In the context of the global amphibian crisis, biomonitoring constitutes a valuable assessment tool to provide critical up to date information on the status and health of amphibians worldwide. The aim of the current study was to evaluate the possible confounding effects of sex, size, and time since capture on enzymatic biomarkers and hematologic parameters of the South American frog Leptodactylus latrans. Frogs were collected by hand between 9 pm and 12 am on two consecutive nights. On the first night, captured frogs were transported for 2 h by car to laboratory installations, maintained overnight in plastic containers, and blood and tissue sampled on the next morning. In contrast, frogs collected on the second night were blood and tissue sampled in the field, immediately after the capture period. Hematological parameters were analyzed, and enzymatic activities of catalase, cholinesterase (ChE), and glutathione S-transferase (GST) were determined in the plasma, liver, kidney, and muscle. A sex difference was observed only for total white blood cell counts (WBC), females exhibiting significantly greater values than males. The packed cell volume (PCV), mean corpuscular hemoglobin concentration (MCHC), WBC, and muscle ChE activity were significantly correlated with snout-vent length (SVL). The correlation was inversed in the case of MCHC, WBC, and muscle ChE, while the correlation was positive between PCV and SVL. Most examined parameters presented similar values when frogs were sampled at night following capture or the next morning. Total red blood cells (RBCs) count, and plasma enzymatic activities of ChE and GST were the only parameters that presented significantly increased values in morning samplings compared with night samplings. Overall, the current study indicates that it is best to sample the frogs as soon as possible after capture if hematologic or plasmatic biomarkers are examined. Nevertheless, it is possible to sample on the next morning if tissular biomarkers are employed. | Instituto de Recursos Biológicos | Fil: Brodeur, Celine Marie Julie. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Recursos Bilológicos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. | Fil: Bahl, María Florencia. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Química. Centro de Investigaciones del Medio Ambiente; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. | Fil: Natale, Guillermo S. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Química. Centro de Investigaciones del Medio Ambiente; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. | Fil: Poliserpi, María Belen. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Recursos Biológicos; Argentina

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. | Fil: Peluso, Julieta. Universidad Nacional de San Martín (UNSAM). Instituto de Investigación e Ingeniería Ambiental; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. | Fil: Aronzon, Carolina Mariel. Universidad Nacional de San Martín (UNSAM). Instituto de Investigación e Ingeniería Ambiental; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET); Argentina. | Fil: Ríos de Molina, María del Carmen. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. | Fil: Rojas, Dante Emanuel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Instituto de Química Biológica; Argentina. | Fil: Cristos, Diego Sebastian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Instituto de Química Biológica; Argentina. | Fil: Pérez Coll, Cristina Silvia. Universidad Nacional de San Martín (UNSAM). Instituto de Investigación e Ingeniería Ambiental; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET); Argentina.

International audience | Environmental contamination is one of the major factors or cofactors affecting amphibian populations. Since 2000, the number of studies conducted in laboratory conditions to understand impacts of chemical exposures increased. They aimed to characterize biological effects on amphibians. This review proposes an overview of biological responses reported after exposures to metals, phytopharmaceuticals or emerging organic contaminants and focuses on endpoints relating to reproduction and development. Due to amphibian peculiar features, these periods of their life cycle are especially critical to pollutant exposures. Despite the large range of tested compounds, the same model species are often used as biological models and morphological alterations are the most studied observations. From the results, the laboratory-to-field extrapolation remained uneasy and exposure designs have to be more elaborated to be closer to environmental conditions. Few studies proposed such experimental approaches. Lastly, gametes, embryos and larvae constitute key stages of amphibian life cycle that can be harmed by exposures to freshwater pollutants. Specific efforts have to be intensified on the earliest stages and notably germ cells.

