South American Toad Rhinella Arenarum Research Articles

Ecotoxicological Characterization of Lithium as a "Timebomb" in Aquatic Systems: Tadpoles of the South American Toad Rhinella arenarum (Hensel, 1867) as Model Organisms.

The aim of this study was to evaluate the acute lethality and chronic sublethal effects of lithium (Li) on Rhinella arenarum tadpoles as model organisms. First a 96 h toxicity assay was performed by exposing tadpoles to Li concentrations from 44.08 to 412.5 mg L-1 to estimate the mortality, and lethal and sublethal effects. Another bioassay was carried out by exposing tadpoles to two environmentally relevant Li concentrations (2.5 and 20 mg L-1) for one and two weeks. The sublethal effects of Li on tadpoles were evaluated by analyzing biochemical, genotoxic, and physiological biomarkers. The mortality in Li-exposed tadpoles increased over time. The median lethal concentration (LC50) ranged from 319.52 (281.21-363.05) mg L-1 at 48 h to 66.92 (52.76-84.89) mg L-1 at 96 h. Exposure to Li at 2.5 and 20 mg L-1 induced alterations in enzymes related to detoxification, antioxidant, and hepatic mechanisms, endocrine disruption of thyroid hormones, genotoxicity, and effects on the physiology of the heart and gastrointestinal systems. Tadpoles exposed to the highest concentration in the chronic bioassay (20 mg L-1 Li), which is the concentration commonly recorded in Li mining sites, showed significant mortality after one week of exposure. These results warn about the high ecotoxicological risk of Li as a contaminant of emerging concern for amphibians.

Toxicity assessment of a tebuconazole-based fungicide on the embryo-larval development of the common south American toad Rhinella arenarum.

Agrochemicals cause diverse effects on aquatic communities, and amphibian species are particularly threatened due the high susceptibility to contamination. Present study evaluates the toxicity of a widely used fungicide tebuconazole (Trigal®) by the assessment of mortality and developmental alterations at acute, subchronic, and chronic exposure during the embryo-larval development of the South American toad Rhinella arenarum. Also, the sensitivity of the different embryonic stages was evaluated with 24-h pulse exposure treatments. The results demonstrated that larvae were more sensitive than embryos at acute exposure (LC50-24 and 96 h = 74.62, 31.92 mg/L and 24.27, 16.81 mg/L for embryos and larvae, respectively). Nevertheless, embryos toxicity increased significantly achieving a sensitivity very similar to larvae at chronic exposure (LC50-168 and 504 h = 13.31, 4.35 mg/L and 14.47, 6.83 mg/L for embryos and larvae, respectively). Embryos exhibited several sublethal effects from 5 mg/L at 96 h onwards, such as delayed development, reduce body size, edemas, tail/axial flexures, weakness, and absence of movements. The teratogenic index at 96 h was 10.13, indicating the severe teratogenic potential of the fungicide. 24-h pulse exposure treatments showed an increased sensitivity in intermediate stages as S.11, S.18, S20, and S.23 (NOEC-96 h = 100, 200, 75, and 20 mg/L, respectively), while stage S.25 was the most sensitive to the fungicide (NOEC-96 h = 5 mg/L). About metamorphic process, tebuconazole caused an acceleration of metamorphosis at the lowest concentration (0.001 mg/L), but also an increase in mortality and in addition, significant differences in the weight in all treatments. The results obtained throughout this work indicate that tebuconazole cause several adverse effects in Rhinella arenarum embryo-larval development.

Gonadal morphogenesis in the South American toad Rhinella Arenarum (Anura, Bufonidae) unveils an extremely delayed rate of sex differentiation.

