Microplastics and TiO2 nanoparticles mixture as an emerging threat to amphibians: A case study on bullfrog embryos.

Emerging contaminants can act as contributing factors to the decline of amphibian populations worldwide. Recently, scientists have drawn attention to the potential ecotoxicity of microplastics and nanomaterials in amphibians, however, their possible effects on embryonic developmental stages are still absent. Thus, the present study analyzed the developmental toxicity of environmentally relevant concentrations of polyethylene microplastics (PE MPs; 60 mg/L) and titanium dioxide nanoparticles (TiO2 NPs; 10 µg/L), isolated or in combination (Mix group) on bullfrog embryos, Aquarana catesbeiana, adapting the Frog Embryo Teratogenesis Assay (FETAX, 96h). Allied to the FETAX protocol, we also analyzed the heart rate and morphometric data. The exposure reduced the survival and hatching rates in groups exposed to TiO2 NPs, and to a lesser extent, also affected the Mix group. TiO2 NPs possibly interacted with the hatching enzymes of the embryos, preventing hatching, and reducing their survival. The reduced effects in the Mix group are due to the agglomeration of both toxicants, making the NPs less available for the embryos. PE MPs got attached to the gelatinous capsule of the chorion (confirmed by fluorescence microscopy), which protected the embryos from eventual direct effects of the microplastics on the hatching and survival rates. Although there were no cardiotoxic effects nor morphometric alterations, there was a significant increase in abdominal edemas in the hatched embryos of the PE MPs group, which indicates that osmoregulation might have been affected by the attachment of the microplastics on the embryos' gelatinous capsule. This study presents the first evidence of developmental toxicity of environmental mixtures of microplastics and nanoparticles on amphibians and reinforces the need for more studies with other amphibian species, especially neotropical specimens that could present bigger sensibility. Our study also highlighted several features of the FETAX protocol as useful tools to evaluate the embryotoxicity of several pollutants on amphibians.

The cardiorespiratory system of miniature frogs.

Anurans of the genus Brachycephalus are among the smallest vertebrates in the world, due to an extreme process of miniaturization. As an example of this process, Brachycephalus species show loss of fingers, loss of the eardrum and middle ear, bone fusions, and the presence of paravertebral plates and parotic plaque. However, no studies addressing the consequences of miniaturization on internal organs, such as the lungs and heart, are currently available. Thus, this study aimed to investigate if overall small body size has affected the cardiorespiratory system. We investigated, via dissections, individuals of four Brachycephaloidea species: Brachycephalus rotenbergae, B. pitanga, Eleutherodactylus johnstonei, and Ischnocnema parva. We observed that B. rotenbergae and B. pitanga present a reduction of the atrial septum and absence of the carotid body. On the other hand, despite being a member of the sister genus to Brachycephalus (both genera belong to the Brachycephalidae), individuals of Ischnocnema present a heart with a complete septum and carotid body; this is also observed in E. johnstonei (Eleutherodactylidae). We observed that B. rotenbergae and B. pitanga have thin skin with a one to two cell thick germ layer, and their lungs likely exhibit lower blood supply when compared to individuals of the E. johnstonei and I. parva species. Based on the observed structures, we suggest that in species of Brachycephalus, respiration is performed mainly through the skin, and their lungs may have a reduced respiratory function.

Exposure to ionizing radiation and liver histopathology in the tree frogs of Chornobyl (Ukraine).

