Carolina M Aronzon | Universidad Nacional de San Martin (original) (raw)

Papers by Carolina M Aronzon

Environmental Toxicology and Pharmacology, 2022

The antibiotic oxytetracycline (OTC) is commonly used in animal production and can enter aquatic ... more 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 (504h-LC50= 64.04mg/L) more sensitive than the larvae (504h-LC50= 97.74mg/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.

Environmental Toxicology, Jan 28, 2010

The acute and short term chronic toxicity of both the herbicide butyl ester of 2,4-Dichlorophenox... more The acute and short term chronic toxicity of both the herbicide butyl ester of 2,4-Dichlorophenoxyacetic acid (2,4-D) and a commercial formulation (CF) were evaluated on Rhinella (= Bufo) arenarum embryos at different developmental stages. Adverse effects were analyzed by means of the isotoxicity curves for lethality, malformations, stage-dependent susceptibility, and ultrastructural features. For all experimental conditions, the CF was more toxic, up to 10 times, than the active ingredient, being the open mouth stage (S.21) the most susceptible to the herbicide. For continuous treatment conditions, the early embryonic development was the most susceptible to 2,4-D and the LC50s for 96 and 168 h were 9.06 and 7.76 mg L(-1) respectively. In addition, both the active ingredient and the CF were highly teratogenic, resulting in reduced body size, delayed development, microcephaly, agenesis of gills, and abnormal cellular proliferation processes as the main adverse effects. According to US EPA, 2,4-D in agricultural scenarios may be up to three times higher than the NOEC values for teratogenic effects reported in this study. Therefore, they might represent a risk for amphibians. This study also points out the relevance of reporting the susceptibility of embryos at different developmental stages to both the active ingredient and the CF of agrochemicals in order to protect nontarget organisms.

Journal of toxicology and environmental health, Feb 25, 2016

The combined effects of two widely used pesticides, endosulfan and cypermethrin, on survival of e... more 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.

Social Science Research Network, 2022

Water Air and Soil Pollution, Apr 13, 2016

Diazinon is an extensively applied organophosphate pesticide, and nonylphenol is one of the major... more Diazinon is an extensively applied organophosphate pesticide, and nonylphenol is one of the major degradation products of nonylphenol polyethoxylates which are commonly used as surfactant in pesticide formulations. Both pollutants are widely distributed and often coexist in agroecosystems, where they might cause toxic effects to wild biota. This study assessed single and joint toxicity of binary mixtures of these organic compounds on the early development of Rhinella arenarum by means of a standardized test. Joint toxicity of diazinon/nonylphenol mixtures were assessed in embryos and larvae exposed to three different proportions at different exposure times. Embryo and larval toxicity was time-dependent, and larvae were significantly more sensitive than embryos to both compounds. For both embryos and larvae, nonylphenol was between 11 and 18 times more toxic than diazinon. Joint toxicity of the chemicals showed a tendency to be significantly higher than the predicted by additivity effects highlighting the threat that diazinon/nonylphenol mixtures represent for Rhinella arenarum populations.

Chemosphere, May 1, 2021

In situ bioassays provide valuable information about the environment and offer more realistic res... more 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.

Environmental Science and Pollution Research, Feb 8, 2020

Copper and nonylphenol are two commonly found chemicals in the aquatic environment, particularly ... more Copper and nonylphenol are two commonly found chemicals in the aquatic environment, particularly in the distribution area of the amphibian Rhinella arenarum. The current work evaluated the lethal toxicity of equitoxic and non-equitoxic binary mixtures of copper and nonylphenol on embryos and larvae of the South America toad by means of the standardized test, AMPHITOX. Joint toxicity of mixtures was assessed in several proportions of these compounds at different exposure times and was analyzed at different level of mortality effect (LC10, LC50 and LC90). Considering the LC50, the equitoxic mixture was always antagonistic independently of the exposure time and the developmental stage. Joint toxicity showed mainly an antagonistic pattern; nonetheless, some time-dependent additive interactions were observed. Regarding the LC10, synergistic interactions were found in embryos and larvae exposed to two different mixture proportions at several exposure times. This highlights the possible synergism of these chemicals at environmentally relevant concentrations. These results point out the relevance of assessing joint toxicity of environmental pollutants for environmental risk assessment.