Globally, amphibian species are experiencing dramatic population declines, and many face the risk of imminent extinction. Endocrine-disrupting chemicals (EDCs) have been recognised as an underappreciated factor contributing to global amphibian declines. In this regard, the use of hormonal growth promotants in the livestock industry provides a direct pathway for EDCs to enter the environment—including the potent anabolic steroid 17β-trenbolone. Emerging evidence suggests that 17β-trenbolone can impact traits related to metabolism, somatic growth, and behaviour in non-target species. However, far less is known about possible effects of 17β-trenbolone on anuran species, particularly during early life stages. Accordingly, in the present study we investigated the effects of 28-day exposure to 17β-trenbolone (mean measured concentrations: 10 and 66 ng/L) on body size, body condition, metabolic rate, and anxiety-related behaviour of tadpoles (Limnodynastes tasmaniensis). Specifically, we measured rates of O₂ consumption of individual tadpoles as a proxy for metabolic rate and quantified their swimming activity and their time spent in the upper half of the water column as indicators of anxiety-related behaviour. Counter to our predictions based on effects observed in other taxa, we detected no effect of 17β-trenbolone on body size, metabolic rate, or behaviour of tadpoles; although, we did detect a subtle, but statistically significant decrease in body condition at the highest 17β-trenbolone concentration. We hypothesise that 17β-trenbolone may induce taxa-specific effects on metabolic function, growth, and anxiety-related behaviour, with anurans being less sensitive to disruption than fish, and encourage further cross-taxa investigation to test this hypothesis.

A pre-diabetes syndrome induced by endocrine disruptors (ED) was recently demonstrated in the model amphibian Silurana (Xenopus) tropicalis and was suggested to be a potential cause of amphibian population decline. However, such effects have not been found in wild type frogs exposed to ED and the capacity of amphibians to physiologically develop diabetes under natural conditions has not been confirmed. This study showed that a high fat diet (HFD) model displaying the important characteristics of mammal HFD models including glucose intolerance, insulin resistance and nonalcoholic fatty liver disease (NAFLD) can be developed with green frogs (Pelophylax spp.). Wild green frogs exposed to 10 μg L⁻¹ benzo [a]pyrene (BaP) for 18 h also displayed several characteristics of the pre-diabetes phenotype previously observed in Xenopus including glucose intolerance, gluconeogenesis activation and insulin resistance. The study results confirmed that metabolic disorders induced by ED in wild green frogs are typical of the pre-diabetes phenotype and could serve as a starting point for field studies to determine the role of ED in the decline of amphibian populations. From an environmental perspective, the response of wild green frogs to different ED (10 μg L⁻¹) suggests that a simple glucose-tolerance test could be used on wild anurans to identify bodies of water polluted with metabolic disruptors that could affect species fitness.

Across the planet, winter de-icing practices have caused secondary salinization of freshwater habitats. Many amphibians are vulnerable because of permeable skin and reliance on small ponds, where salinity can be high. Early developmental stages of amphibians are especially sensitive to salt, and larvae developing in salt-polluted environments must osmoregulate through ion exchange in gills. Though ionoregulation in amphibian gills is generally understood, the role of gill morphology remains poorly described. Yet gill structure should affect ionoregulatory capacity, for instance in terms of available surface area. As larval amphibian gills also play critical roles in gas exchange and foraging, changes in gill morphology from salt pollution potentially affect not only osmoregulation, but also respiration and feeding. Here, we used an exposure experiment to quantify salinity effects on larval gill morphology in wood frogs (Rana sylvatica). We measured a suite of morphological traits on gill tufts—where ionoregulation and gas exchange occur—and on gill filters used in feeding. Larvae raised in elevated salinity developed larger gill tufts but with lower surface area to volume ratio. Epithelial cells on these tufts were less circular but occurred at higher densities. Gill filters showed increased spacing, likely reducing feeding efficiency. Many morphological gill traits responded quadratically, suggesting that salinity might induce plasticity in gills at intermediate concentrations until energetic demands exceed plasticity. Together, these changes likely diminish ionoregulatory and respiratory functionality of gill tufts, and compromise feeding functionality of gill filters. Thus, a singular change in aquatic environment from a widespread pollutant appears to generate a suite of consequences via changes in gill morphology. Critically, these changes in traits likely compound the severity of fitness impacts in populations dwelling in salinized environments, whereby ionoregulatory energetic demands should increase respiratory and foraging demands, but in individuals who possess structures poorly adapted for these functions.