Ionizing radiation has the potential to damage organic molecules and decrease the health and survival of wildlife. The accident at the Chornobyl Nuclear Plant (Ukraine, 1986) led to the largest release of radioactive material to the environment. Among the different organs of a vertebrate, the liver plays a crucial role in detoxification processes, and has been used as a biomarker to investigate cellular damage in ecotoxicological research. Here, we examined the impact of the exposure to the current levels of ionizing radiation present in the Chornobyl Exclusion Zone on the liver of Eastern tree frogs (Hyla orientalis). We quantified the area of melanomacrophage cells and morphological variables of hepatocytes, two cell types often used to estimate damage caused by pollutants in vertebrates. First, we investigated whether these hepatic parameters were indicative of frog (individual) condition. Then, we analyzed the effect of individual absorbed dose rates and ambient radiation levels on frog livers. Most of the studied parameters were correlated with individual body condition (a good predictor of amphibian fitness and survival). We did not detect marked morphological lesions in the liver of frogs captured in medium-high radiation environments. The area occupied by melanomacrophages and the morphology of hepatocytes did not change across a gradient of radiocontamination covering two orders of magnitude. Once accounting for body condition and sampling locality, the area of melanomacrophages was lower in areas with high radiation levels. Finally, the area occupied by melanomacrophages was not linked to dorsal skin coloration. Our results indicate that current levels of radiation experienced by tree frogs in Chornobyl do not cause histopathological damage in their liver. These results agree with previous physiological work in the species in the Chornobyl area, and encourage further molecular and physiological research to fully disentangle the current impact of the Chornobyl accident on wildlife.

Bioaccumulation and ecotoxicological impact of micro(nano)plastics in aquatic and land snails: Historical review, current research and emerging trends.

Microplastics (MPs) and nanoplastics (NPs) are ubiquitous emerging pollutants in the environment. Although MPs/NPs' hazardous effects have been described at different trophic levels, little attention has been given to how they can affect gastropod communities. Thus, the current study aimed to summarize and critically address data available in the scientific literature about micro(nano)plastics' ecotoxicological impact on snails. The analyzed data has evidenced MP/NP bioaccumulation in 40 gastropod species collected in the field; 15 gastropod species were used to assess the potential toxicity of MPs/NPs. Asia accounted for the highest level of MPs/NPs bioaccumulated in gastropods; it was followed by the South American, European and Antarctic continents. MPs/NPs' toxicity depends on their composition, shape and size, as well as on differences in methodological approaches adopted by different studies. Results have shown that MPs/NPs induce several impairments - such as behavioral changes, developmental toxicity, dysbiosis, histopathological alterations, oxidative stress -, generate ecological impairments, as well as act as pollutant vector and increase chiral chemicals' toxicity. Research gaps and recommendations for future research were highlighted to help better understanding MPs/NPs' toxicity in gastropods, given the extremely important role played by them in studies focused on investigating how MPs/NPs can affect invertebrate communities living in terrestrial and aquatic environments.

Effects of lithium and selenium in the tail muscle of American bullfrog tadpoles (Lithobates catesbeianus) during premetamorphosis.

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.

Beach morphodynamics and its relationship with the deposition of plastic particles: A preliminary study in southeastern Brazil.

This study describes the beach profile, characterizes microplastics and correlates their abundance with morphodynamics characteristics on three beaches from the state of São Paulo, Brazil. 745 particles were found in 4 m2 of sediment, mostly styrofoam. Nearly 90% of the fragments were found in Boracéia, the most dissipative beach, while less than 1% were found in Juréia beach, the most reflective one. The chemical composition of microplastics was identified by near-infrared hyperspectral imaging (HSI-NIR). The correlation between the abundance of particles and the slope plus the extension of the sand strip was high, as well as that found with the waves' height. These preliminary results indicate that there might be an intrinsic relation among the morphodynamical forces, the movement and destination of microplastics in marine environments.

Effects of mercury at field estimated concentration in brain of Bombus atratus (Hymenoptera: Bombini).

Although mercury neurotoxic effects are well known in several species, it is poorly studied in bees. Mercury contamination is increasing in several regions of the Brazilian Amazon Rainforest due to illegal and indiscriminate gold mining. Therefore, this study aimed to evaluate the effects of mercury (Hg) in brain Kenyon cells of foraging workers of Bombus atratus exposed to an average concentration (110 ppb) found in pots of honey from native bees of South America and Australia. Twenty forager workers were collected in the field (23° 34' S 47° 31' W), divided into control (n = 10) and exposed (n = 10) groups, and individually kept in special boxes for 48 h. For the exposed group, we offered Hg solution (at 110 ppb) ad libitum, while for the control group we offered water, and for both sucrose syrup at 70%. After the exposure time, the bees were crio-anesthezied at 4 °C. Brains were dissected and processed for morphological, morphometric, and histochemical analyses. Morphological results showed that the Kenyon cells of the Hg-exposed group presented both cytoplasmic vacuolization and nuclear pyknosis, which indicate cell death. These findings were corroborated by the acridine orange staining. Hg exposure also induced significant nuclear chromatin compaction in Kenyon cells. The calyces and peduncles of the mushroom bodies showed disorganization and vacuolization. In summary, these changes may imply in a severe impairment of the cognitive abilities of the bees, which could lead them to the loss of many tasks, such as foraging or even nest founding by the queen.