Among anurans, Bufonids are recognized for their retarded sex differentiation. However, few studies have addressed gonadal morphogenesis in this family. Here, we analyzed the early gonadogenesis in laboratory-reared Rhinella arenarum. Few germ cells were identified in the genital ridge at Gosner stage 26. At metamorphosis, somatic cells and germ cells were observed in the outer region of the undifferentiated gonad, whereas the central region was occupied by stromal tissue. A cortico-medullary organization was first recognized on Day 7 postmetamorphosis. The cortex was composed of germ cells and encompassing epithelial cells, whereas the medulla contained cells presumptively derived from the coelomic epithelium. Medullary somatic cells formed metameric knots along the length of the undifferentiated gonad. Consequently, a series of 12-14 gonomeres became recognizable externally. The first sign of ovarian differentiation was observed on Day 15 postmetamorphosis, when a cavity was formed within each gonomere. In contrast, testes were recognized by a uniform distribution of germ cells and intermingled somatic cells, as the division into cortex and medulla was lost. By Day 50 postmetamorphosis, the gonadal metameric organization was still apparent both in the ovaries and testes. Follicles containing diplotene oocytes were observed within the ovary. In the testis, an incipient lobular architecture was recognized without initiation of meiosis within the seminiferous cords. These observations reveal an extremely delayed gonadal development in R. arenarum, not reported previously for other anuran species. In addition, the late differentiation of the gonads contrasted with the early appearance of follicles in the Bidder's organ. Lastly, we observed that delayed metamorphs exhibited an undifferentiated gonad, demonstrating that gonadogenesis in this species is more dependent on somatic development than on age.

Toxicity characterization and environmental risk assessment of Mancozeb on the South American common toad Rhinella arenarum.

Agricultural activity, especially the increasing use of pesticides, is considered one of the main reasons for the decline of amphibian populations. Mancozeb (MCZ) is one of the most used fungicides worldwide, despite its ancient use and toxicity demonstrated in different taxa. However, there is limited information about the effects of MCZ in amphibians, which are keystones of riparian ecosystems. For species conservation purposes, it is essential to identify the most sensitive developmental period(s) of a given species to a xenobiotic. We evaluated the toxicity of a commercial fungicide of mancozeb, (80% active ingredient) on the early development of the common South American toad Rhinella arenarum (Anura, Bufonidae). Embryos from early blastula (S.4) and larvae from complete operculum (S.25) stages were exposed to a wide range of MCZ concentrations during acute, subchronic and chronic exposure (up to 504h) periods. The toxicity profiles for lethal and sublethal effects were performed. At all exposure times, MCZ was more toxic to embryos, for instance, NOEC 504h were 0.01 and 0.05mg MCZ/L for embryos and larvae, respectively. Thus, embryo sensitivity was 5-fold higher than larvae. A Teratogenic Index of 14 indicated the significant teratogenic potential of this fungicide. Among sublethal effects, embryos exhibited a wide range of abnormalities with high incidence. The ecological risk assessment demonstrated that the estimated Risk Quotient value for Rhinella arenarum embryos at chronic exposure was higher than the Level of Concern value, which warns about the potential risk of MCZ for this native species.

Morphological abnormalities in natural populations of the common South American toad Rhinella arenarum inhabiting fluoride-rich environments

Morphological abnormalities in amphibians may be attributed to contaminants, ultraviolet radiation and trematode parasites, or a synergistic effect between them. In the present study, morphological abnormalities in Rhinella arenarum adults from natural and artificial fluoride-rich environments were identified and evaluated. Three sites were sampled in central Argentina: Los Vallecitos stream (LF-LV), Los Cerros Negros stream (MF-CN), and Decantation ponds (HF-DP), with low (0.33 mg/L), middle (2.03 mg/L) and high (14.0 mg/L) fluoride levels respectively; the latter site is associated with a fluoride mine. Abnormal individuals were photographed and then standard radiographs were taken. Abnormality frequencies and relative percentage of abnormal individuals were calculated for each site. In addition, skeletochronology was used to estimate toad's age. Five abnormality types were identified: syndactyly, ectrodactyly, polydactyly, microphthalmia and ectromelia. Percentages of abnormal individuals per site were: LF-LV = 4%, MF-CN = 21.2% and HF-DP = 6.4%. The MF-CN and HF-DP populations had morphological abnormality frequencies that exceeded the reference value (5%) reported in the literature. The average age did not differ between sites. The results of this study indicate that there is an association between frequency of morphological abnormalities and high fluoride levels.

