larvae exposed to dexamethasone.

Dexamethasone (DEX) is a glucocorticoid highly effective as an anti-inflammatory, immunosuppressant and decongestant drug. In the present study, a preliminary acute toxicity test was assayed in order to determinate DEX median-lethal, lowest-observed-effect and the no-observed-effect concentrations (LC50, LOEC and NOEC, respectively) on the common toad embryos (Rhinella arenarum). Also, morphological and histological abnormalities from five body larval regions, liver melanomacrophages (MM) and glutathione S-transferase (GST) activity were evaluated in the toad larvae to characterize the chronic sublethal effects of DEX (1-1,000 µg L-L). Results of the acute test showed that the LC50 of DEX at 96 h of exposure for the toad embryos (GS 18-20) was 10.720 mg L-g, and the LOEC was 1 µg L-g. In the chronic assay, the larval development and body length were significantly affected. DEX exposition also induced teratogenic effects. Most frequent external abnormalities observed in DEX-treated larvae included abdominal edema and swollen body, abnormal gut coiling and visceral congestion. Intestinal dysplasia was recurrent in cross-section of all DEX-treated larvae. Neural, conjunctive and renal epithelial cells were also affected. Significant increase in liver MM number and size, and GST activity levels were also registered in DEX treatments with respect to controls. The evaluation of a variety of biomarkers provided clear evidence of toad larvae sensitivity to DEX, and the ecotoxicological risk of these pharmaceuticals, commonly found in different water bodies worldwide on aquatic animals.

Differential sensitivity of developmental stages of the South American toad to a fungicide based on fludioxonil and metalaxyl-M.

Agricultural fungicide application in Argentina has increased twice since 2008, with Maxim® XL (2.5% fludioxonil +1% metalaxyl-M) as one of the most used fungicide formulation. The toxicity of this pesticide on Rhinella arenarum was assessed by means of continuous (from embryo and larval development) and 24-h pulse exposure standardized bioassays. Lethality was concentration- and exposure time-dependent. Maxim® XL caused a progressive lethal effect along the bioassays with higher toxicity on embryos than larvae, obtaining 50% lethal concentrations at 96, 336, and 504 h of 10.85, 2.89, and 1.71 mg/L for embryos, and 43.94, 11.79, and 5.76 mg/L for larvae respectively. Lethal 504-h no observed effect concentration values for embryos and larvae were 1 and 2.5 mg/L respectively. A stage-dependent toxicity of Maxim® XL was also demonstrated within the embryo development, with early stages more sensitive than the later ones, and blastula as the most sensitive developmental stage. The risk quotients obtained for chronic risk assessment determined a potential threat for the survival and continuity of R. arenarum populations under these conditions. The results indicate that the levels of the fungicide reaching amphibian habitats could be risky for the early development of this amphibian species. This study also emphasizes the necessity to evaluate the chronic effects of fungicides in pesticide risk assessment.

Monitoring of toxicity of As(V) solutions by AMPHITOX test without and with treatment with zerovalent iron nanoparticles.

Changes in toxicity of As(V) solutions from acute to chronic exposure have been evaluated by the AMPHITOX test. This test employs Rhinella arenarum, a widely distributed toad in Argentine areas. LOEC values were 6.37 and 1.88 mg L-1 for embryos and larvae, respectively, and serious sublethal effects have been observed. Toxicity of As(V) solutions has been also evaluated after treatment with zerovalent iron nanoparticles (nZVI). After 60 min of treatment with nZVI, As(V) removal was 77%, and neither lethal nor sublethal effects were observed. However, nZVI had to be eliminated before the bioassay because they caused adverse effects in both embryos and larvae. This work highlights the high sensitivity of R. arenarum to As(V), the relevance to assess toxicity on different periods of the lifecycle, and the need to expand exposure to As(V) to chronic times. The utility of the test for monitoring toxicity changes in As(V) solutions after nZVI treatment has been also shown.

