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.

Metals, pesticides, and emerging contaminants on water bodies from agricultural areas and the effects on a native amphibian.

In the Paraná River lower basin, an important agro-productive area of Argentina, crop fields and cattle breeding activities are common and may affect water quality. So, the aim of this study was to analyze the impacts of cattle breeding and agricultural activities on a stream from Buenos Aires, through physicochemical parameters (metals, pesticides, and emerging contaminants) and ecotoxicological parameters with Rhinella arenarum larvae, a native amphibian species. Three sites were selected on an ordinary plain stream that goes through agricultural fields and a cattle breeding establishment (upstream -S1-, near -S2- and downstream -S3- the establishment). Physicochemical parameters were measured in situ (in water) and in laboratory (in water and sediment samples: metals, pesticides, ivermectin and oxytetracycline). A semi-static chronic toxicity bioassay (504 h) was performed with water samples, and neurotoxicity, oxidative stress and genotoxicity biomarkers were measured after acute exposure (96 h). According to the index, a degradation in the water quality was observed in all sites. Ivermectin (8.03 mg/kg) and oxytetracycline (1.9 mg/kg) were detected in sediment samples from S2. Pesticides were detected in all sites, mainly in water samples: S1 presented the highest variability (7 residues) and in S3 AMPA, glyphosate and acetochlor concentrations were higher (10.3, 22.4 and 23.8 µg/L). Also, all sites significantly produced lethality at chronic exposure. Lethality at 504h was 40% for S1, 56.66% for S2 and 93.33% for S3. At acute exposure, the oxidative stress biomarkers were altered on R. arenarum larvae exposed to all sites and the neurotoxicity biomarkers were altered on larvae exposed to S1 and S3. Water quality was severely degraded by the surrounding agricultural and cattle breeding activities, which may represent a threat to the ecosystems.

Ecotoxicological assessment of complex environmental matrices from the lower Paraná River basin.

Sediments of aquatic ecosystems constitute the fate of most atmospheric and terrestrial pollutants. Since aquatic organisms, such as amphibians, interact with sediments, the presence of pollutants may affect their survival, growth and reproduction. So, the aim of this study was to evaluate, the sediment and water quality of five sites from the lower basin of the Paraná River (Buenos Aires, Argentina) with different anthropic impacts: Morejón stream (S1), de la Cruz stream upstream (S2) and downstream (S3), Arrecifes river (S4), tributary stream of Arrecifes river (S5). Physicochemical parameters were measured in situ (water) and in laboratory (water and sediment samples). Also, a screening of metals and pesticides was performed. Chronic (504 h) lethal toxicity bioassays were performed exposing Rhinella arenarum larvae to sediment and water samples. Oxidative stress (catalase, superoxide dismutase, glutathione S transferase, reduced glutathione and lipid peroxidation) and genotoxicity (micronuclei test) biomarkers were analyzed at acute (96 h) exposure. According to the calculated water quality index, S1 and S3 showed excellent quality, S2 good quality and, S4 and S5 poor quality. Dissolved oxygen was low in all sites (2.26-5.63 mg/L) and S5 had the highest organic matter content. Copper levels exceeded the limit for the protection of aquatic life in S2 and S4; arsenic levels exceeded its limit in S4; and selenium levels exceeded its limit in S4 and S5. Pesticides were mainly detected in water samples. Sediment from S5 showed higher sulfide and organic matter concentrations. At 504 h, no significant mortality was observed in the control group while S5 caused the greatest mortality (80%), followed by S2 (66.67%), S1 (63.33%), S3 (46.67%) and S4 (43.4%). All samples caused oxidative stress and lipid peroxidation, and samples from S4 also caused genotoxicity. The analysis of sediment and water samples was a suitable approach to assess the effects of water bodies on a native amphibian species.

Acute, chronic and neurotoxic effects of dimethoate pesticide on Rhinella arenarum throughout the development.

