Shedding light on toxicity of SARS-CoV-2 peptides in aquatic biota: A study involving neotropical mosquito larvae (Diptera: Culicidae).

Knowledge about how the COVID-19 pandemic can affect aquatic wildlife is still extremely limited, and no effect of SARS-CoV-2 or its structural constituents on invertebrate models has been reported so far. Thus, we investigated the presence of the 2019-new coronavirus in different urban wastewater samples and, later, evaluated the behavioral and biochemical effects of the exposure of Culex quinquefasciatus larvae to two SARS-CoV-2 spike protein peptides (PSPD-2002 and PSPD-2003) synthesized in our laboratory. Initially, our results show the contamination of wastewater by the new coronavirus, via RT-qPCR on the viral N1 gene. On the other hand, our study shows that short-term exposure (48 h) to a low concentration (40 µg/L) of the synthesized peptides induced changes in the locomotor and the olfactory-driven behavior of the C. quinquefascitus larvae, which were associated with increased production of ROS and AChE activity (cholinesterase effect). To our knowledge, this is the first study that reports the indirect effects of the COVID-19 pandemic on the larval phase of a freshwater invertebrate species. The results raise concerns at the ecological level where the observed biological effects may lead to drastic consequences.

Green toxicology approach involving polylactic acid biomicroplastics and neotropical tadpoles: (Eco)toxicological safety or environmental hazard?

Different and alternative renewable-source materials, commonly called bioplastics, have been proposed due to the high production and consumption of petroleum-derived plastics and to their high toxicity in the biota. However, their toxicological safety has not yet been assessed in a comprehensive way; therefore, their effects on several animal groups remain completely unknown. Thus, we aimed at testing the following hypothesis: the exposure of Physalaemus cuvieri tadpoles to polylaic acid biomicroplastic (PLA BioMP) at environmentally relevant concentrations (760 and 15,020 µg/L) induces physiological changes in them. Based on the collected data, biopolymer uptake changed tadpoles' growth and development features, reduced their lipid reserves (it was inferred by decreased triglyceride levels), as well as increased reactive oxygen and nitric oxide species production after 14-day exposure. The proportional increase in total glutathione levels, and in superoxide dismutase and catalase activity, was not enough to counterbalance the production of reactive species. In addition, the two tested concentrations caused cholinesterase effect, which was marked by increased acetylcholinesterase and butyrylcholinesterase. This finding is indicative of the neurotoxic action of PLA BioMP. To the best of our knowledge, this is the first report on the harmful consequences of exposing amphibian representatives to the herein tested biopolymers. Therefore, this finding encourages further studies and contributes to demystify the idea that bioplastics are "harmless" to the aquatic biota in freshwater environments.

Polyethylene glycol acute and sub-lethal toxicity in neotropical Physalaemus cuvieri tadpoles (Anura, Leptodactylidae).

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.

Can use of hydroxychloroquine and azithromycin as a treatment of COVID-19 affect aquatic wildlife? A study conducted with neotropical tadpole.

The impacts on human health and the economic and social disruption caused by the pandemic COVID-19 have been devastating. However, its environmental consequences are poorly understood. Thus, to assess whether COVID-19 therapy based on the use of azithromycin (AZT) and hydroxychloroquine (HCQ) during the pandemic affects wild aquatic life, we exposed (for 72 h) neotropical tadpoles of the species Physalaemus cuvieri to the water containing these drugs to 12.5 µg/L. We observed that the increase in superoxide dismutase and catalase in tadpoles exposed to AZT (alone or in combination with HCQ) was predominant to keep the production of NO, ROS, TBARS and H2O2 equitable between the experimental groups. In addition, the uptake of AZT and the strong interaction of AZT with acetylcholinesterase (AChE), predicted by the molecular docking analysis, were associated with the anticholinesterase effect observed in the groups exposed to the antibiotic. However, the unexpected increase in butyrylcholinesterase (BChE) in these same groups suggests its constitutive role in maintaining cholinergic homeostasis. Therefore, taken together, our data provide a pioneering evidence that the exposure of P. cuvieri tadpoles to AZT (alone or in combination with HCQ) in a predictably increased environmental concentration (12.5 µg/L) elicits a compensatory adaptive response that can have, in the short period of exposure, guaranteed the survival of the animals. However, the high energy cost for maintaining physiological homeostasis, can compromise the growth and development of animals and, therefore, in the medium-long term, have a general negative effect on the health of animals. Thus, it is possible that COVID-19 therapy, based on the use of AZT, affects wild aquatic life, which requires greater attention to the impacts that this drug may represent.

