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.

Although many polymers are known by their toxicity, we know nothing about the impact of polyethylene glycol (PEG) on anurofauna. Its presence in different products and disposal in aquatic environments turn assessments about its impact on amphibians an urgent matter. Accordingly, we tested the hypothesis that short-time exposure (72 h) of tadpoles belonging to the species Physalaemus cuvieri (Anura, Leptodactylidae) to PEG induces oxidative stress and neurotoxicity on them. We observed that polymer uptake in P. cuvieri occurred after exposure to 5 and 10 mg/L of PEG without inducing changes in their nitrite levels neither at the levels of substances reactive to thiobarbituric acid. However, hydrogen peroxide and reactive oxygen species production was higher in animals exposed to PEG, whose catalase and superoxide dismutase levels were not enough to counterbalance the production of these reactive species. Therefore, this finding suggests physiological changes altering REDOX homeostasis into oxidative stress. In addition, the increased activity of acetylcholinesterase and butyrylcholinesterase, and reduction in superficial neuromasts, confirmed PEG’s neurotoxic potential. To the best of our knowledge, this is the first report on PEG’s biological impact on a particular amphibian species. The study has broadened the understanding about ecotoxicological risks associated with water pollution by these polymers, as well as motivated further investigations on its impacts on amphibians’ health and on the dynamics of their natural populations.

A chemical contaminant of growing concern to freshwater aquatic organisms, including many amphibians, is chloride ion. The salinization of freshwater ecosystems is likely caused, in part, by the application of massive amounts of road de-icing salts to roadways during winter months. The issue of freshwater salinization has become the subject of many toxicity studies and is often investigated in conjunction with other chemical stressors. However, few published studies attempt to investigate the interactions of elevated chloride concentration and increased temperature. Further, no studies have investigated the gap between the recommended feeding conditions typically used in standard toxicity tests and those that may exist in natural amphibian habitats. This study addressed the critical issues of elevated chloride, increased temperature, and variation in food quality. We conducted a 96-h acute toxicity test to investigate acute chloride toxicity as impacted by different diets, as well as a chronic toxicity test to investigate the impacts of chloride, temperature, and resource quality on the survival and development of larval Lithobates sylvaticus (wood frogs). Chloride LC₅₀s ± 1 SE were 3769.22 ± 589.05, 2133.00 ± 185.95, and 2644.69 ± 209.73 mg Cl⁻/L were for non-fed, low-protein diet, and high-protein diet, respectively. For the chronic toxicity study, elevated chloride decreased tadpole survival. Increased temperature, and lower resource quality, were found negatively impacted survival of tadpoles and altered time-to-metamorphosis. This study shows that environmentally relevant concentrations of chloride, temperatures, and the protein content of the diet all exert critical effects on larval wood frogs.

Drugs are excreted from the human body as both original substances and as metabolites and enter aquatic environment through waste water. The aim of this study was to widen the current knowledge considering the effects of waterborne antidepressants with different modes of action—amitriptyline, venlafaxine, sertraline—on embryos of non-target aquatic biota—fish (represented by Danio rerio) and amphibians (represented by Xenopus tropicalis). The tested concentrations were 0.3; 3; 30; 300 and 3000 μg/L in case of amitriptyline and venlafaxine and 0.1; 1; 10; 100 and 1000 μg/L for sertraline. Test on zebrafish embryos was carried out until 144 h post fertilization, while test on Xenopus embryos was terminated after 48 h. Lethal and sublethal effects as well as swimming alterations were observed at higher tested concentrations that are not present in the environment. In contrast, mRNA expression of genes related to heart, eye, brain and bone development (nkx2.5, otx 2, bmp4 and pax 6) seems to be impacted also at environmentally relevant concentrations. In a wider context, this study reveals several indications on the ability of antidepressants to affect non target animals occupying environments which may be contaminated by such compounds.

As a widely used organotin acaricide nowadays, azocyclotin (ACT) could induce thyroidal endocrine disruption in fishes and amphibians, but its dominant disrupting mode remains unknown. In this study, zebrafish were firstly exposed to ACT (0.18–0.36 ng/mL) from 2 hpf (hours post fertilization) to 30 dpf (days post fertilization), and a series of developmental toxicological endpoints and thyroid hormones were measured. Result showed that no developmental toxicity to zebrafish was found in 0.18 and 0.24 ng/mL groups except decreased body weight (30 dpf, 0.24 ng/mL). However, exposed to 0.36 ng/mL ACT led to reductions in heartbeat (48 hpf), hatching rate (72 hpf) and bodyweight (30 dpf). General tendencies of decreases in free T3 but increases in free T4 and reductions in ratio of free T3/T4 were also found, inferring that type II deiodinase (Dio2) was repressed. This inference was confirmed by Western analysis that Dio2 expression reduced by 42.7% after 0.36 ng/mL ACT treatment. Moreover, RNA-Seq analysis implied that exposed to 0.36 ng/mL ACT altered the genome-wide gene expression profiles of zebrafish. Totally 5660 genes (involving 3154 down-regulated and 2596 up-regulated genes) were differentially expressed, and 13 deferentially expressed genes including down-regulated dio2 were significantly enriched in thyroid hormone signaling pathway. Subsequently, an in vitro thyroid receptor-reporter gene assay using GH3 cells was performed to further explore the potential disrupting mechanism. Result showed that luciferase activity slightly increased after exposure to ACT alone or ACT combined with low level T3, but was suppressed when combined with high level T3. It indicted there probably existed a competitive relationship in some extent between ACT and T3 in vivo. Overall, the present study provided preliminary evidences that long-term exposure to trace ACT repressed Dio2 expression, declined T3 and then activated thyroid receptor-mediated signaling, thereby leading to integrated thyroid endocrine disruption in zebrafish larvae.