Among the factors implicated in amphibian global decline, agrochemicals have been gaining increasing attention. In order to evaluate the toxicity of a dimethoate-based insecticide on the early development of an autochthonous amphibian, Rhinella arenarum, continuous and 24 h pulse exposure bioassays were carried out. Lethal and sublethal effects, neurotoxicity and the ecological risk were assessed. Results demonstrate that larvae were more sensitive than embryos with 504 h-LC50 of 12.82 and 16.38 mg L-1, respectively. 24 h pulse experiments showed a high toxicity increment at early embryonic stages, while the sensitivity at later stages was high and constant. Dimethoate caused teratogenesis and several sublethal effects as morphological and behavioral alterations but also disruption in the metamorphic process. About neurotoxicity, dimethoate inhibited the activity of butyrylcholinesterase at 0.5 and 1 mg L-1 exposed larvae. The results obtained in this study as the risk assessment revealed that dimethoate represents a hazard on Rhinella arenarum survival and development but also a potential risk for the continuity of the populations of this species in agroecosystems.

Synergistic effects of glyphosate- and 2,4-D-based pesticides mixtures on Rhinella arenarum larvae.

Glyphosate and 2,4-D are two herbicides commonly used together. Since there is little information about the interactions between these pesticides, the aim of this study was to evaluate the single and joint lethal toxicity of the glyphosate-based herbicide (GBH) ATANOR® (43.8% of glyphosate, isopropylamine salt) and the 2,4-D-based herbicide (2,4-DBH) Así Max 50® (602000 mg/L of 2,4-D) on Rhinella arenarum larvae. Equitoxic and non-equitoxic mixtures were prepared according to the recommendation for their combination and analyzed with a fixed ratio design at different exposure times and levels of lethality (LC10, LC50, and LC90). GBH (504h-LC50=38.67 mg ae/L) was significantly more toxic than 2,4-DBH (504h-LC50=250.31 mg ae/L) and their toxicity was time-dependent. At 48h, the equitoxic mixture toxicity was additive and from the 96h was antagonistic at LC10 and LC50 effect level. The non-equitoxic mixture toxicity was additive at LC10 effect level from the 48h to the 168h, and synergistic from the 240h. At LC50 and LC90 effect level, the mixture interaction resulted synergistic for all exposure times. This is the first study to report the synergistic interactions between GBH and 2,4-DBH on amphibians, alerting about its negative impact on aquatic ecosystems.

Comprehensive assessment of water quality through different approaches: Physicochemical and ecotoxicological parameters.

Traditionally, water quality was assessed by physicochemical parameters. However, a more comprehensive analysis is needed to study the effects of polluted water bodies on key species over time. So, the aim of this study was to monitor through physicochemical and ecotoxicological indicators the surface water quality of four study sites with different land uses from the lower Paraná river basin (Argentina) during spring and summer of two years: Morejón stream (S1), De la Cruz stream upstream (S2), downstream (S3) and Arrecifes river (S4). Physicochemical parameters were measured in situ and in laboratory, and a Water Quality Index (WQI) was calculated. Chronic toxicity bioassays were performed with surface water samples using Rhinella arenarum embryos and larvae. Also, oxidative stress (catalase, superoxide dismutase, glutathione S-transferase, reduced glutathione and lipid peroxidation), neurotoxicity (butyrylcholinesterase) and genotoxicity (micronuclei frequency) biomarkers were measured at acute exposure, and an Integrated Biomarkers Response (IBR) index was calculated. The water quality varied between excellent and bad in S1, good and bad in S2 and S3, and bad and marginal in S4. S1 presented the greatest variability of pesticides and S4 the highest number of metals exceeding the limits for the local protection of aquatic life. Mainly, S4 caused lethality in R. arenarum larvae, reaching a maximum mortality of 83.3% at 504 h of exposure. The lethal toxicity of S1 and S2 varied between periods. Water samples from all sites altered the oxidative stress, neurotoxicity and genotoxicity biomarkers, and the IBR was negatively correlated with the WQI. The IBR reflected the effects of the degraded water quality on the exposed organisms. So, the importance of evaluating both physicochemical and ecotoxicological parameters to analyze integrally the water quality of polluted areas is highlighted. A degradation of the studied water bodies and its negative impact to the native amphibian R. arenarum were observed.

Characterization of acute toxicity, genotoxicity, and oxidative stress of dimethoate in Rhinella arenarum larvae.