Reproductive endpoints of Rhinella arenarum (Anura, Bufonidae): Populations that persist in agroecosystems and their use for the environmental health assessment.

Degradation of the environment by agriculture affects the persistence and health of the amphibian populations. Characteristics related to reproduction of anuran can be used to evaluate the status of populations and as endpoints in environmental health assessment. In this in situ study the aspects related to the amplexus and ovipositions of the bioindicator species Rhinella arenarum that inhabits agroecosystems were analyzed. The hypothesis of this study is that perturbations of agroecosystems have a negative impact on the size of reproductive adults, on the size of ovipositions and eggs, and on the survival of eggs and embryos. Study area is located in the rural landscape of central Argentina. Four sampling sites were selected: C1, C2 and C3 are ponds on agroecosystems; and SM is a reference site that is not affected by agriculture or livestock. Abundance of amplexus pairs, oviposition and tadpoles per site was recorded. Individuals´ snout-vent length (SVL) in amplexus was measured. The fecundity was calculated like number of eggs per oviposition. The eggs' Gosner stage, the diameter eggs and the frequency of dead and abnormal eggs were recorded by oviposition. Killing-power between egg-embryo and egg-tadpole was calculated. The higher phosphate concentration was detected in all agroecosystems and nitrate was detected in C1 and C2. Conductivity, salinity and SDT were higher in C1 site Male SVL from the SM site was lower than the other sites while the largest SVL was of female from the C3 site. The higher frequencies of sprouted eggs and of dead eggs were recorded in the C2 site. Egg diameter was associated with SM and correlated negatively to SVL of the male and female. No correlation between female SVL and oviposition size was recorded. Killing-power in the passage from egg to tadpole classes was higher in the three agroecosystems. The hypothesis of this study was corroborated in part. Reproductive adults in agroecosystems did not have smaller body size. However, in the agroecosystem ponds, the eggs with smaller diameter were registered, the oviposition had higher frequency of abnormal eggs and the higher mortality was registered. This confirms the high sensitivity of the early stages to environmental disturbances and sustains their use as endpoints for the environmental health assessment.

Chronic toxicity of arsenic during Rhinella arenarum embryonic and larval development: Potential biomarkers of oxidative stress and antioxidant response.

The Argentinean autochthonous toad Rhinella arenarum was selected to study the chronic toxicity of arsenic (As) and the biochemical responses elicited by exposure to As in water during embryonic and larval development. Significant decreases in the total reactive antioxidant potential and in catalase activity were observed in individuals exposed chronically to sublethal concentrations of As, which is indicative of an oxidative stress situation. However, an antioxidant response was elicited during chronic exposure to As, as evidenced by the increase in endogenous reduced glutathione content and glutathione-related enzymatic activities such as glutathione S-transferase (GST) and glutathione reductase. This protective response might prevent a deeper decline in the antioxidant system and further oxidative damage. Alternatively, it might be linked to As conjugation with reduced glutathione for its excretion. Considering the sustained increase in GST activity and the decrease in the total antioxidant reactive potential observed, the authors propose them as good candidates to be used as biomarkers during As exposure. Interestingly, glutathione reductase activity was inhibited at a very low concentration of As considered safe for aquatic life. Environ Toxicol Chem 2017;36:1614-1621. © 2016 SETAC.

Combined endosulfan and cypermethrin-induced toxicity to embryo-larval development of Rhinella arenarum.

