Due to the activities inherent to medical care units, the hospital effluent released contains diverse contaminants such as tensoactives, disinfectants, metals, pharmaceutical products and chemical reagents, which are potentially toxic to the environment since they receive no treatment or are not effectively removed by such treatment before entering the drain. They are incorporated into municipal wastewater, eventually entering water bodies where they can have harmful effects on organisms and can result in ecological damage. To determine the toxicological risk induced by this type of eflluents, eight metals and 11 pharmaceuticals were quantified, in effluent from a hospital. Developmental effects, teratogenesis and oxidative stress induction were evaluated in two bioindicator species: Xenopus laevis and Lithobates catesbeianus. FETAX (frog embryo teratogenesis assay–Xenopus) was used to obtain the median lethal concentration (LC50), effective concentration inducing 50% malformation (EC50), teratogenic index (TI), minimum concentration to inhibit growth (MCIG), and the types of malformation induced. Twenty oocytes in midblastula transition were exposed to six concentrations of effluent (0.1, 0.3, 0.5, 0.7, 0.9, 1%) and negative and positive (6-aminonicotinamide) controls. After 96 h of exposure, diverse biomarkers of oxidative damage were evaluated: hydroperoxide content, lipid peroxidation, protein carbonyl content, and the antioxidant enzymes superoxide dismutase and catalase. TI was 3.8 in X. laevis and 4.0 in L. catesbeianus, both exceed the value in the FETAX protocol (1.2), indicating that this effluent is teratogenic to both species. Growth inhibition was induced as well as diverse malformation including microcephaly, cardiac and facial edema, eye malformations, and notochord, tail, fin and gut damage. Significant differences relative to the control group were observed in both species with all biomarkers. This hospital effluent contains contaminants which represents a toxic risk, since these substances are teratogenic to the bioindicators used. The mechanism of damage induction may be associated with oxidative stress.

The presence of chemicals and the destruction of freshwater habitats have been addressed as one of the reasons for the decline in the amphibians’ populations worldwide. Considering the threat that these animals have been suffering in tropical regions, the present study tested if the Brazilian legislation, concerning the permissive levels of lithium and selenium in water bodies and effluents, warrants the protection of aquatic life. To do so, we assessed the metabolic, immunologic, and histopathologic alterations in liver samples of American bullfrog (Lithobates catesbeianus), at the premetamorphic stage, through biomarkers indicative of general energetic status, i.e., glucose, lipid, and protein metabolism using biochemical and histochemical approaches. The immunologic responses were assessed by the quantification of melanomacrophage centres (MMCs); the histopathologic evaluation of the liver sections was also performed. The assay was carried out over 21 days with two periods of sampling (after 7 and 21 days) to assess the effects of exposure over time. The animals were exposed to the considered safe levels of lithium (2.5 mg L⁻¹) and selenium (10 μg L⁻¹), both, isolated and mixed. The exposed animals showed alterations in glucose and lipid metabolism throughout the experiment. The intense presence of MMCs and histopathological responses are compatible with hepatotoxicity. The toxicity expressed by the employed animal model indicates that the Brazilian environmental legislation for the protection of aquatic life needs to be updated. With this study, we intend to provide data for better environmental policies and bring attention to sublethal effects triggered by the presence of contaminants in the aquatic environment.

Expansion of sugarcane crops may have contributed to the increased contamination of native habitats in Brazil. Several species of amphibians inhabit ponds formed in flooded farmlands, where pesticide concentrations are usually high. This study evaluated the ecotoxicological effects of the sugarcane pesticides fipronil and 2,4-D, as well as the fertilizer vinasse (isolated and mixed), on physiological responses of Leptodactylus fuscus and Lithobates catesbeianus tadpoles. In situ assays were conducted in mesocosms with concentrations based on the doses recommended by the manufacturer. Vinasse (1.3% dilution) caused 100% tadpoles’ mortality immediately after its application. Fipronil and/or 2,4-D altered antioxidant and biotransformation responses, induced neurotoxicity and changed lipid contents in tadpoles. A multivariate approach indicated that the mixture of pesticides induced most of the sublethal effects in both tadpole species, in addition to the isolated fipronil in L. fuscus. Fipronil alone increased glucose-6-phosphate dehydrogenase (G6PDH) activity, decreased acetylcholinesterase (AChE) and total lipid contents, and altered some individual lipid classes (e.g., free fatty acids and acetone-mobile polar lipids) in L. fuscus. The interaction between fipronil and 2,4-D in this species were more evident for lipid contents, although enzymatic alterations in G6PDH, AChE and glutathione-S-transferase (GST) were also observed. In L. catesbeianus, the mixture of pesticides reduced triglycerides and total lipids, as well as increased GST and decreased AChE activities. The detoxifying enzyme carboxylesterase was reduced by 2,4-D (alone or in mixture) in both species. Isolated pesticides also modulated specific lipid classes, suggesting their disruptive action on energy metabolism of tadpoles. Our study showed that fipronil, 2,4-D, and vinasse, individually or mixed, can be harmful to amphibians during their larval phase, causing mortality or impairing their functional responses.