Evaluation in situ of genotoxicity and stress in South American common toad Rhinella arenarum in environments related to fluorite mine

Little attention has been paid to the impact of wastewater generated by mining activities on fluoride. In this study, we evaluated the hematology responses of common South American toad Rhinella arenarum inhabiting natural and artificial environments associated with a fluorite mine from central Argentina. We analyzed three sampling stations associated with the fluorite mine: (I) Los Cerros Negros stream (CN), which runs on granitic rock with a high fluorite content; (II) Los Vallecitos stream (LV), which runs on metamorphic rock with low fluorite content; and (III) artificial decantation ponds (DP) containing sediments produced by fluorite flotation process. We calculated frequencies of micronuclei, erythrocyte nuclear abnormalities, mitosis, and immature erythrocytes. In addition, we performed a differential leukocyte count and determined neutrophils/lymphocyte ratio as a stress response estimator. We found high micronucleus (MN) and erythrocyte nuclear abnormality (ENA) frequencies in DP and CN but low frequencies in LV. The neutrophil/lymphocyte ratio was different among sites, with a significant increase in individuals from DP. Values registered in DP could be caused by exposure to mixture of compounds registered in dams that hold wastewater, while high values registered in CN stream might be due to natural concentrations of fluoride. Our results suggest that blood is an effective and non-destructive sensitive indicator for monitoring genotoxic agents in freshwater ecosystems.

Toxicogenomics: new strategies for ecotoxicology studies in autochthonous species II. The 'omic' era in non-model species. Transcriptome analysis for biomarker screening

The emerging field of ecotoxicogenomics aims to combine large-scale approaches to study the responses of organisms to a toxicant. A holistic vision of gene and protein expression in response to toxic exposure contributes to the identification of cellular components, signalling pathways and novel mechanisms of action/response. Native species are preferential for evaluating the impact of contaminants generated by anthropic action. However, biomonitoring using autochthonous species (non-model organisms) is difficult owing to a deficiency of molecular biology data and analytical tools. Our experience in the study of biomarkers in the South American toad Rhinella arenarum revealed many difficulties of finding antibodies or designing probes for it. We performed a transcriptomic study in R. arenarum exposed to two organophosphorus pesticides. We determined that there are specific patterns of gene expression for each organophosphate tested. Thus, a transcriptome approach for biomarker screening seems to be helpful in defining specific gene expression behaviour for a given toxicant.

Toxicogenomics: new strategies for ecotoxicology studies in autochthonous species II. The 'omic' era in non-model species. Transcriptome analysis for biomarker screening

The emerging field of ecotoxicogenomics aims to combine large-scale approaches to study the responses of organisms to a toxicant. A holistic vision of gene and protein expression in response to toxic exposure contributes to the identification of cellular components, signalling pathways and novel mechanisms of action/response. Native species are preferential for evaluating the impact of contaminants generated by anthropic action. However, biomonitoring using autochthonous species (non-model organisms) is difficult owing to a deficiency of molecular biology data and analytical tools. Our experience in the study of biomarkers in the South American toad Rhinella arenarum revealed many difficulties of finding antibodies or designing probes for it. We performed a transcriptomic study in R. arenarum exposed to two organophosphorus pesticides. We determined that there are specific patterns of gene expression for each organophosphate tested. Thus, a transcriptome approach for biomarker screening seems to be helpful in defining specific gene expression behaviour for a given toxicant.

Effects of a fungicide formulation on embryo-larval development, metamorphosis, and gonadogenesis of the South American toad Rhinella arenarum.