Can carbon nanofibers affect anurofauna? Study involving neotropical Physalaemus cuvieri (Fitzinger, 1826) tadpoles.

Although carbon nanotubes' (CNTs) toxicity in different experimental systems (in vivo and in vitro) is known, little is known about the toxic effects of carbon nanofibers (CNFs) on aquatic vertebrates. We herein investigated the potential impact of CNFs (1 and 10 mg/L) by using Physalaemus cuvieri tadpoles as experimental model. CNFs were able to induce nutritional deficit in animals after 48-h exposure to them, and this finding was inferred by reductions observed in body concentrations of total soluble carbohydrates, total proteins, and triglycerides. The increased production of hydrogen peroxide, reactive oxygen species and thiobarbituric acid reactive substances in tadpoles exposed to CNFs has suggested REDOX homeostasis change into oxidative stress. This process was correlated to the largest number of apoptotic and necrotic cells in the blood of these animals. On the other hand, the increased superoxide dismutase and catalase activity has suggested that the antioxidant system of animals exposed to CNFs was not enough to maintain REDOX balance. In addition, CNFs induced increase in acetylcholinesterase and butyrylcholinesterase activity, as well as changes in the number of neuromasts evaluated on body surface (which is indicative of the neurotoxic effect of nanomaterials on the assessed model system). To the best of our knowledge, this is the first report on the impact of CNFs on amphibians; therefore, it broadened our understanding about ecotoxicological risks associated with their dispersion in freshwater ecosystems and possible contribution to the decline in the populations of anurofauna species.

Toxicity of polystyrene nanoplastics and zinc oxide to mice.

The toxicity of zinc oxide (ZnO NPs) and polystyrene nanoplastics (PS NaPs) has been tested in different animal models; however, knowledge about their impact on mice remains incipient. The aim of the current study is to evaluate the effects of these nanomaterials on Swiss mice after their individual exposure to a binary combination of them. The goal was to investigate whether short exposure (three days) to an environmentally relevant dose (14.6 ng/kg, i.p.) of these pollutants would have neurotoxic, biochemical and genotoxic effects on the modelss. Data in the current study have shown that the individual exposure of these animals has led to cognitive impairment based on the object recognition test, although the exposure experiment did not cause locomotor and anxiogenic or anxiolitic-like behavioral changes in them. This outcome was associated with increased nitric oxide levels, thiobarbituric acid reactive species, reduction in acetylcholinesterase activity and with the accumulation of nanomaterials in their brains. Results recorded for the assessed parameters did not differ between the control group and the groups exposed to the binary combination of pollutants. However, both the individual and the combined exposures caused erythrocyte DNA damages associated with hypercholesterolemic and hypertriglyceridemic conditions due to the presence of nanomaterials. Based on the results, the toxicological potential of ZnO NPs and PS NaPs in the models was confirmed and it encouraged further in-depth investigations about factors explaining the lack of additive or synergistic effect caused by the combined exposure to the assessed pollutants.

Nanopolystyrene particles at environmentally relevant concentrations causes behavioral and biochemical changes in juvenile grass carp (Ctenopharyngodon idella).