Science of The Total Environment, Dec 1, 2021

Traditionally, water quality was assessed by physicochemical parameters. However, a more comprehe... more 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.

Ecological Indicators, Oct 1, 2020

Anthropogenic activities may affect wild animal populations. Among them, amphibians are particula... more Anthropogenic activities may affect wild animal populations. Among them, amphibians are particularly sensitive to environmental change. The aim of this study was to evaluate the health condition of Rhinella fernandezae populations at three sites in the Buenos Aires Province, Argentina: a site inside a natural reserve (S1), a site surrounded mainly by industrial activities (S2) and a site surrounded principally by agriculture (S3). Sex and body condition (health status indicators), and micronuclei frequency (genotoxicity biomarker) of R. fernandezae adults were recorded to determine the health status of the local populations. Water samples were also analyzed for metals and pesticides to identify potential correlations with R. fernandezae malformations. Metals and pesticides were detected in the water samples of all three sites, with Al, Cu and Se exceeding the relevant limits for the protection of aquatic life. S1 had the highest overall environmental quality, and S2 had the greatest variety of pesticides. Individuals from S3 had significantly higher micronuclei frequency and females from S3 significantly lower body condition than females from S1, while females from S2 were significantly smaller than those from S1. Worst amphibian-based biological quality was assessed for S3, followed by S2 and S1. The low health statusfitness of females from the impacted sites (especially of the agriculture-surrounded S3 site), indicated by the low body condition and the high micronuclei frequency, may compromise the long-term health of the local amphibian populations. We conclude that action needs to be taken towards the long-term conservation of local amphibian populations from human-induced ecosystem degradation.

Environmental Pollution, Aug 1, 2020

The Paran a River basin is one of the most important in South America and is affected by human ac... more The Paran a 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 ¼ 49 (28 e71.6)%), 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 ¼ 98.72 (60.60e302.52)%), 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.

Chemistry and Ecology, Jun 7, 2016

ABSTRACT Copper (Cu) and diazinon are two widely distributed pollutants; they coexist in agro-eco... more ABSTRACT Copper (Cu) and diazinon are two widely distributed pollutants; they coexist in agro-ecosystems and cause toxicity to wild biota. This study proposes to analyse the joint toxicity of binary mixtures of Cu and diazinon on the early development of the South American toad, Rhinella arenarum by means of a standardised test. Cu was always more toxic than diazinon. Cu was more embryotoxic while the pesticide was more toxic during the larval exposure than during the embryonic period. The different Cu/diazinon mixtures proportions assayed were significantly less toxic than expected by additive effects. Thus, an antagonistic interaction pattern was observed. This pattern was independent of the assayed proportion, the exposure times and the exposure developmental periods. In the risk assessment analysis to establish water quality criteria, the joint toxicity should be considered at different ratios, exposure time and life period for a certain species, in order to preserve wild species.

Environmental Science and Pollution Research, Oct 6, 2021

Glyphosate and 2,4-D are two herbicides commonly used together. Since there is little information... more 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.

Environmental Toxicology and Chemistry, Oct 12, 2011

Copper toxicity in different embryonic and larval stages of the common South American toad Rhinel... more 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 mg Cu 2þ /L and a 168-h LC50 of 19.5 mg Cu 2þ /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 ¼ 51 mg Cu 2þ /L), but in the case of S.28 toxicity increased slightly from a 24-h LC50 of 138.6 mg Cu 2þ /L to a 168-h LC50 of 104 mg Cu 2þ /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.

Journal of Environmental Science and Health Part B-pesticides Food Contaminants and Agricultural Wastes, Jun 10, 2019

Little is known about the effects of polluted water bodies from Buenos Aires Province on the deve... more 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 (LC 50 :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-LC 50 s were 28.12%, 39% and 61% for (S), (P) and (A), respectively. A stagedependent 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.