The combined effects of two widely used pesticides, endosulfan and cypermethrin, on survival of embryo-larval development of the South American toad (Rhinella arenarum) were examined. The toxicity bioassays were performed according to the AMPHITOX test. Embryos and larvae were exposed to mixtures of these pesticides at equitoxic ratios from acute or chronic exposure to evaluate interaction effects. The results were analyzed using both Marking's additive index and combination index (CI)-isobologram methods. Acute (96-h) and intermediate (168-h) toxicity of endosulfan-cypermethrin mixtures remained almost constant for larvae and embryos, but when exposure duration was increased, there was a significant elevation in toxicity, obtaining chronic (240-h) no-observed-effect concentrations (NOEC) values of 0.045 and 0.16 mg/L for embryos and larvae, respectively. These are environmentally relevant concentrations that reflect a realistic risk of this pesticide mixture to this native amphibian species. The toxicity increment with the exposure duration was coincident with the central nervous system development on embryos reaching the larval period, the main target organ of these pesticides. The interactions of the pesticide mixtures at acute and chronic exposure were antagonistic for embryo development (CI > 1), and additive (CI = 1) for larvae, while chronic exposure interactions were synergistic (CI < 1) for both developmental periods. Data indicated that endosulfan-cypermethrin mixtures resulted in different interaction types depending on duration and developmental stage exposed. As a general pattern and considering conditions of overall developmental period and chronic exposure, this pesticide mixture usually applied in Argentine crop fields is synergistic with respect to toxicity for this native amphibian species.

Overcrowding-mediated stress alters cell proliferation in key neuroendocrine areas during larval development in Rhinella arenarum.

Exposure to adverse environmental conditions can elicit a stress response, which results in an increase in endogenous corticosterone levels. In early life stages, it has been thoroughly demonstrated that amphibian larval growth and development is altered as a consequence of chronic stress by interfering with the metamorphic process, however, the underlying mechanisms involved have only been partially disentangled. We examined the effect of intraspecific competition on corticosterone levels during larval development of the toad Rhinella arenarum and its ultimate effects on cell proliferation in particular brain areas as well as the pituitary gland. While overcrowding altered the number of proliferating cells in the pituitary gland, hypothalamus, and third ventricle of the brain, no differences were observed in areas which are less associated with neuroendocrine processes, such as the first ventricle of the brain. Apoptosis was increased in hypothalamic regions but not in the pituitary. With regards to pituitary cell populations, thyrotrophs but not somatoatrophs and corticotrophs showed a decrease in the cell number in overcrowded larvae. Our study shows that alterations in growth and development, produced by stress, results from an imbalance in the neuroendocrine systems implicated in orchestrating the timing of metamorphosis.

Comparative sensitivity among early life stages of the South American toad to cypermethrin-based pesticide.

Cypermethrin is one of the most widely used pesticides due to its low mammalian and bird toxicity, but it is extremely toxic to aquatic organisms. The aim of the present study was to evaluate the toxicity of a commercial formulation of cypermethrin on the embryo-larval development of Rhinella arenarum. An ecological risk assessment based on the hazard quotient (HQ) approach was performed. The results showed that cypermethrin toxicity was stage-dependent and dramatically increased during the larval period. Thus, larvae were more sensitive than embryos, obtaining at the end of the experiment a 336-h median lethal concentration (LC50) of 0.65 µg cypermethrin/L. Cypermethrin exposure caused morphological abnormalities such as general underdevelopment, edema, gill malformations, and behavioral alterations as hyperkinesia and spasmodic contractions. The 168-h teratogenic index was 5, implying a high risk for embryos to be malformed in the absence of significant embryonic lethality. Based on the results of the toxicity effects and the ecological risk assessed (HQ for chronic exposure > level of concern), this pesticide should be considered as a direct (effects on survival) or indirect (severe sublethal effects) risk for conservation purposes of this amphibian in agroecosystems.

Health status of tadpoles and metamorphs of Rhinella arenarum (Anura, Bufonidae) that inhabit agroecosystems and its implications for land use.