Metabolic disorders induced by endocrine disruptors (ED) may contribute to amphibian population declines but no transgenerational studies have evaluated this hypothesis. Here we show that Xenopus tropicalis, exposed from the tadpole stage, to the ED benzo[a]pyrene (BaP, 50 ng.L⁻¹) produced F2 progeny with delayed metamorphosis and sexual maturity. At the adult stage, F2–BaP females displayed fatty liver with inflammation, tissue disorganization and metabolomic and transcriptomic signatures typical of nonalcoholic steato-hepatitis (NASH). This phenotype, similar to that observed in F0 and F1 females, was accompanied by a pancreatic insulin secretory defect. Metabolic disrupted F2–BaP females laid eggs with metabolite contents significantly different from the control and these eggs did not produce viable progeny. This study demonstrated that an ED can induce transgenerational disruption of metabolism and population collapse in amphibians under laboratory conditions. These results show that ED benzo[a]pyrene can impact metabolism over multiple generations and support epidemiological studies implicating environmental EDs in metabolic diseases in humans.

Amphibian larvae are commonly used as indicators of aquatic ecosystem health because they are susceptible to contaminants. However, there is limited information on how species characteristics and trophic position influence contaminant loads in larval amphibians. Importantly, there remains a need to understand whether grazers (frogs and toads [anurans]) and predators (salamanders) provide comparable information on contaminant accumulation or if they are each indicative of unique environmental processes and risks. To better understand the role of trophic position in contaminant accumulation, we analyzed composite tissues for 10 metals from larvae of multiple co-occurring anuran and salamander species from 20 wetlands across the United States. We examined how metal concentrations varied with body size (anurans and salamanders) and developmental stage (anurans) and how the digestive tract (gut) influenced observed metal concentrations. Across all wetlands, metal concentrations were greater in anurans than salamanders for all metals tested except mercury (Hg), selenium (Se), and zinc (Zn). Concentrations of individual metals in anurans decreased with increasing weight and developmental stage. In salamanders, metal concentrations were less correlated with weight, indicating diet played a role in contaminant accumulation. Based on batches of similarly sized whole-body larvae compared to larvae with their digestive tracts removed, our results indicated that tissue type strongly affected perceived concentrations, especially for anurans (gut represented an estimated 46–97% of all metals except Se and Zn). This suggests the reliability of results based on whole-body sampling could be biased by metal, larval size, and development. Overall, our data shows that metal concentrations differs between anurans and salamanders, which suggests that metal accumulation is unique to feeding behavior and potentially trophic position. To truly characterize exposure risk in wetlands, species of different life histories, sizes and developmental stages should be included in biomonitoring efforts.

Novel stressors introduced by human activities increasingly threaten freshwater ecosystems. The annual application of more than 2.3 billion kg of pesticide active ingredient and 22 billion kg of road salt has led to the contamination of temperate waterways. While pesticides and road salt are known to cause direct and indirect effects in aquatic communities, their possible interactive effects remain widely unknown. Using outdoor mesocosms, we created wetland communities consisting of zooplankton, phytoplankton, periphyton, and leopard frog (Rana pipiens) tadpoles. We evaluated the toxic effects of six broad-spectrum insecticides from three families (neonicotinoids: thiamethoxam, imidacloprid; organophosphates: chlorpyrifos, malathion; pyrethroids: cypermethrin, permethrin), as well as the potentially interactive effects of four of these insecticides with three concentrations of road salt (NaCl; 44, 160, 1600 Cl⁻ mg/L). Organophosphate exposure decreased zooplankton abundance, elevated phytoplankton biomass, and reduced tadpole mass whereas exposure to neonicotinoids and pyrethroids decreased zooplankton abundance but had no significant effect on phytoplankton abundance or tadpole mass. While organophosphates decreased zooplankton abundance at all salt concentrations, effects on phytoplankton abundance and tadpole mass were dependent upon salt concentration. In contrast, while pyrethroids had no effects in the absence of salt, they decreased zooplankton and phytoplankton density under increased salt concentrations. Our results highlight the importance of multiple-stressor research under natural conditions. As human activities continue to imperil freshwater systems, it is vital to move beyond single-stressor experiments that exclude potentially interactive effects of chemical contaminants.

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.