The biometric, behavioral and biochemical toxicity of polystyrene nanoplastics (PS NPs) in aquatic freshwater vertebrates and in environmentally relevant concentrations remains poorly known. Thus, using different toxicity biomarkers we tested the hypothesis that the exposure of Ctenopharyngodon idella juveniles to small PS NPs concentrations (0.04 ng/L, 34 ng/L and 34 µg/L), for a short period-of-time, may affect their growth/development, individual and collective behavior, and biochemical parameters. Animals exposed to NPs did not show increased biometric parameters (i.e.: body biomass, total and standard length, peduncle height, head height and visceral somatic and hepatosomatic indices). Despite the lack of damage on the locomotor (open field test) and visual (visual stimulus test) abilities of the evaluated fish, the expected increase in locomotor activity during the vibratory stimulus test was not evident in animals exposed to NPs. Non-exposed animals were the only ones showing increased activity/locomotion time in the presence of the predatory stimulus during the individual anti-predatory response test. The behavior of animals directly confronted with a potential predator has evidenced the influence of NPs on shoals' aggregation and on the distance kept by individuals from the predatory stimulus. These changes were associated with PS NPs accumulation in animals' brains, oxidative stress and increased acetylcholinesterase activity (hepatic and cerebral). Therefore, the current study has confirmed the initial hypothesis and showed that, even at low concentrations, PS NPs can affect the health of C. idella individuals at early life stage.

Effects of polystyrene nanoplastics on Ctenopharyngodon idella (grass carp) after individual and combined exposure with zinc oxide nanoparticles.

The toxicity of polystyrene nanoparticles (PS NPs) and ZnO nanoparticles (ZnO NPs), in combination is poorly known. Thus, the aim of the current study was to evaluate the effects of PS NPs (760 µg/L) on Ctenopharyngodon idella exposed to it, both in separate and in combination with ZnO NPs (760 µg/L), based on behavioral, biochemical and genotoxic biomarkers. Current data have indicated that PS NPs, for a short exposure period (3 days), both in separate and in combination with nanoparticles, have affected animals' response to the mirror test. On the other hand, all treatments have equally induced C. idella inactivity towards alarm substances and DNA damage. There was increased oxidative stress, mainly in groups exposed to PS NPs (in combination, or not, with nanoparticles); although increased, the evaluated antioxidant levels did not appear to be enough to inhibit the effects of treatment-induced production of free radicals. Together, these results are likely co-responsible for the observed changes. The current study did not observe antagonistic, synergistic or additive effect on animals exposed to the combination between PS NPs and ZnO NPs; however, this outcome should not discourage the performance of similar studies focused on assessing the (eco)toxicity of pollutant mixtures comprising nanomaterials.

Toxicity of polystyrene nanoplastics in Ctenopharyngodon idella juveniles: A genotoxic, mutagenic and cytotoxic perspective.

The increased contamination of surface water with plastic waste is proportional to the increased consumption of products that use them as raw material. However, the impact of these residues on aquatic biota remains limited, mainly when it comes to nanoplastics (NPs). Thus, the aim of the current study is to test the hypothesis that the exposure of Ctenopharyngodon idella juveniles to polystyrene nanoplastics (PS NPs) at low concentrations (0.04 ng/L, 34 ng/L and 34 µg/L), for 20 days, leads to DNA damage and has mutagenic and cytotoxic effects on their erythrocytes. Comet assay enabled observing that DNA damage (inferred from the greater tail length, DNA percentage in the tail and Olive tail moment) induced by PS NPs has increased as the pollutant concentrations have increased, as well as that the formation of micronuclei and other nuclear abnormalities was equitable in animals exposed to this pollutant. On the other hand, there were significant changes in erythrocyte shape and size, oxidative stress generation (NO levels, lipid peroxidation, hydrogen peroxide), antioxidant system inhibition (mediated by total hepatic glutathione) and PS NPs accumulation in the liver and brain of animals exposed to higher concentrations of it. Therefore, the current study has confirmed the initial hypothesis and enhanced the knowledge about the genotoxic, mutagenic and cytotoxic potential of PS NPs in freshwater fish at early developmental stage, relating these effects to biochemical changes and significant accumulation of these nanomaterials. Besides, it is a warning about the (eco) toxicological risk represented by these nanopollutants in aquatic environments. CAPSULE: Polystyrene nanoplastics are capable of inducing DNA damage, mutagenic and cytotoxicity changes in fish.