Sublethal toxicity of the formulated fungicide Maxim(®) XL on embryonic, larval and juvenile development of Rhinella arenarum was evaluated by means of standardized bioassays. Maxim(®) XL, one of the most used fungicides in Argentina, is based on a mixture of two active ingredients: Fludioxonil and Metalaxyl-M. Maxim(®) XL exposure induced severe sublethal effects on the embryos, expressed as general underdevelopment, axial flexures, microcephaly, cellular dissociation, abnormal pigmentation, underdeveloped gills, marked edema and wavy tail. As the embryo development advanced, alterations in behavior as spasmodic contractions, general weakness and inanition were observed. Maxim(®) XL did not affect neither the time required to complete metamorphosis nor sex proportions, but gonadal development and differentiation were impaired. Gross gonadal analysis revealed a significant proportion of exposed individuals with underdevelopment of one or both gonads. Histological analysis confirmed that 18% and 10% of the individuals exposed to 0.25 and 2mg/L Maxim(®) XL, respectively, exhibited undifferentiated gonads characterized by a reduced number (or absence) of germ cells. Taking into account the risk evaluation performed by means of Hazard Quotients, this fungicide could be a threat to R. arenarum populations under chronic exposure. This study represents the first evidence of toxic effects exerted by Maxim(®) XL on amphibians. Finally, our findings highlight the properties of this fungicide that might jeopardize non-target living species exposed to it in agricultural environments.

Acute toxicity of arsenic and oxidative stress responses in the embryonic development of the common South American toad Rhinella arenarum.

Arsenic (As), a natural element of ecological relevance, is found in natural water sources throughout Argentina in concentrations between 0.01 mg/L and 15 mg/L. The autochthonous toad Rhinella arenarum was selected to study the acute toxicity of As and the biochemical responses elicited by the exposure to As in water during its embryonic development. The median lethal concentration (LC50) value averaged 24.3 mg/L As and remained constant along the embryonic development. However, As toxicity drastically decreased when embryos were exposed from heartbeat-stage on day 4 of development, suggesting the onset of detoxification mechanisms. Given the environmental concentrations of As in Argentina, there is a probability of exceeding lethal levels at 1% of sites. Arsenic at sublethal concentrations caused a significant decrease in the total antioxidant potential but generated an increase in endogenous glutathione (GSH) content and glutathione S-transferase (GST) activity. 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 GSH for its excretion. The authors conclude that toad embryos are more sensitive to As during early developmental stages and that relatively high concentrations of this toxic element are required to elicit mortality, but oxidative stress may be an adverse effect at sublethal concentrations.

Biochemical biomarkers of sublethal effects in Rhinella arenarum late gastrula exposed to the organophosphate chlorpyrifos

We determined the biochemical and molecular effects of the organophosphate insecticide chlorpyrifos (CPF) in the late gastrula embryonic stage of the South American toad Rhinella arenarum continuously exposed from fertilization (24 h). Our objective was to evaluate these responses as potential biomarkers at low, sublethal levels of the toxicant. We first established the EC50 for embryo arrest in 21.3 mg/L, with a LOEC of 16 mg/L. At 4 mg/L CPF, some embryos were unable to complete the dorsal lip of the blastopore and the yolk plug became blur, probably because of abnormal cell migration. Acetylcholinesterase activity, the specific biomarker for organophosphates, was unaffected by any of the tested concentrations of CPF (2–14 mg/L). In turn, 2 mg/L CPF increased the reduced glutathione levels and inhibited glutathione-S-transferase activity, suggesting an oxidative stress and antioxidant response. Catalase was induced by CPF exposure at higher concentrations (8 and 14 mg/L). We also studied transcription factor c-Fos as a signaling event related to development in early embryogenesis. Analysis of nuclear c-Fos protein showed two bands, both enhanced in embryos exposed to 2 and 8 mg/L CPF. While nuclear Erk protein was practically unaffected, Mek protein levels were induced by the OP. Transcription factor c-Fos may be then linking oxidative stress with developmental alterations observed due to CPF exposure. These molecular and biochemical responses observed in R. arenarum gastrula at sublethal CPF exposures may replace non-responsive AChE as very early biomarkers in toad gastrula.