Metamorphic acceleration following the exposure to lithium and selenium on American bullfrog tadpoles (Lithobates catesbeianus).

To regulate the presence of contaminants in Brazilian water, the Brazilian Environmental Council (CONAMA) promulgates regulations regarding the concentrations of given compounds that are supposed to be safe for aquatic life. Considering these regulations, this study tested the effects of considered safe levels of lithium (2.5 mgL-1) and selenium (0.01 mgL-1), isolated and mixed, on the American bullfrog (Lithobates catesbeianus) tadpoles. The evaluation was done through the use of biomarkers of larval development as total wet weight (TWW), snout-vent-length (SVL), hind-limb-length (HLL), activity level (AL), histologic evaluation of the thyroid gland and the mortality rate. The tadpoles were allocated into four groups (n = 20 each): a control group (CT); a group exposed to lithium (LI), a group exposed to selenium (SE), and a group exposed to both lithium and selenium (SELI). The whole assay was carried out over 21 days, with two rounds of data collection (on 7th and 21st day) to evaluate the responses over time. A statistical reduction in the AL was observed in the tadpoles from the LI and SELI groups after 7 days of exposure, the same pattern was observed after 21 days. Histological analyses of the thyroid gland showed signs of up-regulation (i.e. statistic reduction in number and area of the follicles, as well a significant reduction in the area of the gland) in all exposed groups, which represents an endocrine response as an adaptative strategy to deal with polluted aquatic environment. The stress triggered by the polluted medium is discussed.

Metabolic, immunologic, and histopathologic responses on premetamorphic American bullfrog (Lithobates catesbeianus) following exposure to lithium and selenium.

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.

Hepatotoxicity of the anionic surfactant linear alkylbenzene sulphonate (LAS) in bullfrog tadpoles.

The liver of anurans play an important role in metabolism, including detoxification, the biotransformation of molecules, and the storage of metabolites. Surfactants are part of domestic and industrial effluents. The effects of linear alkylbenzene sulfonate (LAS) on anuran liver remain unknown, however, some studies have evaluated the effects of LAS on the skin, gills, heart, testes, and liver of fishes. Here, we tested the hypothesis that LAS is hepatotoxic, promoting morphometric alterations in hepatocytes along with inflammation in the tissue, altering hepatic catabolism. We evaluated the effects of a LAS concentration that is considered environmentally safe in Brazilian inland waters on the liver of Lithobates catesbeianus tadpoles, including studies on morphology, morphometry, immunology, and metabolism. LAS exposure promoted enlargement of liver sinusoids and vacuolization of hepatocytes. Exposure to LAS also increased the area of mast cells and melanomacrophages (MMs). Additionally, LAS exposure increased hemosiderin inside MMs, suggesting alterations in the catabolism and storage of iron. Hepatocyte size increased after exposure to LAS, suggesting cytotoxic effects. Integrative analyses (i.e., morphometric, metabolic, and immunological) demonstrated hepatotoxic effects of LAS. These types of studies are key to understanding the negative effects of these substances on tadpole health, as these liver alterations impair anuran homeostasis.

Cardiac biomarkers as sensitive tools to evaluate the impact of xenobiotics on amphibians: the effects of anionic surfactant linear alkylbenzene sulfonate (LAS).