Perturbations of water bodies near agricultural and livestock systems can affect embryonic and larval stages of anurans and negatively impact adult populations and structure of amphibian communities. This study is focused on early development of Rhinella arenarum, for which body growth, abnormalities in the oral disc and genetic damage on erythrocytes were analyzed to establish the impact of agroecosystems on local populations of amphibians. Tadpoles and metamorphs of R. arenarum were collected in three agroecosystems (namely, C1, C2, and C3) and in a site without agricultural and livestock activities (SM) from central Argentina. Egg masses of C1 were extracted for breeding tadpoles under laboratory conditions (Lab). Tadpoles were in small size and lighter in weight in C1 and C2. Metamorphs were shorter and lighter in weight in C1 and C3. In SM and Lab samples, no tadpoles with abnormal LTRF (labial tooth row formula) or without labial teeth were observed. In C1, the highest frequency of abnormal LTRF was recorded and was the only site in which tadpoles without labial teeth were found. In C1 and C2 the tadpoles had highest micronucleus frequencies and nuclear abnormalities. C1 can be considered as the site with the highest anthropogenic perturbation and with less healthy tadpoles. Livestock practices such as alternating cattle between parcel and keeping a buffer between crops and water bodies, would allow a better development of the first aquatic stages that are essential for the conservation of the anuran populations.

Effect of estradiol on apoptosis, proliferation and steroidogenic enzymes in the testes of the toad Rhinella arenarum (Amphibia, Anura).

Estrogens inhibit androgen production and this negative action on amphibian steroidogenesis could be related to the regulation of steroidogenic enzymes. Estrogens are also involved in the regulation of amphibian spermatogenesis by controlling testicular apoptosis and spermatogonial proliferation. The Bidder's organ (BO) is a structure characteristic from the Bufonidae family and in adult males of Rhinella arenarum it is one of the main sources of plasma estradiol (E2). The purpose of this study is to analyze the effect of E2 on testicular steroidogenic enzymes, apoptosis and proliferation in the toad R. arenarum. For this purpose, testicular fragments were treated during 24h with or without 2 or 20nM of E2. After treatments, the activities of cytochrome P450 17α-hydroxylase-C17-20 lyase (CypP450c17) and 3ß-hydroxysteroid dehydrogenase/isomerase (3ß-HSD/I) were measured by the transformation of radioactive substrates into products, and CypP450c17 expression was determined by Western blot analysis. Apoptosis in testicular sections was detected with a commercial fluorescent kit based on TUNEL method, and proliferation was evaluated by BrdU incorporation. Results indicate that E2 has no effect on CypP450c17 protein levels or enzymatic activity, while it reduces 3ß-HSD/I activity during the post reproductive season. Furthermore, although E2 has no effect on apoptosis during the pre and the post reproductive seasons, it stimulates testicular apoptosis during the reproductive season, mostly in spermatocytes. Finally, E2 has no effect on testicular proliferation all year long. Taken together, these results suggest that E2 is involved in the regulation of testicular steroidogenesis and spermatogenesis.

Developmental toxicity and risk assessment of nonylphenol to the South American toad, Rhinella arenarum.

The toxicity of Nonylphenol, an emerging pollutant, on the common South American toad Rhinella arenarum was stage and time dependent, thus Median Lethal Concentrations (LC50) for acute (96h), short-term chronic (168h) and chronic exposure (336h) were 1.06; 0.96 and 0.17mgNP/L from embryonic period (S.4), whereas for exposure from larvae (S.25), LC50 remained constant at 0.37mgNP/L from 96h to 168h, decreasing to 0.11mgNP/L at 336h. NOEC-168h for exposure from embryos was 0.025mgNP/L. The Teratogenic Potential (NOEC-lethality/NOEC-sublethal effects) was 23 times higher than the threshold value, indicating a high risk for embryos to be malformed in absence of significant lethality and representing a threat for the species conservation. By comparing with other amphibians, the early development of R. arenarum was very sensitive to NP. The results highlight the relevance of extending the exposure time and look for the most sensitive stage in order to perform the bioassays for conservation purposes.

Induction of micronuclei and nuclear abnormalities in tadpoles of the common toad (Rhinella arenarum) treated with the herbicides Liberty® and glufosinate-ammonium.