Toxicity of the insecticide chlorpyrifos to the South American toad Rhinella arenarum at larval developmental stage

Chlorpyrifos (CPF) is an insecticide widely used for pest control in the fruit-productive region of North Patagonia, Argentina, where it is found in superficial waters. The aim of this study was to establish the toxic effects of CPF in Rhinella arenarum toad larvae as a potentially exposed species. We determined the 96h-LC50 (1.46±0.27mg/L), the LOEC (0.81mg/L, LC10) and NOEC (0.43mg/L, LC1) for CPF lethality as endpoint. We also analyzed biochemical biomarkers in larvae exposed to sublethal CPF concentrations. The IC50 for cholinesterase was 0.113±0.026mg/L, one order of magnitude lower than the LC50. Carboxylesterase activity was inhibited, buffering OP toxicity on cholinesterase. Reduced glutathione increased after 24h as an antioxidant response, and decreased at 96h together with catalase activity, due to oxidative stress. These biochemical effects suggest that environmentally relevant CPF concentrations pose a threat to R. arenarum larvae progression.

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.

Synergy between glyphosate- and cypermethrin-based pesticides during acute exposures in tadpoles of the common South American Toad Rhinella arenarum

The herbicide glyphosate and the insecticide cypermethrin are key pesticides of modern management in soy and corn cultures. Although these pesticides are likely to co-occur in ephemeral ponds or aquatic systems supporting amphibian wildlife, the toxicological interactions prevailing in mixtures of these two pesticides have been little studied. The current study evaluated the toxicity of equitoxic and non-equitoxic binary mixtures of glyphosate- and cypermethrin-based pesticides to tadpoles of the common South American toad, Rhinella arenarum. Two different combinations of commercial products were tested: glyphosate Glifosato Atanor®+cypermethrin Xiper® and glyphosate Glifoglex®+cypermethrin Glextrin®. When tested individually, the formulations presented the following 96h-LC50s: Glifosato Atanor® 19.4mg aeL−1 and Glifoglex 72.8mg aeL−1, Xiper® 6.8mgL−1 and Glextrin® 30.2mgL−1. Equitoxic and non-equitoxic mixtures were significantly synergic in both combinations of commercial products tested. The magnitude of the synergy (factor by which toxicity differed from concentration addition) was constant at around twofold for all tested proportions of the glyphosate Glifoglex®+cypermethrin Glextrin® mixture; whereas the magnitude of the synergy varied between 4 and 9 times in the glyphosate Glifosato Atanor®+cypermethrin Xiper® mixture. These results call for more research to be promptly undertaken in order to understand the mechanisms behind the synergy observed and to identify and quantify the extent of its environmental impacts.

Toxicity of endosulfan on embryo‐larval development of the South American toad Rhinella arenarum

Endosulfan is a widely used pesticide despite its extreme toxicity to a variety of taxa and its worldwide ban. The aim of the present study was to evaluate the acute and chronic toxicity of endosulfan on the embryonic-larval development of the common South American toad Rhinella arenarum. The results showed that lethal and sublethal effects increased with concentration and exposure time. The sensitivity to endosulfan increased during the larval period, the complete operculum stage (S.25) being the most sensitive (504-h median lethal concentration [LC50] = 0.01 mg endosulfan/L; 10% lethal concentration [LC10] = 0.004 mg endosulfan/L). Endosulfan exposure caused morphological abnormalities such as general underdevelopment, edema, gill malformations, and cellular dissociation as well as neurotoxicity. Our results also showed that larvae exposed to concentrations of 0.005 mg endosulfan/L and 0.01 mg endosulfan/L completed metamorphosis earlier than controls, but with underdevelopment. The 240-h teratogenic index was 6.13, implying a high risk for embryos to be malformed in the absence of significant embryonic lethality. Because the hazard quotients for chronic exposure were over 1, the level of concern value and toxicity endpoints obtained in the present study for R. arenarum occurred at concentrations lower than the levels of endosulfan reported in the environment, this pesticide should be considered a potential risk for this species.

