Long-term exposure of zebrafish juveniles to carbon nanofibers at predicted environmentally relevant concentrations: Outspreading warns about ecotoxicological risks to freshwater fish.

Although carbon-based nanomaterials (CNMs) toxicity has already been demonstrated in some animal models, little is known about the impact of carbon nanofibers (CNFs) on aquatic vertebrates. Thus, we aimed to evaluate the possible effects of long-term exposure of zebrafish (Danio rerio) juveniles (90 days) to CNFs in predicted environmentally relevant concentrations (10 ng/L and 10 µg/L). Our data revealed that exposure to CNFs did not affect the growth and development of the animals, in addition to not having induced locomotor alterations or anxiety-like behavior. On the other hand, we observed that zebrafish exposed to CNFs showed a response deficit to the vibratory stimulus test, alteration in the density of neuromasts recorded in the final ventral region, as well as an increase in thiobarbituric acid reactive substances levels and a reduction in total antioxidant activity, nitric oxide, and acetylcholinesterase activity in the brain. Such data were directly associated with a higher concentration of total organic carbon in the brain, which suggests the bioaccumulation of CNFs. Furthermore, exposure to CNFs induced a picture suggestive of genomic instability, inferred by the increased frequency of nuclear abnormalities and DNA damage in circulating erythrocytes. Although the individual analyses of the biomarkers did not point to a concentration-dependent effect, the principal component analysis (PCA) and the Integrated Biomarker Response Index (IBRv2) indicate a more prominent effect induced by the higher CNFs concentration (10 µg/L). Therefore, our study confirms the impact of CNFs in the studied model (D. rerio) and sheds light on the ecotoxicological risks of these nanomaterials to freshwater fish. Based on the ecotoxicological screening provided by our study, new horizons are opened for investigations into the mechanisms of action of CNFs, which will help understand the magnitude of the impact of these materials on aquatic biota.

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.

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.

Trophic transfer of carbon nanofibers among eisenia fetida, danio rerio and oreochromis niloticus and their toxicity at upper trophic level.

Although the toxicity of carbon-based nanomaterials has already been demonstrated in several studies, their transfer in the food chain and impact on the upper trophic level remain unexplored. Thus, based on the experimental food chain "Eisenia fetida → Danio rerio → Oreochromis niloticus", the current study tested the hypothesis that carbon nanofibers (CNFs) accumulated in animals are transferred to the upper trophic level and cause mutagenic and cytotoxic changes. E. fetida individuals were exposed to CNFs and offered to D. rerio, which were later used to feed O. niloticus. The quantification of total organic carbon provided evidence of CNFs accumulation at all evaluated trophic levels. Such accumulation was associated with higher frequency of erythrocyte nuclear abnormalities such as constricted erythrocyte nuclei, vacuole, blebbed, kidney-shaped and micronucleated erythrocytes in Nile tilapia exposed to CNFs via food chain. The cytotoxic effect was inferred based on the smaller size of the erythrocyte nuclei and on the lower "nuclear/cytoplasmic" area ratio in tilapia exposed to CNFs via food chain. Our study provided pioneering evidence about CNFs accumulation at trophic levels of the experimental chain, as well as about the mutagenic and cytotoxic effect of these materials on O. niloticus.

Biomicroplastics versus conventional microplastics: An insight on the toxicity of these polymers in dragonfly larvae.