Genotoxic analyses are commonly used in ecotoxicological studies as early biomarkers to investigate the potential effects of environmental contaminants on biological models. Several pollutants can induce DNA damage and, therefore, counting micronuclei and other nuclear abnormalities are efficient tools to evaluate genotoxicity. Some pollutants such as 4-nonylphenol (NP), a detergent used mainly in industries, and Cyproterone Acetate (CPA), an antiandrogenic medicine, have already shown genotoxic effects on some vertebrates. However, although amphibians are considered bioindicators of environmental quality and their populations are declining worldwide, the effects of these compounds on anurans are not yet known and, therefore, we believe that it is important to investigate such effects on anurans. Since water contamination is one of the ultimate causes of amphibian decline, ecotoxicological studies are important to discuss the appropriate solutions to avoid species extinction. Thus, this study investigates the genotoxic effects on Rana catesbeiana tadpoles and juveniles after being exposed to 1, 10 and 100 μg/L NP and 0.025, 0.25 and 2.5 ng/L CPA, by counting the nuclear abnormalities after exposure. The laboratory experiments lasted 28 days. The experimental conditions were the same except for the water volume since tadpoles and juveniles exhibit different habits at different developmental stages. Compared to juveniles, tadpoles were more susceptible to both compounds as indicated by the increased nuclear abnormalities observed in the highest NP concentration and all tested CPA concentrations. The juveniles, on the other hand, responded only to the two highest CPA concentrations. We concluded that CPA, even at very low concentrations, is extremely harmful to both anuran developmental stages and, particularly, to tadpoles. The significant effects observed on tadpoles is an important outcome of this study since 100 μg/L or higher NP concentrations are frequently detected in the environment.

The amphibian populations have faced a drastic decline over the past decades. This decline has been associated with the presence of contaminants in the environment, among other environmental stressors. The present study tested the responses following the exposure to lithium (2.5 mg L⁻¹) and selenium (10μg L⁻¹), both isolated and as a mixture, on the metabolic status of the tail muscle of premetamorphic American bullfrog (Lithobates catesbeianus) through the assessment of the total protein content, mobilization of glucose and triglycerides, and the activity of lactate dehydrogenase (LDH). The exposure followed a 21-day assay with two sampling periods (on the 7th and 21st day after the onset of exposure) to evaluate the effects over time. The group exposed to the mixture presented a statistically decreased LDH activity (P < 0.05) in both sampling periods. The presence of selenium elicited a statistically significant increase (P < 0.05) in the glucose mobilization after 7 days of exposure. After 21 days, the animals exposed to selenium presented levels of glucose mobilization comparable to the control group. The mobilization of glucose and triglycerides remained similar to the control group for the animals exposed to lithium and to the mixture in both periods of sampling (P > 0.05). The total protein content did not show any statistical difference in the treated groups throughout the experiment (P > 0.05). The presented results highlight the importance of the assessment of mixtures that can occur in the environment, since the combination of contaminants may elicit distinct toxicity compared with the effects triggered by the chemicals isolated.

Animals have a long history of assessing ecosystem responses to environmental disturbances, and amphibians stand out for presenting themselves as good animal model and bioindicators of environmental quality. The main purpose of the present work was to investigate the cellular effects of contamination of waters of the Marrecas River, located in the southwest of the state of Paraná, Brazil. Therefore, the objective of the present study was to investigate and monitor the cytotoxic and mutagenic effect, with bullfrog tadpoles, and to discuss these effects with land use along this hydrographic basin. Mutagenic effects were determined by micronucleus assay, and cytotoxicity by other nuclear changes, such as segmented cells, binucleated cells, cells with buds and reniform cells. Water samples were obtained at nine sites along the Marrecas River, covering areas with rural and urban hydrological contribution. For each site, four samples were collected, along the years 2017 and 2018, encompassing the four seasons (summer, autumn, winter, and spring). The results showed mutagenic and cytotoxic effect in four sampling sites, and only cytotoxic effect in other four sites. These effects may be due, possibly, to the use of different agrochemicals across the hydrographic basin region, which have predominant hydrological contributions from crops. Data of this study indicate the presence of cytotoxic and mutagenic contaminants in the waters of the Marrecas River, which can generate environmental problems on the river fauna/flora, and can also affect the local population health.