Behavioral and biochemical consequences of Danio rerio larvae exposure to polylactic acid bioplastic.

The literature has largely shown the toxicity of petroleum-based PLA biomicroplastics (PLABioMPs) and encouraged the production of alternative materials to replace their use, such as biopolymers. However, knowledge concerning the effects of biopolymers on aquatic organisms remains under development. The hypothesis that the acute exposure (five days) to polylactic acid (PLA) biopolymers may lead to behavioral and biochemical changes and to their accumulation in Danio rerio larvae was tested. Based on the results, PLA biomicroplastics (PLA BioMPs) at concentration of 3 and 9 mg/L decreased swimming distance and speed of larvae in the open field test. This outcome suggests effects on animals' locomotor and exploration activities. Larvae's longer immobility time and greater permanence in the peripheral zone of the apparatus is indicative of anxiety-like behavior caused by the exposure to PLA BioMPs. Zebrafish larvae accumulated PLA BioMPs and their acetylcholinesterase activity was inhibited by their presence, which reinforces the accumulative potential of biopolymers and their direct or indirect role as anxiogenic agents, even at sublethal concentrations. The decreased activity of acetylcholinesterase reinforces the neurotoxic action in groups exposed to PLA BioMPs. The current study has confirmed the initial hypothesis and is an insight about the toxicity of these biopolymers in D. rerio larvae, since it deepens the discussion about the environmental risk of these substances in freshwater ecosystems.

Ingestion of tannery effluent as a risk factor to the health of birds: A toxicological study using Coturnix coturnix japonica as a model system.

Tannery industries generate large amounts of tannery effluents (TE), which have been considered highly toxic to various groups of animals. However, the identification and characterization of the impact of this mix of pollutants on birds is still highly incipient. So, our goal was to evaluate the possible biological changes of Coturnix coturnix japonica, exposed for 45 days, to different dilutions of TE (1.4%, 3.1% and 6.5%), using behavioural biomarkers, mutagenics and egg production. When submitted to the behavioural tests, quails that ingested TE presented behaviour compatible with an anxiolytic effect in the open field test; absence of emotional reactivity in the object recognition test; reduced rates of predation of Tenebrio molitor larvae (potential prey); as well as an anti-predatory defensive response deficit when confronted, especially with Felis catus males (potential predator). In addition, we observed increased biomass of the liver, increased feed conversion index and lower feed efficiency index; mutagenic effect of TE (inferred by the increase of nuclear erythrocyte abnormalities); reduced productive performance and egg quality, in addition to different staining patterns of the eggs produced by quails from the control group. Therefore, our study confirms the toxicity of TE in C. coturnix japonica, even in small dilutions. While behavioural changes demonstrate the neurotoxic potential of the pollutant, the other alterations suggest that the mechanisms of action of its chemical constituents are not selective, that is, they act systemically, acting synergistic, antagonistic or additively, causing harmful effects in animals.

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.

The intake of water containing a mix of pollutants at environmentally relevant concentrations leads to defensive response deficit in male C57Bl/6J mice.

Previous studies have individually confirmed the toxic effects from different pollutants on mammals. However, effects resulting from the exposure of these animals to multi-pollutant mixes have not been studied so far. Thus, the aim of the current study is to assess the effect from the chronic exposure (105days) of C57Bl/6J mice to a mix of pollutants on their response to potential predators. In order to do so, the following groups were formed: "control", "Mix 1× [compounds from 15 pollutants identified in surface waters at environmentally relevant concentration (ERC)]", "Mix 10×" and "Mix 25×" (concentrations 10 and 25 times higher than the ERC). From the 100th experimental day on, the animals were subjected to tests in order to investigate whether they showed locomotor, visual, olfactory and auditory changes, since these abilities are essential to their anti-predatory behavior. Next, the animals' behavior towards potential predators (Felis catus and Pantherophis guttatus) was assessed. The herein collected data did not show defensive response from any of the experimental groups to the predatory stimulus provided by P. guttatus. However, the control animals, only, presented anti-predatory behavior when F. catus was introduced in the apparatus, fact that suggests defensive response deficit resulting from the treatments. Thus, the current study is pioneer in showing that the chronic intake of water containing a mix of pollutants (even at low concentrations) leads to behavioral disorders able to affect the survival and population dynamics of mammalian species at ecological level.

