Exposure of adult zebrafish (Danio rerio) to SARS-CoV-2 at predicted environmentally relevant concentrations: Outspreading warns about ecotoxicological risks to freshwater fish.

While the multifaceted social, economic, and public health impacts associated with the COVID-19 pandemic are known, little is known about its effects on non-target aquatic ecosystems and organisms. Thus, we aimed to evaluate the potential ecotoxicity of SARS-CoV-2 lysate protein (SARS.CoV2/SP02.2020.HIAE.Br) in adult zebrafish (Danio rerio) at predicted environmentally relevant concentrations (0.742 and 2.226 pg/L), by 30 days. Although our data did not show locomotor alterations or anxiety-like or/and anxiolytic-like behavior, we noticed that exposure to SARS-CoV-2 negatively affected habituation memory and social aggregation of animals in response to a potential aquatic predator (Geophagus brasiliensis). An increased frequency of erythrocyte nuclear abnormalities was also observed in animals exposed to SARS-CoV-2. Furthermore, our data suggest that such changes were associated with a redox imbalance [↑ROS (reactive oxygen species), ↑H2O2 (hydrogen peroxide), ↓SOD (superoxide dismutase), and ↓CAT (catalase)], cholinesterasic effect [↑AChE (acetylcholinesterase) activity], as well as the induction of an inflammatory immune response [↑NO (nitric oxide), ↑IFN-γ (interferon-gamma), and ↓IL-10 (interleukin-10)]. For some biomarkers, we noticed that the response of the animals to the treatments was not concentration-dependent. However, principal component analysis (PCA) and the "Integrated Biomarker Response" index (IBRv2) indicated a more prominent ecotoxicity of SARS-CoV-2 at 2.226 pg/L. Therefore, our study advances knowledge about the ecotoxicological potential of SARS-CoV-2 and reinforces the presumption that the COVID-19 pandemic has negative implications beyond its economic, social, and public health impacts.

Multiple endpoints of polyethylene microplastics toxicity in vascular plants of freshwater ecosystems: A study involving Salvinia auriculata (Salviniaceae).

More recently, the number of studies on the impacts of microplastics (MPs) on plants has drawn attention considerably. However, many of these studies focused on terrestrial plants, with vascular plants from freshwater ecosystems being little studied. Thus, we aimed to evaluate the possible effects of exposure of Salvinia auriculata, for 28 days, to different concentrations of polyethylene MPs (PE MPs - diameter: 35.46 ± 18.17 µm) (2.7 ×108 and 8.1 ×108 particles/m3), using different biomarkers. Our data indicated that exposure to PE MPs caused alterations in plant growth/development (inferred by the lower floating frond number, "root" length, and the number of "roots"), as well as lower dispersion of individuals in the experimental units. Plants exposed to PE MPs also showed lower epidermal thickness (abaxial leaf face) and a longer length of the central leaf vein and vascular bundle area. Ultrastructural analyses of S. auriculata exposed to MPs revealed rupture of some epidermal cells and trichomes on the adaxial and abaxial, leaf necrosis, and chlorosis. In the "roots", we observed dehydrated filamentous structures with evident deformations in plants exposed to the pollutants. Both on the abaxial leaf face and on the "roots", the adherence of PE MPs was observed. Furthermore, exposure to PE MPs induced lower chlorophyll content, cell membrane damage, and redox imbalance, marked by reduced catalase and superoxide dismutase activity and increased production of reactive oxygen and nitrogen species as well as malondialdehyde. However, in general, we did not observe the dose-response effect for the evaluated biomarkers. The values of the integrated biomarker response index, the principal component analysis (PCA) results and the hierarchical clustering analysis confirmed the similarity between the responses of plants exposed to different PE MPs concentrations. Therefore, our study sheds light on how PE MPs can affect S. auriculata and reinforces that putting these pollutants in freshwater environments might be hazardous from an ecotoxicological point of view.

