Assessment of multiple biomarkers in Lithobates catesbeianus (Anura: Ranidae) tadpoles exposed to zinc oxide nanoparticles and zinc chloride: integrating morphological and behavioral approaches to ecotoxicology.

The ecotoxicological risk to vertebrates posed by zinc oxide nanoparticles (ZnO NPs) is still poorly understood, especially in animals with a biphasic life cycle, which have aquatic and terrestrial phases, such as amphibians. In the present study, we investigated whether acute exposure (7 days) to ZnO NPs and zinc chloride (ZnCl2) at three environmentally relevant concentrations (0.1, 1.0, and 10 mg L-1) induces changes in the morphology, chondrocranium, and behavior of the tadpoles of Lithobates catesbeianus (Anura: Ranidae). Tadpoles exposed to both forms of Zn did not undergo any morphological or behavioral changes at the lowest concentrations (0.1 and 1.0 mg L-1). However, the animals exposed to the highest concentration (10 mg L-1) lacked oral disc structures, were smaller in size, had a longer tail, and presented changes in the position and coiling of the intestine and malformations of the chondrocranium in comparison with the control group. This indicates that ZnO NPs and ZnCl2 altered the development of the tadpoles, causing delays in their metamorphosis and even reducing individual fitness. The tadpoles exposed to both forms of Zn at 10 mg L-1 also had reduced mobility, especially in the presence of conspecifics. Based on these findings, we emphasize the importance of studying morphological, skeletal, and behavioral biomarkers to evaluate the toxic effects of metal-based nanoparticles in amphibians.

Microplastic ingestion induces behavioral disorders in mice: A preliminary study on the trophic transfer effects via tadpoles and fish.

In this study, the hypothesis that polyethylene microplastics (MPs) can accumulate in animals, reach the upper trophic level and trigger behavioral changes was tested. Physalaemus cuvieri tadpoles were exposed to MPs (for 7 days) and fed on tambatinga fish for the same period. Subsequently, these fish were given as food to Swiss mice. The MP amount in animals' liver was quantified and results have evidenced its accumulation at all assessed trophic levels [tadpole: 18,201.9 particles/g; fish: 1.26 particles/g; mice receiving tambatingas who had fed on tadpoles exposed to MPs: 57.07 particles/g and mice receiving water added with MPs: 89.12 particles/g). Such accumulation in the last group was associated with shorter traveled distance, slower locomotion speed and higher anxiety index in the open field test. Mice receiving tambatingas who had fed on tadpoles exposed to MPs were confronted to a potential predator and showed responses similar to those of animals who had ingested water added with MPs (lack of defensive social aggregation and reduced risk assessment behavior). Thus, results have preliminarily confirmed the initial hypothesis about how MPs in water can reach terrestrial trophic levels and have negative impact on the survival of these animals.

Environmental impacts of COVID-19 treatment: Toxicological evaluation of azithromycin and hydroxychloroquine in adult zebrafish.

One of the most impact issues in recent years refers to the COVID-19 pandemic, the consequences of which thousands of deaths recorded worldwide, are still inferior understood. Its impacts on the environment and aquatic biota constitute a fertile field of investigation. Thus, to predict the impact of the indiscriminate use of azithromycin (AZT) and hydroxychloroquine (HCQ) in this pandemic context, we aim to assess their toxicological risks when isolated or in combination, using zebrafish (Danio rerio) as a model system. In summary, we observed that 72 h of exposure to AZT and HCQ (alone or in binary combination, both at 2.5 µg/L) induced the reduction of total protein levels, accompanied by increased levels of thiobarbituric acid reactive substances, hydrogen peroxide, reactive oxygen species and nitrite, suggesting a REDOX imbalance and possible oxidative stress. Molecular docking analysis further supported this data by demonstrating a strong affinity of AZT and HCQ with their potential antioxidant targets (catalase and superoxide dismutase). In the protein-protein interaction network analysis, AZT showed a putative interaction with different cytochrome P450 molecules, while HCQ demonstrated interaction with caspase-3. The functional enrichment analysis also demonstrated diverse biological processes and molecular mechanisms related to the maintenance of REDOX homeostasis. Moreover, we also demonstrated an increase in the AChE activity followed by a reduction in the neuromasts of the head when zebrafish were exposed to the mixture AZT + HCQ. These data suggest a neurotoxic effect of the drugs. Altogether, our study demonstrated that short exposure to AZT, HCQ or their mixture induced physiological alterations in adult zebrafish. These effects can compromise the health of these animals, suggesting that the increase of AZT and HCQ due to COVID-19 pandemic can negatively impact freshwater ecosystems.