Issues in toxicology, 2017

Amphibians at early stages of their life cycle constitute a very valuable ecotoxicological tool f... more Amphibians at early stages of their life cycle constitute a very valuable ecotoxicological tool for toxicity studies as indicators of environmental quality for wildlife protection purposes. AMPHITOX was developed in 1999 in order to cope with the increasing diversity of chemical and physicochemical agents and cover the needs for a sensitive, cost-effective and customized assay for different exposure times and developmental stage toxicity tests. Based on a set of bioassays using amphibian embryos, a large number of toxicity studies with both environmental samples and physicochemical agents (single or in combination) were included. More recently, the growing concern about the chronic effects of xenobiotics in environmentally relevant concentrations led us to expand the exposure conditions of classic, acute (up to 96 h of exposure), short-term chronic (168 h) and chronic (14 days) AMPHITOX tests with young larvae, to evaluate noxious effects on specific stages of the early life cycle (pulse-exposures) and exposure periods that cover whole stages of development (embryo–larval and metamorphosis). This last test has even included post-exposure effects to assess alterations on metamorphosis, gonadogenesis and sexual differentiation. In this contribution, the basic conditions of AMPHITOX test and some case studies are included to illustrate both basic and customized potential of this toxicity test. The versatility of this test has been challenged with different conditions, customizing it to the toxicity of the sample, exposure conditions, and developmental stage.

Journal of Hazardous Materials, Dec 1, 2013

Lethal and sublethal toxicity of the major chemical used in epoxide compounds, epichlorohydrin (E... more Lethal and sublethal toxicity of the major chemical used in epoxide compounds, epichlorohydrin (ECH) was evaluated on the early life cycle of the common South American toad, Rhinella arenarum (Anura, Bufonidae). The stages evaluated were (according to Del Conte and Sirlin): early blastula (S.3-S.4), gastrula (S.10-S.12), rotation (S.15), tail bud (S.17), muscular response (S.18), gill circulation (S.20), open mouth (S.21), opercular folds (S.23) and complete operculum (S.25). The LC50 and EC50 values for lethal and sublethal effects were calculated. The early blastula was the most sensitive stage to ECH both for continuously and pulse-exposures (LC50-24h=50.9 mg L(-1)), while S.20 was the most resistant (LC50-24h=104.9 mg L(-1)). Among sublethal effects, early blastula was also the most sensitive stage (LOEC-48 h=20 mg L(-1)) and it has a Teratogenic Index of 2.5, which indicates the teratogenic potential of the substance. The main abnormalities were persistent yolk plugs, cell dissociation, tumors, hydropsy, oral malformations, axial/tail flexures, delayed development and reduced body size. ECH also caused neurotoxicity including scarce response to stimuli, reduction in the food intake, general weakness, spasms and shortening, erratic or circular swimming. Industrial contamination is considered an important factor on the decline of amphibian populations. Considering the available information about ECH's toxicity and its potential hazard to the environment, this work shows the first results of its developmental toxicity on a native amphibian species, Rhinella arenarum.