The assessment of micronucleated erythrocytes (ME) in blood represents a widely used method for the detection of chromosomal damage by chemical agents, such as herbicides that may occur as water contaminants. We investigated the changes in some circulating blood-cell parameters of tadpoles of the common toad (Rhinella arenarum) that were exposed during 48 or 96 h to three sub-lethal concentrations (3.75, 7.5, and 15 mg/L) of a commercial formulation of a glufosinate-ammonium (GLA)-based herbicide (Liberty(®), LY(®)) as well as to the corresponding active ingredient GLA. The frequency of ME and other erythrocyte nuclear abnormalities (ENA, i.e., lobed nuclei, binucleates or segmented nuclei, kidney-shaped nuclei, notched nuclei, and picnotic nuclei) were evaluated and compared with positive (cyclophosphamide, CP, 40 mg/L) and negative (de-chlorinated tap water) controls. The results indicate that the exposure of R. arenarum tadpoles to LY(®) induces a concentration-dependent increase in ME frequency. The ENA frequency at 48 h was also significantly higher than that in the negative control group for all the chemicals assayed (CP, LY(®) and GLA) whereas at 96 h, increases in ENA over the negative control group were found only for CP and GLA (7.5 mg/L). Our study demonstrates that the commercial formulation of a GLA-based herbicide induces micronucleus formation in R. arenarum tadpoles, in contrast to the active ingredient. According to these results, the inert ingredients of the commercial formulation played an important role in the production of genotoxic damage in erythrocytes of amphibian tadpoles.

Effectiveness evaluation of glyphosate oxidation employing the H(2)O(2)/UVC process: toxicity assays with Vibrio fischeri and Rhinella arenarum tadpoles.

The H(2)O(2)/UVC process was applied to the photodegradation of a commercial formulation of glyphosate in water. Two organisms (Vibrio fischeri bacteria and Rhinella arenarum tadpoles) were used to investigate the toxicity of glyphosate in samples M(1,) M(2), and M(3) following different photodegradation reaction times (120, 240 and 360 min, respectively) that had differing amounts of residual H(2)O(2). Subsamples of M(1), M(2), and M(3) were then used to create samples M(1,E), M(2,E) and M(3,E) in which the H(2)O(2) had been removed. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities were measured in tadpoles to determine possible sub-lethal effects. In V. fischeri, M(1,E), which was collected early in the photodegradation process, caused 52% inhibition, while M(3,E), which was collected at the end of the photodegradation process, caused only 17% inhibition. Survival of tadpoles was 100% in samples M(2), M(3), and in M(1,E), M(2,E) and M(3,E). The lowest percentages of enzymatic inhibition were observed in samples without removal of H(2)O(2): 13.96% (AChE) and 16% (BChE) for M(2), and 24.12% (AChE) and 13.83% (BChE) for M(3). These results show the efficiency of the H(2)O(2)/UVC process in reducing the toxicity of water or wastewater polluted by commercial formulations of glyphosate. According to the ecotoxicity assays, the conditions corresponding to M(2) (11 ± 1 mg a.e. L(-1) glyphosate and 11 ± 1 mg L(-1) H(2)O(2)) could be used as a final point for glyphosate treatment with the H(2)O(2)/UV process.