Differential toxicity and uptake of Diazinon on embryo-larval development of Rhinella arenarum

Diazinon, an anti-cholinesterase organophosphate, is an extensively used pesticide. The main objective of this work was to assess the lethal and sublethal effects of Diazinon and its comparison with the uptake by embryos and larvae of the common South American toad Rhinella arenarum by means of standardized bioassays during acute (96h), short-term chronic (168h) and chronic (504h) exposures. Toxicity resulted time- and stage-dependent, thus the lethal concentration 50 for 96h, 168h and 504h were 27.2; 20.1 and 6.8mgDiazinonL−1 for embryos and 8, 6.7 and 1.9mgDiazinonL−1 for larvae. It is noteworthy the remarkable differences found in the concentration which caused lethality with those causing adverse effects on development such as malformations (teratogenic effects). Therefore, the teratogenic index from 144h was greater than two; the main adverse effects were axial flexures, irregular borders, wavy tail, microcephaly, malformed mouth and adhesive structures, gut miscoiling, underdeveloped gills, cloacal edema, desquamation and severe hydropsy. Moreover, the characteristic sublethal effect of Diazinon on larvae was abnormal behavior related to neurotoxicity with a NOEC-168h of 4.5mgDiazinonL−1. Diazinon contents in R. arenarum were time-dependent and significantly related to exposure concentration for both embryos and larvae. Diazinon contents were also stage-dependent, as it was up to 27times higher for organisms exposed from blastula stage onwards than early larvae. These facts and the Hazard Quotients, a numerical expression of ecological risk, of 2.73, which is above USEPA’s Level of Concern, showed the threat that Diazinon represents for R. arenarum populations.

Stage-dependent toxicity of bisphenol a onRhinella arenarum(anura, bufonidae) embryos and larvae

The acute and chronic toxicity of bisphenol A (BPA) was evaluated on the common South American toad Rhinella arenarum embryos and larvae by means of continuous and pulse exposure treatments. Embryos were treated continuously from early blastula (S.4) up to complete operculum (S.25), during early larval stages and by means of 24 h pulse exposures of BPA in concentrations ranging between 1.25 and 40 mg L(-1) , in order to evaluate the susceptibility to this compound in different developmental stages. For lethal effects, S.25 was the most sensitive and gastrula was the most resistant to BPA. The Teratogenic Index for neurula, the most sensitive embryonic stage for sublethal effects was 4.7. The main morphological alterations during early stages were: delayed or arrested development, reduced body size, persistent yolk plug, microcephaly, axial/tail flexures, edemas, blisters, waving fin, underdeveloped gills, mouth malformations, and cellular dissociation. BPA caused a remarkable narcotic effect from gill circulation stage (S.20) onwards in all the organisms exposed after 3 h of treatment with 10 mg L(-1) BPA. After recovering, the embryos exhibited scarce response to stimuli, erratic or circular swimming, and spasmodic contractions from 5 mg L(-1) onwards. Our results highlight the lethal and sublethal effectsof BPA on R. arenarum embryos and larvae, in the last case both at structural and functional levels.

Stage‐dependent susceptibility to copper in Rhinella arenarum embryos and larvae

Copper toxicity in different embryonic and larval stages of the common South American toad Rhinella arenarum was evaluated by means of continuous and 24-h pulse treatments in 12 different developmental stages. Lethal concentrations (LC) of 10, 50, and 90% of continuous treatment with Cu from early blastula (S.4), complete operculum (S.25), and hind limb bud (S.28) stages were plotted from 24 to 168 h, resulting from S.4 in a 24-h LC50 of 137 µg Cu(2+) /L and a 168-h LC50 of 19.5 µg Cu(2+) /L. This result was in agreement with pulse treatments that showed a high resistance to Cu at blastula and gastrula stages, whereas the organogenic period, between muscular response (S.18) and open mouth (S.21), was very susceptible to this metal. Continuous treatments from S.25 showed no significant differences along exposure time (168-h LC50 = 51 µg Cu(2+) /L), but in the case of S.28 toxicity increased slightly from a 24-h LC50 of 138.6 µg Cu(2+) /L to a 168-h LC50 of 104 µg Cu(2+) /L, pointing out that, although the larval period was significantly more resistant to Cu, there was also a remarkable stage-dependent susceptibility to this metal. Copper teratogenic potential was approximately two, and main adverse effects were reduced body size, axial flexure, microcephaly, acephaly, mouth malformations, agenesis of or underdeveloped gills, agenesis of or underdeveloped tail, and hydropsy. The results are discussed considering Cu toxicity mechanisms, an evolutionary perspective, and environmental protection.