Plant-ZnO nanoparticles interaction: An approach to improve guinea grass (Panicum maximum) productivity and evaluation of the impacts of its ingestion by freshwater teleost fish.

We aimed to evaluate the possible effects of the application of zinc oxide nanoparticles [ZnO NPs; 68.96 ± 33.71 nm; at 100 and 500 mg/kg in a soil mixture of the Typic Dystrophic Red Latosol type and sand (2:1 ratio)] in the cultivation of Panicum maximum (until 125 days), using different biomarkers in addition to evaluating the uptake of Zn by the plants. Furthermore, we assessed the possible transfer of ZnO NPs from P. maximum leaves to zebrafish and their potential. Plants cultivated in substrates with ZnO NPs at 500 mg/kg showed reduced germination rate and growth. However, at 100 mg/kg, plants showed higher biomass and productivity, associated with higher Zn uptake, without inducing oxidative and nitrosative stress. Zinc content in zebrafish was not associated with ingesting leaves of P. maximum cultivated in substrate containing ZnCl2 or ZnO NPs or with genotoxic, mutagenic, and biochemical effects. In conclusion, ZnO NPs (at 100 mg/kg) are promising in the cultivation of P. maximum, and their ingestion by zebrafish did not cause changes in the evaluated biomarkers. However, we recommend that studies with other animal models be conducted to comprehensively assess the ecotoxicological hazard associated with applying ZnO NPs in soil.

Toxicity assessment of SARS-CoV-2-derived peptides in combination with a mix of pollutants on zebrafish adults: A perspective study of behavioral, biometric, mutagenic, and biochemical toxicity.

The dispersion of SARS-CoV-2 in aquatic environments via the discharge of domestic and hospital sewage has been confirmed in different locations. Thus, we aimed to evaluate the possible impacts of zebrafish (Danio rerio) exposure to SARS-CoV-2 peptide fragments (PSPD-2001, 2002, and 2003) alone and combined with a mix of emerging pollutants. Our data did not reveal the induction of behavioral, biometric, or mutagenic changes. But we noticed an organ-dependent biochemical response. While nitric oxide and malondialdehyde production in the brain, gills, and muscle did not differ between groups, superoxide dismutase activity was reduced in the "PSPD", "Mix", and "Mix+PSPD" groups. An increase in catalase activity and a reduction in DPPH radical scavenging activity were observed in the brains of animals exposed to the treatments. However, the "Mix+PSPD" group had a higher IBRv2 value, with NO levels (brain), the reduction of acetylcholinesterase activity (muscles), and the DPPH radical scavenging activity (brain and muscles), the most discriminant factors for this group. The principal component analysis (PCA) and hierarchical clustering analysis indicated a clear separation of the "Mix+PSPD" group from the others. Thus, we conclude that exposure to viral fragments, associated with the mix of pollutants, induced more significant toxicity in zebrafish adults than in others.

Exposure to polystyrene nanoplastics induces an anxiolytic-like effect, changes in antipredator defensive response, and DNA damage in Swiss mice.

Although the in vivo toxicity of nanoplastics (NPs) has already been reported in different model systems, their effects on mammalian behavior are poorly understood. Thus, we aimed to evaluate whether exposure to polystyrene (PS) NPs (diameter: 23.03 ± 0.266 nm) alters the behavior (locomotor, anxiety-like and antipredator) of male Swiss mice, induces brain antioxidant activity, and erythrocyte DNA damage. For this, the animals were exposed to NPs for 20 days at different doses (6.5 ng/kg and 6500 ng/kg). Initially, we did not observe any effect of pollutants on the locomotor activity of the animals (inferred via open field test and Basso mouse scale for locomotion). However, we noticed an anxiolytic-like behavior (in the open field test) and alterations in the antipredatory defensive response of mice exposed to PS NPs, when confronted with their predator potential (snake, Pantherophis guttatus). Furthermore, such changes were associated with suppressing brain antioxidant activity, inferred by lower DPPH radical scavenging activity, reduced total glutathione content, as well as the translocation and accumulation of NPs in the brain of the animals. In addition, we noted that the treatments induced DNA damage, evaluated via a single-cell gel electrophoresis assay (comet assay) applied to circulating erythrocytes of the animals. However, we did not observe a dose-response effect for all biomarkers evaluated and the estimated accumulation of PS NPs in the brain. The values of the integrated biomarker response index and the results of the principal component analysis (PCA) and the hierarchical clustering analysis confirmed the similarity between the responses of animals exposed to different doses of PS NPs. Therefore, our study sheds light on how PS NPs can impact mammals and reinforce the ecotoxicological risk associated with the dispersion of these pollutants in natural environments and their uptake by mammals.