Aquatic Toxicology, Dec 1, 2022

The lower Salado River basin receive agricultural, industrial and domestic waste water. So, the a... more The lower Salado River basin receive agricultural, industrial and domestic waste water. So, the aim was to evaluate the quality of three sampling sites that belong to the Salado River basin (S1: Cululú stream; S2: Salado River, at Esperanza City, S3: Salado River at Santo Tomé City) based on physicochemical parameters, metals and pesticides analyses and ecotoxicity on Rhinella arenarum larvae. R. arenarum larvae (Gosner Stage-GS-25) were chronically exposed (504h) to complex matrixes of surface water and sediment samples of each site for the determination of the survival rate. Biomarkers of oxidative stress, neurotoxicity and genotoxicity were analyzed in R. arenarum larvae (GS. 25) after exposure (96h) to the complex matrix of water and sediment. The water quality index showed a marginal quality for all sites, influenced mainly by low dissolved oxygen, high total suspended solid, phosphate, nitrite, conductivity, Pb, Cr and Cu levels. Metal concentrations were higher in sediment than in water samples (̃34-35000 times). In total, thirty different pesticides were detected in all water and sediment samples, S1 presented the greatest variety (26). Glyphosate and AMPA were detected in sediments from all sites, being higher in S3. N,N-Diethyl-meta-toluamide (DEET) and atrazine were detected in all water samples. Greatest mortality was observed in larvae exposed to samples from S1 from 288h (43.3%), reaching a maximum value of 50% at 408h. Oxidative stress and genotoxicity were observed in larvae exposed to S1 and S3 matrix samples. Neurotoxicity was observed in larvae exposed to all matrix samples. The integrated biomarker response index showed that larvae exposed to S1 and S3 were the most affected. According to the physicochemical data and the ecotoxicity assessment, this important river basin is significantly degraded and may represent a risk to aquatic biota, especially for R. arenarum larvae.

International Journal of Environment and Health, 2017

The toxicities of endosulfan and diazinon were comparatively analysed on the early development of... more The toxicities of endosulfan and diazinon were comparatively analysed on the early development of the South American toad, Rhinella arenarum. Pesticides varied greatly in their effects on survival, with concentration-, time-and stage-dependent sensitivity. Endosulfan was 227 times more toxic than diazinon. The larval period was the most sensitive for both pesticides (LC-504 h: 0.01 mg L-1 and 1.92 mg L-1 for endosulfan and diazinon, respectively). Endosulfan toxicity increased 587 and 60 times from acute to chronic exposure in treatments with embryos and larvae, respectively, while diazinon toxicity increased about four and five times, respectively. Both pesticides caused important neurotoxic effects expressed as behavioural disturbances. Risk evaluation assessed by hazard quotient (HQ) was over 1, the level of concern (LOC), for both pesticides at chronic exposure periods and even at acute endosulfan exposure. Results obtained in the present study highlight that these pesticides should be considered potential threats for this species.

Environmental Toxicology and Pharmacology, 2022

The antibiotic oxytetracycline (OTC) is commonly used in animal production and can enter aquatic ... more 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 (504h-LC50= 64.04mg/L) more sensitive than the larvae (504h-LC50= 97.74mg/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.

Environmental Toxicology, Jan 28, 2010

The acute and short term chronic toxicity of both the herbicide butyl ester of 2,4-Dichlorophenox... more The acute and short term chronic toxicity of both the herbicide butyl ester of 2,4-Dichlorophenoxyacetic acid (2,4-D) and a commercial formulation (CF) were evaluated on Rhinella (= Bufo) arenarum embryos at different developmental stages. Adverse effects were analyzed by means of the isotoxicity curves for lethality, malformations, stage-dependent susceptibility, and ultrastructural features. For all experimental conditions, the CF was more toxic, up to 10 times, than the active ingredient, being the open mouth stage (S.21) the most susceptible to the herbicide. For continuous treatment conditions, the early embryonic development was the most susceptible to 2,4-D and the LC50s for 96 and 168 h were 9.06 and 7.76 mg L(-1) respectively. In addition, both the active ingredient and the CF were highly teratogenic, resulting in reduced body size, delayed development, microcephaly, agenesis of gills, and abnormal cellular proliferation processes as the main adverse effects. According to US EPA, 2,4-D in agricultural scenarios may be up to three times higher than the NOEC values for teratogenic effects reported in this study. Therefore, they might represent a risk for amphibians. This study also points out the relevance of reporting the susceptibility of embryos at different developmental stages to both the active ingredient and the CF of agrochemicals in order to protect nontarget organisms.

Journal of toxicology and environmental health, Feb 25, 2016

The combined effects of two widely used pesticides, endosulfan and cypermethrin, on survival of e... more 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.