The exposure to water with cigarette residue changes the anti-predator response in female Swiss albino mice.

Recent studies have shown that cigarette consumption affects much more than human health. Smoked cigarette butt (SCB) disposal into the environment can bring little-known negative biological consequences to mammals, since it contains many organic and inorganic toxic chemical constituents. Thus, we aim at assessing whether the ingestion of water with leached SCB for 60 days by female Swiss mice changes their defensive behavioral response to potential predators (cats and snakes). We worked with the following groups of animals: control (pollutant-free water), water with environmental concentration of SCB (1.9 µg/L of nicotine), and concentration 1000 times higher (EC1000×). Our data show that the treatments did not cause locomotor, visual, auditory, and olfactory deficit in the animals. However, we observed that the animals exposed to the pollutants did not present behavioral differences in the test session with or without the snake. On the other hand, animals in all groups showed defensive behavior when the test was conducted with the cat in the apparatus. However, female mice presented weaker response than the control. Thus, our data point towards the potential neurotoxic damage caused to mice who have ingested water with SCB residues, even at low concentrations.

Short-term social memory deficits in adult female mice exposed to tannery effluent and possible mechanism of action.

The accumulated organic residues in tannery-plant courtyards are an eating attraction to small rodents; however, the contact of these animals with these residues may change their social behavior. Thus, the aim of the present study is to investigate whether the exposure to tannery effluent (TE) can damage the social recognition memory of female Swiss mice, as well as to assess whether vitamin C supplementation could provide information about how TE constituents can damage these animals' memory. We have observed that resident females exposed to TE (without vitamin supplementation) did not explore the anogenital region, their body or chased intruding females for shorter time or with lower frequency during the retest session of the social recognition test, fact that indicates social recognition memory deficit in these animals. Such finding is reinforced by the confirmation that there was no change in the animals' olfactory function during the buried food test, or locomotor changes in females exposed to the pollutant. Since no behavioral change was observed in the females exposed to TE and treated with vitamin C (before or after the exposure), it is possible saying that these social cognitive impairments seem to be directly related to the imbalance between the cellular production of reactive oxygen species and the counteracting antioxidant mechanisms (oxidative stress) in female mice exposed to the pollutant (without vitamin supplementation). Therefore, the present study evidences that the direct contact with tannery effluent, even for a short period-of-time, may cause short-term social memory deficits in adult female Swiss mice.

Protective effect of vitamin C in female Swiss mice dermally-exposed to the tannery effluent.

Previous studies involving the oral exposure of mice to tannery effluents have found neurotoxic effects. However, studies about the effects the dermal exposure to pollutant have on the cognitive function of females have not been found in the literature. Thus, the aim of the current study is to investigate whether the dermal exposure of female Swiss mice to tannery effluents (2 h/day for 20 days) can cause cognitive impairment, as it was already evidenced in male Swiss mice. Furthermore, based on the administration of vitamin C (before or after the exposure to the xenobiotic), the current study also aims to assess the protective effect of vitamin C in female Swiss mice dermally-exposed to the tannery effluent. Female Swiss mice exposed to the tannery effluent (without vitamin supplementation) have shown lower novel object recognition index during the test session of the novel object recognition task, and they have descended significantly faster from the inhibitory avoidance platform when they were compared to mice belonging to the other groups, therefore evidencing memory deficit. However, the test performance of females receiving vitamin C was similar to that of control animals. Thus, the current study confirms the initial hypothesis that the dermal exposure to the pollutant, even for a short period, causes cognitive deficit in female Swiss mice. The herein presented findings also provide evidence that the mechanisms of action of the tannery effluent in these animals are related to oxidative damages in specific brain regions directed to the formation of short memory to perform aversive and object recognition tasks.