The mining sector has great importance to the economic activity in Brazil. However, it is also responsible for several environmental impacts such as the rupture of the Fundão dam (Mariana, Brazil) that resulted in the spillage of 50 million m³ of mining tailings in the Doce River Basin. This study evaluated the acute and chronic effects of Fundão tailings on growth, development, respiration rates, swimming performance, and avoidance behavior of Lithobates catesbeianus tadpoles. Results showed that 96-h exposure to different dilutions (10, 25, 50, 75, and 100%) of a stock solution containing mining tailings (50 g/L) caused no mortality of tadpoles; however, the most concentrated solution decreased the swimming speed of the animals. After 16 days, tadpoles exposed to 25, 50, and 100% treatments had both swimming speed and distance traveled reduced. Oxygen consumption was also decreased in tadpoles exposed to the 100% solution after 20 days. Avoidance test indicated that tadpoles avoided lower tailing concentrations, but a reduced avoidance response was attested to the higher concentrations, probably due to the toxic effects of the residues that prevented animals’ displacement. Chemical analysis confirmed the occurrence of cadmium (Cd), lead (Pb), iron (Fe), manganese (Mn), zinc (Zn), and aluminum (Al) in Fundão tailings and its presence in the mouth and inside the intestine of treated tadpoles indicated the ingestion of metals by these organisms. This study showed that even presenting low lethal toxicity, long-term exposure to mining tailings from Fundão dam caused morphophysiological and behavioral damage in tadpoles.

Identifying the relative importance of stressors is critical for effectively managing and conserving freshwater aquatic ecosystems. However, variability in natural ecosystems and the potential for multiple stressors make understanding the effects of stressors challenging in the field. To address these challenges, we assessed four common stressors in the northeastern USA including acidification (pH), climate change (water temperature), salinization (Na and Cl), and nutrient addition using laboratory mesocosms. Each stressor was evaluated independently, with ten mesocosms assigned across a gradient of concentrations for each stressor (total N = 40). We then monitored the effects of the stressors on a model community consisting of periphyton, zooplankton, Northern watermilfoil (Myriophyllum sibericum), American ribbed fluke snail (Pseudosuccinea columella), and larval American bullfrogs (Lithobates catesbeianus). Aquatic stressors varied in the strength of their effects on community structure: Nutrient addition was the least influential stressor, with no significant effects. Acidification influenced periphyton biomass, but not higher trophic levels. Water temperature influenced primary productivity and survival of amphibian larvae, but not intermediate trophic levels. Finally, road salt led to decreases in productivity for all trophic levels included in our model systems. Our results support the findings of prior research, although the effects of acidification and nutrient addition were less pronounced in our study. Importantly, we found that road salt had the most far-reaching effects on a model aquatic community. Given that road salt is the most easily managed of the stressors we compared, our results indicate that improving the condition of freshwater aquatic ecosystems in the northeastern USA may be a feasible objective.

Environmental pollution plays an important role in amphibian population decline. Contamination with endocrine disrupting chemicals (EDCs) is particularly worrying due to their capacity to adversely affect organisms at low doses. We hypothesized that exposure to EDCs such as 4-nonylphenol (NP) and cyproterone acetate (CPA) could trigger responses in the liver and gonads, due to toxic and endocrine disrupting effects. Growth rate may also be impaired by contamination. We investigated sublethal effects of a 28-day exposure to three different concentrations of NP and CPA on liver pigmentation, gonadal morphology, body mass, and length of tadpoles and juveniles Lithobates catesbeianus. Liver pigmentation and the gonadal morphologies of treated tadpoles did not differ from control, but growth rate was impaired by both pollutants. Juveniles treated with 10 μg/L NP and 0.025 and 0.25 ng/L CPA displayed increased liver melanin pigmentation, but gonadal morphologies, sex ratios, and body mass were not affected after treatments. The increase in liver pigmentation may be related to defensive, cytoprotective role of melanomacrophages. The decreased growth rate in tadpoles indicates toxic effects of NP and CPA. Thus, contamination with NP and CPA remains a concern and sublethal effects of different dosages of the compounds on native species should be determined.

This study sought to analyze the effects of the herbicide quinclorac on body condition indices; plasma levels of corticosterone, glucose, and uric acid; activity of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST); and levels of lipid peroxidation (LPO) in the liver and caudal muscle of American bullfrog (Rana catesbeiana) tadpoles. After a 7-day acclimation period, animals were exposed to four concentrations (0.05, 0.1, 0.2, and 0.4 μg/L) of herbicide for a further 7 days. Then, blood samples were obtained, animals were euthanized, and the liver and caudal muscle resected. Levels of corticosterone and uric acid were reduced in tadpoles exposed to the highest concentration of herbicide, and this reduction was preceded by an increase in glucose levels. In the liver tissue, LPO was increased after exposure to 0.1 μg/L quinclorac, followed by a return to baseline values in the remaining concentrations; this response was accompanied by an increase in SOD and GST and reduction of tissue protein levels. At the highest concentration, a reduction in activity of all enzymes was observed, with protein returning to control-like levels. In muscle, SOD and GST levels declined with exposures up to 0.1 g/L and 0.4 μg/L, respectively, whereas LPO decreased in animals exposed to 0.1 μg/L. These results suggest participation of nonenzymatic antioxidant defenses, as demonstrated by the reduction in uric acid levels. Exposure to the range of quinclorac concentrations used in this study slowed body mass and length gain, reduced corticosterone levels, and modulated antioxidant defenses. Graphical abstract ᅟ