Ecología trófica en larvas de Rhinella arenarum (Anura: Bufonidae) en agroecosistemas y sus posibles implicaciones para la conservación / Trophic ecology in tadpoles of Rhinella arenarum (Anura: Bufonidae) in agroecosystems and their possible implications for conservation

Trophic ecology in tadpoles of Rhinella arenarum (Anura: Bufonidae) in agroecosystems and their possible implications for conservation. The progress of the agriculture border has led an important loss of natural habitats, with significant consequences for biodiversity. In this sense, the studies in anuran amphibian tadpoles inhabiting these environments are relevant, because the larval stage is a phase of population regulation. The aim of this study was to analyze the body condition and diet in Rhinella arenarum, tadpoles, an anuran species widely distributed in South America and that inhabit agroecosystems. Three sites were sampled, two agroecosystems with different alteration degrees (C1 and C2) and an uncultured (SM) third place. The captured tadpoles were anesthetized, fixed and preserved in formaldehyde (10%). Subsequently, body measurements were made and the complete intestine was removed and analyzed for food items under a binocular microscope. The diet in R. arenarum tadpoles has a dominance of algae Bacillariophyceae, followed by Cyanophyceae. In particular, the class Bacillariophyceae, due to the presence of the genus Navicula, Nitzschia, Gomphonema and Hantzschia, was important in the diet of the anurans in those agroecosystems. Class Cyanophyceae, mainly represented by genus Oscillatoria and Euglenophyceae represented by Euglena and Strombomonas, were predominant in the diet of the anurans in SM. Some differences in the total items consumed by tadpole were observed between the studied sites. Tadpoles that inhabit the modified sites (C1 and C2) recorded a significantly smaller amount of food. Moreover, the tadpoles that inhabit these sites showed a lower body condition. The presence of certain algae associated with eutrophic environments, could indicate some pollution in agroecosystems (C1 and C2). Food resources would be lesser in places with strong agricultural activity, possibly with a greater degree of eutrophication. A smaller food amount could have consequences at population level for the short and long time terms, because of its impact on individual growth. Larval diet is suggested as a potential bioindicator of environmental health for these areas. Rev. Biol. Trop. 60 (2): 771-779. Epub 2012 June 01.

El crecimiento de la agricultura produce perdida del habitat natural, con consecuencias para la biodiversidad de los anfibios. Se analizo la dieta y condición corporal de larvas de anuros de Rhinella arenarum que habitan agroecosistemas. Los muestreos fueron realizados en dos agroecosistemas y en un tercer sitio, no afectado por cultivos. Las larvas capturadas fueron anestesiadas, fijadas y preservadas en formaldehido, se realizaron medidas corporales y se analizo la dieta de las larvas. Se registro diversidad en la oferta alimenticia proporcionada por los distintos ambientes. La dieta tuvo una predominancia de algas Bacillarophyceae, seguidas por Cyanophyceae. Particularmente, los géneros Navicula, Nitzschia, Hantzschia y Gomphonema (clase Bacillarophyceae), fueron importantes en los agroecosistemas. Los géneros Osillatoria, Euglena y Strombomonas (clases Cyanophyceae y Euglenophyceae), predominaron en la dieta de las larvas de anuros en el sitio menos alterado. Las larvas de los sitios más alterados consumen menor cantidad de alimento y registran una menor condición corporal. La presencia de determinadas algas, indicarían un mayor grado de contaminación en los agroecosistemas. La eutrofización de lagunas podría alterar la disponibilidad de alimento para larvas de anuros que podría tener consecuencias poblacionales negativas. Se sugiere el análisis de dietas larvarias como un potencial bioindicador de salubridad ambiental.

Sublethal effects of atrazine on embryo-larval development of Rhinella arenarum (Anura: Bufonidae).