Short-term exposure of the mayfly larvae (Cloeon dipterum, Ephemeroptera: Baetidae) to SARS-CoV-2-derived peptides and other emerging pollutants: A new threat for the aquatic environments.

The input of SARS-CoV-2 or its fragments into freshwater ecosystems (via domestic or hospital sewage) has raised concerns about its possible impacts on aquatic organisms. Thus, using mayfly larvae [Cloeon dipterum (L.), Ephemeroptera: Baetidae] as a model system, we aimed to evaluate the possible effects of the combined short exposure of SARS-CoV-2-derived peptides (named PSPD-2001, PSPD-2002, and PSPD-2003 - at 266.2 ng/L) with multiple emerging pollutants at ambient concentrations. After six days of exposure, we observed higher mortality of larvae exposed to SARS-CoV-2-derived peptides (alone or in combination with the pollutant mix) and a lower-body condition index than those unexposed larvae. In the "PSPD" and "Mix+PSPD" groups, the activity of superoxide dismutase, catalase, DPPH radical scavenging activity, and the total thiol levels were also lower than in the "control" group. In addition, we evidenced the induction of nitrosative stress (inferred by increased nitrite production) and reduced acetylcholinesterase activity by SARS-CoV-2-derived peptides. On the other hand, malondialdehyde levels in larvae exposed to treatments were significantly lower than in unexposed larvae. The values of the integrated biomarker response index and the principal component analysis (PCA) results confirmed the similarity between the responses of animals exposed to SARS-CoV-2-derived peptides (alone and in combination with the pollutant mix). Although viral peptides did not intensify the effects of the pollutant mix, our study sheds light on the potential ecotoxicological risk associated with the spread of the new coronavirus in aquatic environments. Therefore, we recommend exploring this topic in other organisms and experimental contexts.

Toxicity induced via ingestion of naturally-aged polystyrene microplastics by a small-sized terrestrial bird and its potential role as vectors for the dispersion of these pollutants.

In recent years, there has been a growing number of studies on the impact of microplastics (MPs) on biota. However, its effects on birds' health are poorly understood. Thus, we aimed to evaluate the possible effects of ingestion of naturally-aged MPs by Coturnix Coturnix japonica (11 and 22 MP particles/day/bird, once a day, for 9 days), from different toxicity biomarkers. At the end of the experiment, it was found that the ingested MPs in birds showed a significant reduction in body biomass. Also, an increase in malondialdehyde production in the liver, brain, intestine, and gizzard of the birds, as well as a suppressive effect on hepatic nitric oxide production and superoxide dismutase activity in the liver and intestine were observed. Cerebral catalase activity was reduced in birds exposed to MPs and the cholinesterasic effect (marked by increased acetylcholinesterase activity) was observed in the muscle and brain of these animals. Despite these differences, through the main component analysis, hierarchical clustering analysis, and integrated biomarker response assessment, we observed similar toxicological effects in birds exposed to different amounts of MPs. In addition, the size of MPs was reduced, and their shape was altered as they transited through the gastrointestinal system, which probably explains their accumulation in the liver of birds. An expressive number of MPs are released through the feces of the birds throughout the experiment. As far as we know, this is the first report that associates MPs ingestion by small-sized terrestrial birds with biochemical alterations viz., predictive of oxidative stress, redox imbalance, and cholinesterasic effect, in addition to shedding light on the potential role of these birds as vectors for dispersal of MPs in natural environments.