Toxicity and trophic transfer of polyethylene microplastics from Poecilia reticulata to Danio rerio.

The potential transfer of microplastics (MPs) between vertebrates belonging to the same taxonomic group, and the impact of such a transfer on higher trophic levels remains little explored. An experimental food chain with two fish species was installed to test the hypothesis that polyethylene MPs (PE MPs) can accumulate in animals and cause behavioral, mutagenic and cytotoxic changes at upper trophic levels. Poecilia reticulata fry were exposed to MPs for 48 h and, subsequently, offered (as food) to Danio rerio adults for 10 days to simulate an upper level food chain. PE MPs quantification in fry and in different Danio rerio tissues evidenced their accumulation at the two assessed trophic levels. This finding suggested their absorption, adherence and translocation from one organism to another. The accumulation seen in D. rerio directly exposed to MPs was associated with behavioral disorders at upper trophic level. These animals presented behavior suggestive of anti-predatory response deficit when they were confronted with a potential aquatic predator (Geophagus brasiliensis). This finding was inferred through lower school cohesion, shallower school depth and shorter distance from the potential predator. In addition, animals exposed to MPs recorded higher nuclear abnormality rates and changes in the size and shape of erythrocytes and in their nuclei; this outcome has suggested mutagenic and cytotoxic effects, respectively. Based on the current results, MPs are transferred through a food chain that only involves two vertebrates. MPs enter the vertebrates' organs, change their behavior and induce mutagenic and cytotoxic processes in animals, which can cause significant ecological consequences in freshwater ecosystems.

How much are microplastics harmful to the health of amphibians? A study with pristine polyethylene microplastics and Physalaemus cuvieri.

Microplastics (MPs) are critical emerging pollutants found in the environment worldwide; however, its toxicity in aquatic in amphibians, is poorly known. Thus, the aim of the present study is to assess the toxicological potential of polyethylene microplastics (PE MPs) in Physalaemus cuvieri tadpoles. According to the results, tadpoles' exposure to MP PE at concentration 60 mg/L for 7 days led to mutagenic effects, which were evidenced by the increased number of abnormalities observed in nuclear erythrocytes. The small size of erythrocytes and their nuclei area, perimeter, width, length, and radius, as well as the lower nucleus/cytoplasm ratio observed in tadpoles exposed to PE MPs confirmed its cytotoxicity. External morphological changes observed in the animal models included reduced ratio between total length and mouth-cloaca distance, caudal length, ocular area, mouth area, among others. PE MPs increased the number of melanophores in the skin and pigmentation rate in the assessed areas. Finally, PE MPs were found in gills, gastrointestinal tract, liver, muscle tissues of the tail and in the blood, a fact that confirmed MP accumulation by tadpoles. Therefore, the present study pioneering evidenced how MPs can affect the health of amphibians.

Do predictive environmentally relevant concentrations of ZnO nanoparticles induce antipredator behavioral response deficit in Swiss mice?