Atrazine (ATR), one of the most widely used herbicides in the world, affects not only target organisms but also the biota in general. Here, the teratogenic and neurotoxic effects of ATR on Rhinella arenarum (South American toad) embryos, and larvae were evaluated by means of standardized bioassays during acute and chronic exposures. The herbicide had a significant incidence of malformations, with a Teratogenic Index (TI) of 3.28. The main effects were delayed development, reduced body size, microcephaly, axial flexures, wavy tail and edema. In addition, delayed development, reduced development of forelimbs, and edema were recorded at metamorphosis stages. Scanning electron microscopy allowed observing different degrees of cellular dissociation and persistent cilliar cells in specific regions like the adhesive structure and tail fin. Results obtained by ATR 24 h pulse exposures at six developmental stages pointed out blastula as the most susceptible developmental stage both for immediate and delayed adverse effects. A noteworthy recovery capacity from acute toxic effects was recorded from the neural plate stage onwards. Regarding neurotoxic effects, abnormal, and erratic swimming and spasmodic contractions were recorded. Both the teratogenic and neurotoxic effects reported in this study demonstrate the importance of evaluating sublethal effects in non-target organisms as they could imply reduced fitness of individuals and eventually a population decline. The Hazard Quotients (HQ) for ATR ranged from 0.14 to 10.80, and the fact that some of these values are above USEPA's level of concern indicate that ATR is likely a risk to R. arenarum.

[Trophic ecology in tadpoles of Rhinella arenarum (Anura: Bufonidae) in agroecosystems and their possible implications for conservation]. / Ecología trófica en larvas de Rhinella arenarum (Anura: Bufonidae) en agroecosistemas y sus posibles implicaciones para la conservación.

The progress of the agriculture border has led an important loss of natural habitats, with significant consequences for biodiversity. In this sense, the studies in anuran amphibian tadpoles inhabiting these environments are relevant, because the larval stage is a phase of population regulation. The aim of this study was to analyze the body condition and diet in Rhinella arenarum, tadpoles, an anuran species widely distributed in South America and that inhabit agroecosystems. Three sites were sampled, two agroecosystems with different alteration degrees (C1 and C2) and an uncultured (SM) third place. The captured tadpoles were anesthetized, fixed and preserved in formaldehyde (10%). Subsequently, body measurements were made and the complete intestine was removed and analyzed for food items under a binocular microscope. The diet in R. arenarum tadpoles has a dominance of algae Bacillariophyceae, followed by Cyanophyceae. In particular, the class Bacillariophyceae, due to the presence of the genus Navicula, Nitzschia, Gomphonema and Hantzschia, was important in the diet of the anurans in those agroecosystems. Class Cyanophyceae, mainly represented by genus Oscillatoria and Euglenophyceae represented by Euglena and Strombomonas, were predominant in the diet of the anurans in SM. Some differences in the total items consumed by tadpole were observed between the studied sites. Tadpoles that inhabit the modified sites (C1 and C2) recorded a significantly smaller amount of food. Moreover, the tadpoles that inhabit these sites showed a lower body condition. The presence of certain algae associated with eutrophic environments, could indicate some pollution in agroecosystems (C1 and C2). Food resources would be lesser in places with strong agricultural activity, possibly with a greater degree of eutrophication. A smaller food amount could have consequences at population level for the short and long time terms, because of its impact on individual growth. Larval diet is suggested as a potential bioindicator of environmental health for these areas.

Sublethal and lethal effects on Rhinella arenarum (Anura, Bufonidae) tadpoles exerted by the pirimicarb-containing technical formulation insecticide Aficida.

Acute toxicity, genotoxicity, and cytotoxicity of the pirimicarb-containing commercial-formulation carbamate insecticide Aficida(R) (50% pirimicarb) were evaluated on Rhinella arenarum (Anura, Bufonidae) tadpoles exposed under laboratory conditions. Lethal and sublethal effects were employed as bioassays for acute toxicity, whereas micronuclei (MNi) induction and alterations in the ratio erythrocytes:erythroblasts were employed as end-points for genotoxicity and cytotoxicity, respectively. Cr(VI) (23 mg L(-1)) and cyclophosphamide (40 mg L(-1)) were employed as positive controls for toxicity and geno-cytotoxicity assays, respectively. In Gosner stage 25 (STD25), the results revealed mean values of 402.0 and 223.6 mg Aficida L(-1) for LC-50(24)(h) and LC-50(96)(h), respectively. When STD37-39 tadpoles were exposed, the LC-50(24)(h) and LC-50(96)(h) reached values of 239.4 and 181.7 mg Aficida L(-1), respectively. Sublethal effects revealed a mean EC-50(96)(h) of 133.85 and 104.2mg Aficida in those STD25 and STD37-39 treated tadpoles, respectively. The results demonstrated that in 48-h-exposed tadpoles, a MNi increase was found only in those 80.0 mg L(-1) Aficida-treated individuals. When tadpoles were exposed to Aficida for 96h, only the 160 mg L(-1)-treated individuals showed a significant increase in MNi frequency. Concentrations ranging from 80.0 to 250.0mg Aficida L(-1) resulted in cellular cytotoxicity, revealed by a decreased proportion of circulating erythrocytes and an enhancement of erythroblasts. Accordingly, this species could provide a suitable and useful experimental model for biomonitoring aquatic ecosystems.