Toxicological impact of SARS-CoV-2 on the health of the neotropical fish, Poecilia reticulata.

There have been significant impacts of the current COVID-19 pandemic on society including high health and economic costs. However, little is known about the potential ecological risks of this virus despite its presence in freshwater systems. In this study, we aimed to evaluate the exposure of Poecilia reticulata juveniles to two peptides derived from Spike protein of SARS-CoV-2, which was synthesized in the laboratory (named PSPD-2002 and PSPD-2003). For this, the animals were exposed for 35 days to the peptides at a concentration of 40 µg/L and different toxicity biomarkers were assessed. Our data indicated that the peptides were able to induce anxiety-like behavior in the open field test and increased acetylcholinesterase (AChE) activity. The biometric evaluation also revealed that the animals exposed to the peptides displayed alterations in the pattern of growth/development. Furthermore, the increased activity of superoxide dismutase (SOD) and catalase (CAT) enzymes were accompanied by increased levels of malondialdehyde (MDA), reactive oxygen species (ROS) and hydrogen peroxide (H2O2), which suggests a redox imbalance induced by SARS-CoV-2 spike protein peptides. Moreover, molecular docking analysis suggested a strong interaction of the peptides with the enzymes AChE, SOD and CAT, allowing us to infer that the observed effects are related to the direct action of the peptides on the functionality of these enzymes. Consequently, our study provided evidence that the presence of SARS-CoV-2 viral particles in the freshwater ecosystems offer a health risk to fish and other aquatic organisms.

Hazardous effects of road-side soils on the redox and cholinesterasic homeostasis of mound-building termite (Cornitermes cumulans).

For our knowledge, the roadside soils end up being the deposit of various residues discarded by drivers or passengers, plus, that coming from the runoff of rainwater. Basically, we do not know the impacts that this pollution causes on animals which inhabit these environments. Thus, in this study, our objective was to evaluate how the presence of plastic microfibers (MPFs), organic compounds and heavy metals affect the redox and cholinesterase homeostasis of mound-building termite [Cornitermes cumulans (workers) adults]. As a result, we noticed that MPFs were present in all sampled areas, being higher in road area (RA). Regardless of the presence of these pollutants, animals sampled in the RA were those in which we observed greater production of reactive oxygen species (ROS), hydrogen peroxide (H2O2) and nitric oxide (NO) (via nitrite), whose higher activities of antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), was not able to counterbalance the oxidative stress suggested by the evaluated biomarkers. Moreover, we observed increase in acetylcholinesterase (AChE) activity in these same animals, which suggests a cholinesterasic effect. Such alterations were positively correlated with the contamination of soil samples by Cd, Pb, Zn, Fe and Cu, as well as with the presence of the 11,10-guaiane-type sesquiterpenoid compound, identified only in the RA. Thus, our unique study reveals that the contamination of roadside soils constitutes an additional environmental stressor to populations of C. cumulans, which reinforces the need for greater attention and further investigation to be given to the pollution of these environments.

From carrion-eaters to plastic material plunderers: Toxicological impacts of plastic ingestion on black vultures, Coragyps atratus (Cathartiformes: Cathartidae).