Amphibian populations have been experiencing a drastic decline worldwide. Aquatic contaminants are among the main factors responsible for this decline, especially in the aquatic environment. The linear alkylbenzene sulfonate (LAS) is of particular concern, since it represents 84% of the anionic surfactants' trade. In Brazil, the maximal LAS concentration allowed in fresh waters is 0.5mgL-1, but its potential harmful effects in amphibians remain unknown. Therefore, this study aimed to analyze the effects of a sublethal concentration of LAS (0.5mgL-1) for 96h on sensitive cardiac biomarkers of bullfrog tadpoles, Lithobates catesbeianus (Shaw, 1802). For this, we measured the activity level (AL - % of animals), in situ heart rate (fH - bpm), relative ventricular mass (RVM - % of body mass), in vitro myocardial contractility and cardiac histology of the ventricles. Tadpoles' AL and fH decreased in LAS group. In contrast, the RVM increased, as a result of a hypertrophy of the myocardium, which was corroborated by the enlargement of the nuclear measures and the increase of myocytes' diameters. These cellular effects resulted in an elevation of the in vitro contractile force of ventricle strips. Acceleration in the contraction (TPT - ms) also occurred, although no alterations in the time to relaxation (THR -ms) were observed. Therefore, it can be concluded that even when exposed to an environmentally safe concentration, this surfactant promotes several alterations in the cardiac function of bullfrog tadpoles that can impair their development, making them more susceptible to predators and less competitive in terms of reproduction success. Thus, LAS concentrations that are considered safe by Brazilian by regulatory agencies must be revised in order to minimize a drastic impact over amphibian populations. This study demonstrates the relevance of employing cardiac biomarkers at different levels (e.g., morphological, physiological and cellular) to evaluate effects of xenobiotics in tadpoles.

Hepatic effects of the clomazone herbicide in both its free form and associated with chitosan-alginate nanoparticles in bullfrog tadpoles.

The use of agrochemicals in agriculture is intense and most of them could be carried out to aquatic environment. Nevertheless, there are only few studies that assess the effects of these xenobiotics on amphibians. Clomazone is an herbicide widely used in rice fields, where amphibian species live. Thus, those species may be threatened by non-target exposure. However, nanoparticles are being developed to be used as a carrier system for the agrochemicals. Such nanoparticles release the herbicide in a modified way, and are considered to be more efficient and less harmful to the environment. The aim of this study was to comparatively evaluate the effect of clomazone in its free form and associated with nanoparticles, in the liver of bullfrog tadpoles (Lithobates catesbeianus) when submitted to acute exposure for 96 h. According to semi-quantitative analysis, there was an increase in the frequency of melanomacrophage centres, in the accumulation of eosinophils and in lipidosis in the liver of experimental groups exposed to clomazone - in its free form and associated with nanoparticles - in comparison with the control group, and the nanotoxicity of chitosan-alginate nanoparticles. The increase of melanomacrophage centres in all exposed groups was significant (P < 0.0001) in comparison to control group. Therefore, the results of this research have shown that exposure to sublethal doses of the herbicide and nanoparticles triggered hepatic responses. Moreover, these results provided important data about the effect of the clomazone herbicide and organic nanoparticles, which act as carriers of agrochemicals, on the bullfrog tadpole liver.

Cardiac adaptations of bullfrog tadpoles in response to chytrid infection.

The chytrid fungus Batrachochytrium dendrobatidis (Bd) can result in heart failure in Bd-susceptible species. Since Bd infection generally does not cause mortality in North American bullfrogs, the aim of this work was to verify whether this species presents any cardiac adaptation that could improve the tolerance to the fungus. Thus, we analyzed tadpoles' activity level, relative ventricular mass, ventricle morphology, in loco heart frequency, and in vitro cardiac function. The results indicate that infected animals present an increase in both ventricular relative mass and in myofibrils' incidence, which accompanied the increase in myocytes' diameter. Such morphological alterations enabled an increase in the in vitro twitch force that, in vivo, would result in elevation of the cardiac stroke volume. This response requires much less energy expenditure than an elevation in heart frequency, but still enables the heart to pump a higher volume of blood per minute (i.e., an increase in cardiac output). As a consequence, the energy saved in the regulation of the cardiac function of Bd-infected tadpoles can be employed in other homeostatic adjustments to avoid the lethal effect of the fungus. Whether other species present this ability, and to what extent, remains uncertain, but such possible interspecific variability might explain different mortality rates among different species of Bd-infected amphibians.