Vitellogenesis in Bufo arenarum: identification, characterization and immunolocalization of high molecular mass lipovitellin during oogenesis.

Vitellogenin (Vtg), a large lipoglycophosphoprotein, is the most important precursor of the yolk proteins, and the major source of nutrients for the developing embryo in oviparous species. After its uptake by the oocytes, Vtg is converted into lipovitellins (high and light) and phosvitin, which are deposited into crystalline yolk platelets. We describe here the presence of two high molecular mass lipovitellin isoforms in Bufo arenarum mature oocytes with masses of 113 and 100 kDa, respectively. The amino acid sequence analysis of p113 and p100 peptides showed a high sequence homology between both polypeptides and the complete reported sequences of Xenopus laevis vitellogenin. Using specific antibodies, we determined that the Vtg uptake begins early during oogenesis, at the previtellogenic stage, and continues until oocytes have reached their mature status. In addition, we found that large endocytic vesicles mediate Vtg uptake in stage I oocytes, and that the size of the endocytic vesicles declines with oogenesis progression. In terms of the Vtg protein trafficking, we detected the Vtg precursor (190 kDa) in the liver of estradiol-injected females. Finally, we propose a subclassification of B. arenarum stage II oocytes into three physiologically and morphologically distinct periods (early, mid and late).

Changes in the antioxidant metabolism in the embryonic development of the common South American toad Bufo arenarum: differential responses to pesticide in early embryos and autonomous-feeding larvae.

Amphibians may be critically challenged by aquatic contaminants during their embryonic development. Many classes of compounds, including organophosphorus pesticides, are able to cause oxidative stress that affects the delicate cellular redox balance regulating tissue modeling. We determined the progression of antioxidant defenses during the embryonic development of the South American common toad, Bufo arenarum. Superoxide dismutase (SOD) and catalase (CAT) activities were high in the unfertilized eggs, and remained constant during the first stages of development. SOD showed a significant increase when the gills were completely active and opercular folds began to form. Reductase (GR) activity was low in the oocytes and increased significantly when gills and mouth were entirely developed and the embryos presented a higher exposure to pro-oxidant conditions suggesting an environmental control. Reduced glutathione (GSH) content was also initially low, and rose continuously pointing out an increasing participation of GSH-related enzymes in the control of oxidative stress. GSH peroxidases and GSH-S-transferases showed relatively high and constant activities, probably related to lipid peroxide control. B. arenarum embryos have plenty of yolk platelets containing lipids, which provide the energy and are actively transferred to the newly synthesized membranes during the early embryonic development. Exposure to the pro-oxidant pesticide malathion during 48 h did not significantly affect the activity of antioxidant enzymes in early embryos, but decreased the activities of CAT, GR, and the pool of GSH in larvae. Previous work indicated that lipid peroxide levels were kept low in malathion-exposed larvae, thus we conclude that oxidative stress is overcome by the antioxidant defenses. The increase in the antioxidant metabolism observed in the posthatching phase of development of B. arenarum embryo, thus constitutes a defense against natural and human-generated pro-oxidants present in the aquatic environment.