Social Science Research Network, 2022

Water Air and Soil Pollution, Apr 13, 2016

Diazinon is an extensively applied organophosphate pesticide, and nonylphenol is one of the major... more Diazinon is an extensively applied organophosphate pesticide, and nonylphenol is one of the major degradation products of nonylphenol polyethoxylates which are commonly used as surfactant in pesticide formulations. Both pollutants are widely distributed and often coexist in agroecosystems, where they might cause toxic effects to wild biota. This study assessed single and joint toxicity of binary mixtures of these organic compounds on the early development of Rhinella arenarum by means of a standardized test. Joint toxicity of diazinon/nonylphenol mixtures were assessed in embryos and larvae exposed to three different proportions at different exposure times. Embryo and larval toxicity was time-dependent, and larvae were significantly more sensitive than embryos to both compounds. For both embryos and larvae, nonylphenol was between 11 and 18 times more toxic than diazinon. Joint toxicity of the chemicals showed a tendency to be significantly higher than the predicted by additivity effects highlighting the threat that diazinon/nonylphenol mixtures represent for Rhinella arenarum populations.

Chemosphere, May 1, 2021

In situ bioassays provide valuable information about the environment and offer more realistic res... more 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.

Environmental Science and Pollution Research, Feb 8, 2020

Copper and nonylphenol are two commonly found chemicals in the aquatic environment, particularly ... more Copper and nonylphenol are two commonly found chemicals in the aquatic environment, particularly in the distribution area of the amphibian Rhinella arenarum. The current work evaluated the lethal toxicity of equitoxic and non-equitoxic binary mixtures of copper and nonylphenol on embryos and larvae of the South America toad by means of the standardized test, AMPHITOX. Joint toxicity of mixtures was assessed in several proportions of these compounds at different exposure times and was analyzed at different level of mortality effect (LC10, LC50 and LC90). Considering the LC50, the equitoxic mixture was always antagonistic independently of the exposure time and the developmental stage. Joint toxicity showed mainly an antagonistic pattern; nonetheless, some time-dependent additive interactions were observed. Regarding the LC10, synergistic interactions were found in embryos and larvae exposed to two different mixture proportions at several exposure times. This highlights the possible synergism of these chemicals at environmentally relevant concentrations. These results point out the relevance of assessing joint toxicity of environmental pollutants for environmental risk assessment.

Science of The Total Environment, Dec 1, 2021

Traditionally, water quality was assessed by physicochemical parameters. However, a more comprehe... more 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.

Ecological Indicators, Oct 1, 2020

Anthropogenic activities may affect wild animal populations. Among them, amphibians are particula... more Anthropogenic activities may affect wild animal populations. Among them, amphibians are particularly sensitive to environmental change. The aim of this study was to evaluate the health condition of Rhinella fernandezae populations at three sites in the Buenos Aires Province, Argentina: a site inside a natural reserve (S1), a site surrounded mainly by industrial activities (S2) and a site surrounded principally by agriculture (S3). Sex and body condition (health status indicators), and micronuclei frequency (genotoxicity biomarker) of R. fernandezae adults were recorded to determine the health status of the local populations. Water samples were also analyzed for metals and pesticides to identify potential correlations with R. fernandezae malformations. Metals and pesticides were detected in the water samples of all three sites, with Al, Cu and Se exceeding the relevant limits for the protection of aquatic life. S1 had the highest overall environmental quality, and S2 had the greatest variety of pesticides. Individuals from S3 had significantly higher micronuclei frequency and females from S3 significantly lower body condition than females from S1, while females from S2 were significantly smaller than those from S1. Worst amphibian-based biological quality was assessed for S3, followed by S2 and S1. The low health statusfitness of females from the impacted sites (especially of the agriculture-surrounded S3 site), indicated by the low body condition and the high micronuclei frequency, may compromise the long-term health of the local amphibian populations. We conclude that action needs to be taken towards the long-term conservation of local amphibian populations from human-induced ecosystem degradation.

Environmental Pollution, Aug 1, 2020

The Paran a River basin is one of the most important in South America and is affected by human ac... more The Paran a 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 ¼ 49 (28 e71.6)%), 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 ¼ 98.72 (60.60e302.52)%), 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.