There is a great concern worldwide about the global decline of amphibians, particularly by agrochemical pollution. The aim of this study was to assess oxidative stress and genotoxicity of a commercial formulation of the insecticide dimethoate in Rhinella arenarum larvae, using sublethal biomarkers. The 24- and 96-h LC50 values of dimethoate to R. arenarum were 48.81 and 38.86 mg L-1, while the 96-h no observed effect concentration (NOEC) value was 20 mg L-1. For sublethal biomarker assays, R. arenarum larvae were exposed to 1.25, 2.5, and 5% of the 96-h NOEC (0.25, 0.5, and 1 mg L-1 dimethoate, respectively). After 96 h of exposure, inhibition of catalase (CAT) and glutathione S-transferase (GST) activities were registered. Also, an increased superoxide dismutase (SOD) activity was observed in larvae exposed to the highest concentration (1 mg L-1). Lipid peroxidation by increased thiobarbituric acid reactive substance levels in larvae exposed to 0.5 and 1 mg L-1 was detected. No differences in micronuclei frequency between treatments and negative control were observed. These results demonstrate the oxidative toxicity of dimethoate at sublethal concentrations in Rhinella arenarum larvae. The disruption of defense mechanisms may contribute to a deleterious impact on amphibian populations from habitats exposed to this organophosphorus insecticide.

In situ exposure of amphibian larvae (Rhinella fernandezae) to assess water quality by means of oxidative stress biomarkers in water bodies with different anthropic influences.

In situ bioassays provide valuable information about the environment and offer more realistic results than usual laboratory experiments. The aim of this study was to evaluate the quality of water bodies from the lower Paraná River basin, the second most important in South America, through analysis of physiochemical parameters, metals and pesticides and in situ exposure of Rhinella fernandezae larvae to assess oxidative stress biomarkers. The sites were: S1(Morejón stream, reference); S2, S3(De la Cruz stream upstream and downstream, respectively) and S4(Arrecifes River). In all sites, dissolved oxygen was low, atrazine was detected and Cu was higher than the limit for aquatic life protection. According to the water quality index, S2, S3 and S4 presented bad water quality, while S1 good water quality. Larvae were exposed in situ for 96h in order to analyze: lipid peroxidation(TBARS) as oxidative damage, antioxidant enzymatic (catalase-CAT-, superoxide dismutase-SOD- and glutathione s-transferase-GST-) and non-enzymatic defenses (reduced glutathione-GSH-). Larvae exposed in the most impacted sites (S2, S3 and S4) presented oxidative stress since the levels of TBARS were around 2 times higher than in S1. Also, the other oxidative stress biomarkers were altered in larvae exposed at S2, S3 and S4. These results highlight the importance of analyzing oxidative stress biomarkers during in situ exposures since they are useful tools for documenting the extent of exposure at sublethal levels. The complex pollution of the water bodies affected the exposed larvae, which may jeopardize the native populations.

Developmental Toxicity Assessment of a Chlorothalonil-Based Fungicide in a Native Amphibian Species.

The toxicity of a commercial formulation of the fungicide chlorothalonil in sensitive stages of the native amphibian Rhinella arenarum (Ra) was assessed by means continuous treatments from embryo and larval development and 24-h pulse exposures evaluating acute and chronic lethal and sublethal effects and stage-dependent sensitivity. A risk assessment of chlorothalonil in Ra development also was performed. The results of continuous exposure in embryos showed a significant toxicity increase with exposure time, whereas sensitivity of larvae remained relatively constant through time (24 and 504-h LC50 = 0.86 and 0.04 mg L-1, and 0.37 and 0.34 mg L-1 for embryos and larvae respectively). Embryos exhibited several sublethal effects, such as delayed development, tail/axial flexures, edemas, and behavioral alterations. The 96-h NOEC values for lethal and sublethal effects were 0.025 and 0.01 mg L-1 respectively, so the 96-h Teratogenic Index was 2.5, which indicates the severe teratogenic potential of the fungicide. For 24-h pulse exposure experiments, S.21 and S.23 were the most sensitive stages for lethality (504-h NOEC = 0.05 mg L-1), whereas earlier stages exhibited severe morphological alterations. The results obtained in this study and the ecological risk evaluation highlight the severe toxicity of chlorothalonil threatening the continuity of Ra populations.