The toxicity of zinc oxide nanoparticles (ZnO NPs) has been addressed in several studies; however, their effect on the mammalian group, even at environmentally relevant concentrations, remains poorly understood. The aims of the present study are to expose female Swiss mice to ZnO NP concentrations commonly faced by mammals who enter aquatic systems to perform different ecological functions and to assess the possible effects of such particles on their behavior. The test animals were placed in water added with ZnO NPs for 3 min, 2 times/day, for 21 days. Two experimental groups were set, NP1x, composed of animals subjected to ZnO NP concentration of 760 µg/L; and NP50x (control), which encompassed animals subjected to 38,000 µg/L. Based on field test results (OF), the contact with NPs did not induce locomotor deficits or anxiogenic and anxiolytic effect on the animal models. However, models exposed to NPs were not able to recognize the predatory threat posed by the presence of Pantherophis guttatus and Arapaima gigas; on the other hand, animals in the control group, who were not exposed to ZnO NPs, did not present antipredator behavioral response deficit. Furthermore, mice exposed to NPs were unable to distinguish real predators from plastic copies, and it suggests antipredator behavioral response deficit. High Zn concentrations in blood, liver, brain and skin samples are associated with deficit caused by the exposure to ZnO NPs. To the best of our knowledge, the current study is in the first to evidence that ZnO NPs induce changes in antipredator behavioral responses, even under ephemeral conditions and at low concentrations. However, the exposure to ZnO NPs can be a risk to the health of the assessed individuals and to the dynamics of their populations if the present antipredator behavioral response test results are extrapolated to the ecological context.

Developmental toxicity in zebrafish exposed to polyethylene microplastics under static and semi-static aquatic systems.

Different studies have reported the ecotoxicological effects of polyethylene microplastics (PE MPs) on aquatic organisms; however, little is known about their toxicity in the early life stages of aquatic vertebrates living in freshwater ecosystems. Thus, the aim of the current study is to evaluate the toxicity of PE MPs throughout the development of Danio rerio after their static and semi-static exposure to different concentrations of these pollutants (6.2, 12.5, 25, 50 and 100 mg/L) - models were monitored at different time-periods, namely: 24, 48, 72, 96, 120 and 144 h. Based on the collected data, small PE MP concentrations have harmful effects on D. rerio embryos and larvae; the magnitude and characteristics of these effects depend on the adopted exposure system, which can be static or semi-static. PE MPs had negative effect on embryos' hatching rate in both exposure systems. However, the early hatching observed during the exposure through the static system could explain the lower larval survival rate after egg hatching. Nevertheless, PE MPs induced significant changes in various morphometric parameters. The present study is the first to assess the addressed topic; therefore, it is recommended to carry out future investigations to broaden the knowledge about PE MP toxicity.

How leachates from wasted cigarette butts influence aquatic life? A case study on freshwater mussel Anodontites trapesiali.

There are several reports on the damage smoking causes to human health available in the literature, but little is known about the environmental and biological consequences from inappropriate cigarette butt (CB) disposal in urban and natural environments. The immunotoxic, morphotoxic and mutagenic potential of leachates from cigarette butts (LCB) diluted at environmentally relevant rates (LCB1x: 1.375%; LCB10x: 13.75%) was evaluated in adult representatives of the bivalve species Anodontites trapesialis, which was adopted as model organism. Type II hyalinocytes and granulocytes (phagocytic cells) frequency increased in the hemolymph of subjects exposed to the pollutant for 14 days. Based on this outcome, LCB chemical constituents did not induce immunotoxic effects. The treatments also did not seem to have any impact on the subjects' hemocitary morphometry parameters: diameter, area, perimeter, circularity and nucleus - cytoplasm ratio. However, subjects in groups LCB1x and LCB10x recorded a larger number of hyalinocytes with some nuclear abnormality such as micronucleus, blebbed nucleus, asymmetric constriction nucleus, and nuclear multilobulation and binucleation. The association between these abnormalities and the treatments was confirmed by the Cr, Ni, Pb, Zn, Mn and Na bioaccumulation in tissue samples of the bivalve models exposed to LCB. To the best of our knowledge, this is the first report on LCB mutagenicity in representatives of a freshwater bivalve group. Given the chemical complexity of the addressed pollutants, it is imperative to develop further investigations about the topic.

Sub-lethal effects induced by a mixture of different pharmaceutical drugs in predicted environmentally relevant concentrations on Lithobates catesbeianus (Shaw, 1802) (Anura, ranidae) tadpoles.