Chemistry and Ecology, Jun 7, 2016

ABSTRACT Copper (Cu) and diazinon are two widely distributed pollutants; they coexist in agro-eco... more ABSTRACT Copper (Cu) and diazinon are two widely distributed pollutants; they coexist in agro-ecosystems and cause toxicity to wild biota. This study proposes to analyse the joint toxicity of binary mixtures of Cu and diazinon on the early development of the South American toad, Rhinella arenarum by means of a standardised test. Cu was always more toxic than diazinon. Cu was more embryotoxic while the pesticide was more toxic during the larval exposure than during the embryonic period. The different Cu/diazinon mixtures proportions assayed were significantly less toxic than expected by additive effects. Thus, an antagonistic interaction pattern was observed. This pattern was independent of the assayed proportion, the exposure times and the exposure developmental periods. In the risk assessment analysis to establish water quality criteria, the joint toxicity should be considered at different ratios, exposure time and life period for a certain species, in order to preserve wild species.

Environmental Science and Pollution Research, Oct 6, 2021

Glyphosate and 2,4-D are two herbicides commonly used together. Since there is little information... more 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.

Environmental Toxicology and Chemistry, Oct 12, 2011

Copper toxicity in different embryonic and larval stages of the common South American toad Rhinel... more 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 mg Cu 2þ /L and a 168-h LC50 of 19.5 mg Cu 2þ /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 ¼ 51 mg Cu 2þ /L), but in the case of S.28 toxicity increased slightly from a 24-h LC50 of 138.6 mg Cu 2þ /L to a 168-h LC50 of 104 mg Cu 2þ /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.

Journal of Environmental Science and Health Part B-pesticides Food Contaminants and Agricultural Wastes, Jun 10, 2019

Little is known about the effects of polluted water bodies from Buenos Aires Province on the deve... more 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 (LC 50 :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-LC 50 s were 28.12%, 39% and 61% for (S), (P) and (A), respectively. A stagedependent 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.

Issues in toxicology, 2017

Amphibians at early stages of their life cycle constitute a very valuable ecotoxicological tool f... more Amphibians at early stages of their life cycle constitute a very valuable ecotoxicological tool for toxicity studies as indicators of environmental quality for wildlife protection purposes. AMPHITOX was developed in 1999 in order to cope with the increasing diversity of chemical and physicochemical agents and cover the needs for a sensitive, cost-effective and customized assay for different exposure times and developmental stage toxicity tests. Based on a set of bioassays using amphibian embryos, a large number of toxicity studies with both environmental samples and physicochemical agents (single or in combination) were included. More recently, the growing concern about the chronic effects of xenobiotics in environmentally relevant concentrations led us to expand the exposure conditions of classic, acute (up to 96 h of exposure), short-term chronic (168 h) and chronic (14 days) AMPHITOX tests with young larvae, to evaluate noxious effects on specific stages of the early life cycle (pulse-exposures) and exposure periods that cover whole stages of development (embryo–larval and metamorphosis). This last test has even included post-exposure effects to assess alterations on metamorphosis, gonadogenesis and sexual differentiation. In this contribution, the basic conditions of AMPHITOX test and some case studies are included to illustrate both basic and customized potential of this toxicity test. The versatility of this test has been challenged with different conditions, customizing it to the toxicity of the sample, exposure conditions, and developmental stage.