Oxidative stress and genotoxicity in Rhinella arenarum (Anura: Bufonidae) tadpoles after acute exposure to Ni-Al nanoceramics.

The employ of nanomaterials (NMs) has exponentially grown due to the large number of technological advances in industrial, pharmaceutical and medical areas. That is the case of alumina (Al) nanoparticles which are extensively employed as support in heterogeneous catalysis processes. However, these NMs can cause great toxicity because of their ubiquitous properties, such as extremely small size and high specific surface area. So, it is required to assess the potential deleterious effects of these NMs on living organisms. In the present study, we analyze the oxidative stress and genotoxic potential of a nanoceramic catalyst Ni/-Al2O3 (NC) and the NMs involved in their synthesis, -Al2O3 support (SPC) and NiO/-Al2O3 precursor (PC) on Rhinella arenarum larvae. Biomarkers of oxidative stress and genotoxic damage were measured in tadpoles exposed to 5 and 25 mg/L of each NMs for 96 h. The results indicated an inhibition of catalase activity in tadpoles exposed to both concentrations of PC and to 25 mg/L of SPC and NC. Moreover, both exposure concentrations of PC and NC significantly inhibited superoxide dismutase activity. Exposure to the three NMs caused inhibition of glutathione S-transferase activity, but there were no significant variations in reduced glutathione levels. Oxidative stress damage (lipid peroxidation) was observed in tadpoles treated with 25 mg/L PC, while the other treatments did not produce alterations. The MNs frequency significantly increased in larvae exposed to 25 mg/L PC indicating irreversible genotoxic damage. The results show that these NMs exert genotoxic effects and antioxidant defense system disruption in R. arenarum larvae.

Integrated analysis of the quality of water bodies from the lower Paraná River basin with different productive uses by physicochemical and biological indicators.

The Paraná River basin is one of the most important in South America and is affected by human activities that take place on its margins. In particular, the De la Cruz stream flows through an industrial pole and the Arrecifes River goes mainly through agricultural fields. The aim of this study was to evaluate the water quality of the De la Cruz stream (S1) and the Arrecifes River (S2) by means of physicochemical parameters, including metals and pesticides concentrations. Since amphibians are good indicators of environmental quality, bioassays with Rhinella arenarum were carried on. For lethal and sublethal parameters, embryos and larvae were exposed to a dilution gradient of water samples and AMPHITOX Solution (AS) as negative control for 504 h. For the determination of oxidative stress biomarkers (Catalase -CAT-, Glutathione S-Transferase -GST-, Reduced Glutathione -GSH-, and lipid peroxidation -TBARS-), embryos and larvae were exposed to undiluted water samples and AS. For the determination of micronuclei, larvae at hind limb bud stage (S.28) were exposed to undiluted water samples, simultaneously with negative and positive controls (AS and cyclophosphamide 40 mg/L, respectively). Dissolved oxygen was low in both sites and the copper levels exceeded the Argentine limit for the protection of aquatic life. In embryos exposure, water sample from S1 caused lethal effects (504h-LC50 = 49 (28-71.6)%), increased TBARS levels, and GST and CAT activities. In larvae exposure, water sample from this site decreased CAT activity, while the water sample from S2 caused important lethal effects (504h-LC50 = 98.72 (60.60-302.52)%), low GSH levels and increased GST activity. Water samples from both sites induced higher micronuclei frequency than the negative control. This study alerts about the degradation of water quality of the studied sites including lethal and sublethal effects in R. arenarum that can jeopardize the native populations of this species.

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 504 h) periods. The toxicity profiles for lethal and sublethal effects were performed. At all exposure times, MCZ was more toxic to embryos, for instance, NOEC 504 h were 0.01 and 0.05 mg 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.

Assessment of environmental quality of water bodies next to agricultural areas of Buenos Aires province (Argentina) by means of ecotoxicological studies with Rhinella arenarum.