The increasing consumption of medications by humans has negative effects such as the increased disposal of these compounds in the environment. Little is known about how the disposal of a "drug mix" (DM) in aquatic ecosystems can affect their biota. Thus, we evaluated whether the exposure of Lithobates casteibeianus tadpoles to a DM composed of different medication classes (antibiotic, anti-inflammatory, antidepressant, anxiolytic, analgesic, and antacid drugs)-at environmentally relevant concentrations-may change their oral morphology, trigger behavioral disorders, and have mutagenic effects on erythrocyte cells. Based on our data, animals exposed to the DM showed changes in mandibular sheath pigmentation, dentition, and swimming activity, as well as atypical behavior in the social aggregation test [with co-specific and interspecific (Physalaemus cuvieri) individuals] and antipredatory defensive response deficit (chemical stimulus from Odonata larvae), after 15 exposure days. The mutagenic analysis revealed higher frequency of nuclear abnormalities in the erythrocytes of tadpoles exposed to the DM (e.g., multilobulated, blebbed, kidney-shaped, notched nucleus, binuclear, and micronucleated erythrocytes). Given the chemical complexity of the DM, we assumed that several organic functions may have been affected, either by the isolated, synergistic, antagonistic, or additive action of DM compounds. Finally, our study confirms the toxicological potential of DM in L. catesbeianus tadpoles, with emphasis to impacts that can affect the fitness of individuals and their natural populations. Thus, we suggest that more attention should be given to the disposal of medications in the environment and reinforce the need of improving water and sewage treatment systems.

Depression, anxiety-like behavior, and memory impairment in mice exposed to chitosan-coated zein nanoparticles.

The advent of biotechnology provided the synthesis of nanoproducts with diverse applications in the field of medicine, agriculture, food, among others. However, the toxicity of many nanoparticles (NP) currently used, which can penetrate natural systems and impact organisms, is not known. Thus, in this study, we evaluated whether the short exposure (5 days) to low concentrations of chitosan-coated zein nanoparticles (ZNP-CS) (0.2 ng/kg, 40 ng/kg, and 400.00 ng/kg) was capable of causing behavioral alterations compatible with cognitive deficit, as well as anxiety and depression-like behavior in Swiss mice. However, we observed an anxiogenic effect in the animals exposed to the highest ZNP-CS concentration (400.00 ng/kg), without locomotor alterations suggestive of sedation or hyperactivity in the elevated plus maze (EPM) test. We also observed that the ZNP-CS caused depressive-like behavior, indicated by the longer immobile time in the tail suspension test and the animals exposed to ZNP-CS presented deficit in recognition of the new object, not related to locomotor alteration in this test. To the best of our knowledge, this is the first report of the neurotoxicity of ZNP in a mammal animal model, contributing to the biological safety assessment of these nanocomposites.

Behavioral toxicity of tannery effluent in zebrafish (Danio rerio) used as model system.

The ecotoxicity of untreated tannery effluent (UTE) in several animal models has been reported; however, its effects on fish behavior, and neurotoxicity, remain unknown. Thus, the hypothesis that the chronic exposure to UTE can induce behavioral changes in adult zebrafish (Danio rerio) representatives, even when it is highly diluted in water, was tested. Animals exposed to 0.1% and 0.3% UTE for 30 days showed behavioral changes in visual social preference tests through their co-specific and antipredator defensive responses, which had indicated neurotoxic actions. Zebrafish exposed to UTE appeared to have not co-specific preference when it is paired with Poecilia sphrenops. In addition, only animals in the control group showed aversive behavior in the presence of the herein used predatory stimulus (Oreochromis niloticus). However, Cr, Na and Mg bioaccumulation was higher in zebrafish exposed to 0.1% and 0.3% UTE, although anxiogenic and anxiolytic effects were not observed in the models exposed to UTE in the novel tank diving or aggressiveness-increase-in-the-mirror tests. This outcome allowed associating the exposure to the pollutant and bioaccumulation with the observed behavioral changes. The present study is pioneer in scientifically evidencing the sublethal impact caused by chronic exposure to UTE in experimental environment simulating realistic aquatic pollution conditions. Accordingly, results in the current research should motivate further investigations to broaden the knowledge about the real magnitude of UTE biological impacts on the aquatic biota.