Despite plastic ingestion has already been reported in several bird species, its physiological impacts have been little inspected, especially in representatives of the Cathartidae family. Thus, in this study, we aimed to identify, characterize, and evaluate the effects arising from the ingestion of plastic materials by Coragyps atratus adults, that captured in landfill areas. Herein, a total of 51 individuals were captured, the frequency of plastic intake being higher than 40%. The plastic materials consisted mainly of low-density polyethylene and film-type polystyrene, as well as presenting irregular shapes and diameters between 10 and 30 mm. Biochemically, we observed in animals that contained plastics in the stomach ("plastic" group) high production of reactive oxygen species (ROS), hydrogen peroxide (H2O2) and malondialdehyde (MDA) - especially in the intestine, muscle and brain - whose activity of catalase (CAT) and superoxide dismutase (SOD) was not sufficient to counteract the oxidative stress. Moreover, in the liver of these same animals, we observed high production of nitrite and nitrate, suggesting a hepatic nitrosative stress. Plus, we observed a cholinesterase effect in animals from the "plastic" group, marked by increased activity of butyrylcholinesterase (BChE) (in the brain) and muscle and cerebral acetylcholinesterase (AChE). On the other hand, the biochemical changes perceived were not significantly correlated with the identified plastic material concentrations (2.808 ± 0.598 items/g of stomach content and 0.276 ± 0.070 items/g of stomach - fresh weight), body condition of the animals, size, and shape of the identified plastic materials. Hence, our study sheds the light on the toxicity of plastics deposited in landfills and their ingestion by C. atratus, which reinforces the hypothesis that these materials are harming the health of these birds and, consequently, the dynamics of their populations.

Multiple endpoints of polylactic acid biomicroplastic toxicity in adult zebrafish (Danio rerio).

Although the toxicity of conventional microplastic types (i.e., petroleum derivatives) in different organisms is already known, knowledge about the effects of alternative biopolymers on aquatic vertebrates remains incipient. Thus, the aim of the present study is to test the hypothesis that the exposure of adult Danio rerio individuals to this pollutant for 30 days is enough to cause polylactic acid biomicroplastics (BioMPs of PLA) accumulation in their bodies, which leads to behavioral/neurotoxic, biochemical, and morphological changes. Based on our results, PLA BioMPs at concentrations of 2.5 and 5 mg/L accumulated in the liver, brain, gills and carcass of the assessed animals. However, such an accumulation was not able to cause locomotor damages or to trigger anxiety-like behavior in them. On the other hand, it was enough to cause behavioral changes (in shoal) predictive of co-specific social interaction and anti-predatory defensive response deficit likely related to cholinergic changes inferred by increased acetylcholinesterase activity and REDOX imbalance. This imbalance was featured by increased production of reactive species. We observed that the treatments have affected animals' pigmentation pattern. Therefore, our study highlights the toxicological potential of the herein assessed biopolymer, and this finding puts in check the innocuousness of this material, as well as expands our knowledge about how PLA BioMPs can affect the ichthyofauna in freshwater environments.

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.

Anti-cancer drugs in aquatic environment can cause cancer: Insight about mutagenicity in tadpoles.

Cyclophosphamide (Cyc) and 5-fluorouracil (5-FU) are two of the most used antineoplastic drugs (AD) in the world. However, their discharge in the environment became a yet-unknown environmental issue that has impact on some groups of animals, such as amphibians. We assessed tadpoles (Lithobates catesbeianus) exposed to environmental concentrations (EC) of Cyc and 5-FU to evaluate whether they can cause morphological and mutagenic changes in them. We defined the following groups: control, positive control (50 mg/L of Cyc), EC-Cyc-I (0.2 µg/L), EC-Cyc-II (0.5 µg/L), EF-Cyc (2.0 µg/L), EC-5-FU-I (13.0 µg/L), EC-5-FU-II (30.4 µg/L) and EF-5-FU (123.5 µg/L). EC groups presented predictive AD concentrations in 10% and 25% hospital-effluent dilutions in water. EF groups met gross hospital-effluent concentrations. Based on our data, ADs caused intestinal changes and influenced the interocular distance in tadpoles after 30-day exposure. We also observed the aneugenic and clastogenic effect of ADs due to the higher frequency of micronucleated and binucleated erythrocytes, and blebbed, multilobulated, notched and kidney-shaped nuclei in animals exposed to them. Based on such changes, we assume that Cyc and 5-FU can trigger malignant cell transformation processes, and cancer, in animals exposed to them, even at low concentrations. Our study is the first to describe that Cyc and 5-FU, spread in the environment, cause damages in non-target organisms opposite to their original end.