Little is known about the effects of polluted water bodies from Buenos Aires Province on the development of native fauna. Ecotoxicological quality of water bodies from agricultural sites was evaluated by means of standardized laboratory bioassays with embryos and larvae of the native amphibian Rhinella arenarum. The organisms were acutely and chronically exposed to surface water samples from streams of Arrecifes (A), Pergamino (P) and Salto (S) districts that represent the most important agricultural core from the region. Lethal, sublethal and genotoxic effects were assessed. Water sample from (A) caused chronic toxicity (LC50:45.35%) in embryos, followed by (S) and the water sample from (P) was not toxic. In larvae, an inversion of the toxicity pattern was found. Thus, the 504 h-LC50s were 28.12%, 39% and 61% for (S), (P) and (A), respectively. A stage-dependent sensitivity was registered, being larvae more affected than embryos. Significant genotoxic effects, estimated by micronucleus test were observed in the larvae exposed to water samples from all sites. The present study warns about environmental degradation of surface waters next to agricultural areas of Buenos Aires Province. This fact jeopardizes R. arenarum populations in this area.

Lethality, neurotoxicity, morphological, histological and cellular alterations of Ni-Al nanoceramics on the embryo-larval development of Rhinella arenarum.

Alumina nanoparticles (NP-Al2O3) are widely used but their environmental effects are unknown, so they can become potentially dangerous. The aim of this study was to evaluate the toxicity of a nanoceramic catalyst Ni/γ-Al2O3 (NC) and NPs involved in their synthesis, γ-Al2O3 support (SPC) and NiO/γ-Al2O3 precursor (PC) on Rhinella arenarum embryo-larval development. The NPs toxicity significantly increased over time obtaining a similar sensitivity to PC and NC (336 h-LC50 = 4.03 and 5.11 mg/L respectively) and very low sensitivity to SPC (336 h-LC50 = 90.83 mg/L). Embryos exposed to SPC and PC exhibited general underdevelopment, axial flexures and behavioral alterations. Pharyngeal and intestinal epithelia alterations at the level of cell surface as dissociation, apoptosis and numerous lysosomes were observed at light and transmission electronic microscopy. Images of scanning electron microscope with backscattered electron detector revealed the presence of nickel in the intestinal epithelium. The increased toxicity of PC could be due to the presence of Ni as oxide which could interfere with vital functions such as breathing and feeding. Taking into account the exponential production and use of these NPs it is expected that their pollution levels will considerably increase and amphibians will be more exposed and at higher risk.

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.

Monitoring the ecotoxicity of γ-Al2O3 and Ni/γ-Al2O3 nanomaterials by means of a battery of bioassays.

The increasing application of nanoparticles (NPs) to a variety of new technologies has become a matter of concern due to the potential toxicity of these materials. Many questions about the fate of NPs in the environment and the subsequent impact on ecosystems need to be answered. The aim of this work was to evaluate the ecotoxicity of two alumina-based nanoceramics, γ-Al2O3 (NC) and Ni/ γ-Al2O3 (NiNC) by means of three different standardized tests: Biochemical Oxygen Demand (BOD5), bioassay with luminescent bacteria (Vibrio fischeri; Microtox), and bioassay on amphibian larvae (Rhinella arenarum) (AMPHITOX). BOD5 values of a very biodegradable mixture (glucose/glutamic acid) decreased with the addition of NiNC(43.8%) and NC (31.6%) with respect to control samples (52.9%). Microtox test results indicated that NiNC presents higher toxicity than NC, with EC50s values of 16.1% and 29.9% respectively; a reduced toxicity was observed, however, in presence of organic matter, thus obtaining EC50s of 37.8% and 19.4%. The results of AMPHITOX test showed a significant increase in the toxicity of both substances over time, the NiNC toxicity being greater than that of NC. The values of 96h-LC50 and 504h-LC50 determined for NiNC were 1.58 and 0.83mg/L, respectively, and 14.5 and 10.5mg/L for NC samples. Amphibian larvae exhibited collapsed cavities, edema, axial flexures, and behavioral alterations as hyperkinesia and reduced movements. These results evidence the vulnerability of wildlife to xenobiotics and the need to develop specific standardized ecotoxicity tests in order to help environmental sustainability and natural species conservation.

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.

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.