Journal of Hazardous Materials, Dec 1, 2013

Lethal and sublethal toxicity of the major chemical used in epoxide compounds, epichlorohydrin (E... more Lethal and sublethal toxicity of the major chemical used in epoxide compounds, epichlorohydrin (ECH) was evaluated on the early life cycle of the common South American toad, Rhinella arenarum (Anura, Bufonidae). The stages evaluated were (according to Del Conte and Sirlin): early blastula (S.3-S.4), gastrula (S.10-S.12), rotation (S.15), tail bud (S.17), muscular response (S.18), gill circulation (S.20), open mouth (S.21), opercular folds (S.23) and complete operculum (S.25). The LC50 and EC50 values for lethal and sublethal effects were calculated. The early blastula was the most sensitive stage to ECH both for continuously and pulse-exposures (LC50-24h=50.9 mg L(-1)), while S.20 was the most resistant (LC50-24h=104.9 mg L(-1)). Among sublethal effects, early blastula was also the most sensitive stage (LOEC-48 h=20 mg L(-1)) and it has a Teratogenic Index of 2.5, which indicates the teratogenic potential of the substance. The main abnormalities were persistent yolk plugs, cell dissociation, tumors, hydropsy, oral malformations, axial/tail flexures, delayed development and reduced body size. ECH also caused neurotoxicity including scarce response to stimuli, reduction in the food intake, general weakness, spasms and shortening, erratic or circular swimming. Industrial contamination is considered an important factor on the decline of amphibian populations. Considering the available information about ECH's toxicity and its potential hazard to the environment, this work shows the first results of its developmental toxicity on a native amphibian species, Rhinella arenarum.

Aquatic Toxicology, Dec 1, 2022

The lower Salado River basin receive agricultural, industrial and domestic waste water. So, the a... more The lower Salado River basin receive agricultural, industrial and domestic waste water. So, the aim was to evaluate the quality of three sampling sites that belong to the Salado River basin (S1: Cululú stream; S2: Salado River, at Esperanza City, S3: Salado River at Santo Tomé City) based on physicochemical parameters, metals and pesticides analyses and ecotoxicity on Rhinella arenarum larvae. R. arenarum larvae (Gosner Stage-GS-25) were chronically exposed (504h) to complex matrixes of surface water and sediment samples of each site for the determination of the survival rate. Biomarkers of oxidative stress, neurotoxicity and genotoxicity were analyzed in R. arenarum larvae (GS. 25) after exposure (96h) to the complex matrix of water and sediment. The water quality index showed a marginal quality for all sites, influenced mainly by low dissolved oxygen, high total suspended solid, phosphate, nitrite, conductivity, Pb, Cr and Cu levels. Metal concentrations were higher in sediment than in water samples (̃34-35000 times). In total, thirty different pesticides were detected in all water and sediment samples, S1 presented the greatest variety (26). Glyphosate and AMPA were detected in sediments from all sites, being higher in S3. N,N-Diethyl-meta-toluamide (DEET) and atrazine were detected in all water samples. Greatest mortality was observed in larvae exposed to samples from S1 from 288h (43.3%), reaching a maximum value of 50% at 408h. Oxidative stress and genotoxicity were observed in larvae exposed to S1 and S3 matrix samples. Neurotoxicity was observed in larvae exposed to all matrix samples. The integrated biomarker response index showed that larvae exposed to S1 and S3 were the most affected. According to the physicochemical data and the ecotoxicity assessment, this important river basin is significantly degraded and may represent a risk to aquatic biota, especially for R. arenarum larvae.

International Journal of Environment and Health, 2017

The toxicities of endosulfan and diazinon were comparatively analysed on the early development of... more The toxicities of endosulfan and diazinon were comparatively analysed on the early development of the South American toad, Rhinella arenarum. Pesticides varied greatly in their effects on survival, with concentration-, time-and stage-dependent sensitivity. Endosulfan was 227 times more toxic than diazinon. The larval period was the most sensitive for both pesticides (LC-504 h: 0.01 mg L-1 and 1.92 mg L-1 for endosulfan and diazinon, respectively). Endosulfan toxicity increased 587 and 60 times from acute to chronic exposure in treatments with embryos and larvae, respectively, while diazinon toxicity increased about four and five times, respectively. Both pesticides caused important neurotoxic effects expressed as behavioural disturbances. Risk evaluation assessed by hazard quotient (HQ) was over 1, the level of concern (LOC), for both pesticides at chronic exposure periods and even at acute endosulfan exposure. Results obtained in the present study highlight that these pesticides should be considered potential threats for this species.