Reproductive Ecology of the Common South American Toad Rhinella arenarum (Anura: Bufonidae): Reproductive Effort, Clutch Size, Fecundity, and Mate Selection

Understanding the causes of population declines is difficult when the ecology of the organisms themselves is insufficiently known. The common South American toad, Rhinella arenarum, inhabits a wide variety of habitats. Their populations are abundant; however, life-history characteristics of the adult, including reproductive traits, are poorly known. We studied the reproductive ecology of R. arenarum for several populations in central Argentina during two breeding seasons (2007–08). Breeding activity was compared for the two seasons with respect to variation in habitat variables. Females deposited a mean of 23,226.67 eggs per clutch (N = 10), and snout–vent length did not show any significant effect on the number of eggs. Spawning females lost 27% of their mass when spawning occurred, and the heavier females had greater reproductive output. We found a significantly positive relationship between amplexed male and female snout–vent lengths, indicating size-assortative mating. Females in good body and somatic condition produced larger clutches independent of snout–vent length. Because clutch hydration in ponds occurs, reproductive effort as a function of body loss mass of spawning females is a more appropriate estimate of reproductive output for R. arenarum. Trends in the number of egg strings over several breeding periods can provide information about changes in population size. Long-term studies designed to monitor populations are needed to determine the impacts and consequences of environmental changes.

Comparative susceptibility to atrazine of three developmental stages of Rhinella arenarum and influence on metamorphosis: Non-monotonous acceleration of the time to climax and delayed tail resorption

Acute and subchronic toxicity of atrazine was evaluated in embryos (stage 4) and in premetamorphosis (stage 25) and prometamorphosis (stage 38–39) larvae of the common South American toad Rhinella arenarum (Anura: bufonidae). The influence of atrazine on the last stages of metamorphosis was also examined by exposing prometamorphosis larvae until completion of metamorphosis. Results obtained revealed that larvae in premetamorphosis are more sensitive than larvae in prometamorphosis and that these are, in turn, more sensitive than embryonic stages. Indeed, concentrations of atrazine as high as 30 mg/L had little effects on embryonic stages, the embryos surviving and developing in a similar manner as controls. LC50s of premetamorphosis larvae equaled 27.16, 7.03 and 2.32 mg/L of atrazine after 4, 14 and 21 days of exposure, respectively, compared to LC50s values of 18.27 and 14.43 mg/L after 14 and 21 days of exposure for larvae in prometamorphosis. In experiments with premetamorphosis larvae, the range of tested concentrations was extended to very low concentrations (down to 0.0001 mg/L) to examine whether recent findings of greater mortality at lower doses than at higher doses were also observed in R. arenarum but no such pattern was found. Exposure of prometamorphosis larvae to concentrations of atrazine of 10 mg/L and above widely prevented completion of metamorphosis and caused important mortality. Alternatively, whereas all animals eventually completed metamorphosis when exposed to concentrations of atrazine between 0.1 and 5 mg/L, the timings of metamorphosis were altered starting from 0.1 mg/L, the lowest concentration tested. Indeed, a significant decrease in the time needed for 50% of the larvae to reach the metamorphic climax (stage 42) was observed within this range of atrazine concentrations, the response presenting a U-shaped non-monotonic dose–response curve. Larvae exposed to these concentrations of atrazine also needed significantly more time for completing tail resorption, this effect being equivalent at all concentrations. Overall, the combination of these two different facets of atrazine influence on metamorphosis resulted in a significant acceleration of metamorphosis at 1 mg/L and a significant increase in the duration of metamorphosis at 5 mg/L, whereas no significant difference was observed with 0.1 mg/L.