The toxicological safety of products developed as alternative for conventional plastics (i.e., petroleum derivatives) inevitably demands conducting (eco)toxicological studies. Thus, the aim of the current study was to evaluate the biochemical toxicity of polyethylene microplastics (PE MPs) (representative of conventional MPs) and polylactic acid biomicroplastics (PLA BioMPs) in Aphylla williamsoni larvae used as experimental models. Animals subjected to short exposure to both pollutants (48 h), at environmentally relevant concentration (6 mg/L). At the end of the experiment, different toxicity biomarkers were evaluated. To assess the possible impact of pollutants on the nutritional status of the animals, the total protein, total soluble carbohydrate and triglyceride levels were determined. However, we did not observe differences between the groups, which suggests that PE MPs and PLA BioMPs did not affect the animals' energy metabolism, inducing them to a nutritional deficit. However, larvae exposed to PLA BioMPs have shown increased nitrite and lipid peroxidation levels, which supports the hypothesis that these pollutants increase oxidative stress processes in the animals evaluated, which can affect the animals' physiological homeostasis from different changes. In addition, the decrease in superoxide dismutase activity and of total thiols levels, in these same animals, is suggestive of the impact of PLA BioMPs on the antioxidant defenses, causing a REDOX imbalance, never before reported. On the other hand, decreased AChE activity was only observed in larvae exposed to PLA BioMPs, which demonstrates the anticholinergic activity of the tested polymers; the consequences of which include changes in different neurophysiological functions. Thus, the current study has helped improving the scientific knowledge about impacts caused by PLA BioMPs on freshwater ecosystems, as well as corroborated assertions about the risks posed by such biopolymers on these environments.

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.

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.

Cigarette butt leachate as a risk factor to the health of freshwater bivalve.

The toxicity caused by smoking to human health has been demonstrated in several scientific studies. However, little attention has been given to damages caused to aquatic biota when cigarette butts (CB) are disposed of on water surface. Thus, the main aim of the current study is to evaluate the behavioural toxicity of cigarette butt leachates (CBL) in freshwater bivalve species Anodontites trapesialis exposed to different environmentally-relevant dilutions (CBL1x = 1.375%, CBL10x: 13.75%). There were significant CBL effects on the burrowing performance of the evaluated bivalves, after 14 exposure days. Animals exposed to CBL presented higher latency to foot emission and to start the burrowing process, as well as larger number of cycles required for burial. In addition, there were lower burrowing angle and burrowing rate index in CBL-exposed bivalves than in the unexposed ones. Chemical analyses performed on the muscle tissues of animals exposed to both CBL dilutions evidenced the bioaccumulation of several metals at high concentrations in CBL (Cr, Ni, Pb, Mn, Zn and Na); this outcome enabled associating these metals with behavioural changes observed in CBL-exposed groups. Thus, the current study firstly reports that even highly-diluted CBL concentrations can induce behavioural changes in freshwater bivalves, as well as that CBL extrapolation to natural environments can lead to several damages to the fitness of living organisms and to the dynamics of their population.

Analysis of ZnO nanoparticle-induced changes in Oreochromis niloticus behavior as toxicity endpoint.

The toxicity of zinc oxide nanoparticles (ZnO NPs) has been investigated in different animal models. However, concentrations tested in most studies are often much higher than the ones potentially identified in the environment. Therefore, such toxicity limits the application of these studies to evaluate ecotoxicological risks posed by these nanopollutants. Thus, the aim of the current study is to evaluate the impacts of ZnO NPs (at environmentally relevant concentrations - 760 µg/L and 76,000 µg/L, for 72 h) on the behavioral responses of Oreochromis niloticus (Nile tilapia) exposed to it. Results did not evidence harmful effects of NPs on animals' locomotor abilities (evaluated through open-field and light-dark transition tests), or anxiety-predictive behavior. On the other hand, Zn bioaccumulation in the body tissues of the analyzed tilapias was correlated to changes in eating behavior (motivated by ration pellets), as well as to deficits in antipredatory defensive behavior (under individual and collective conditions). Tilapia exposed to ZnO NPs recorded lower avoidance, flight and territorialist behavior rates when they were individually confronted with potential predators (Salminus brasiliensis). However, collectively exposed animals were unable to recognize their predators, as well as to differentiate them from artificial baits ("false predators"). The present study is the first to report biological impacts resulting from the short exposure of fish-group representatives to ZnO NPs. Thus, we believe that it may be relevant to improve the knowledge about ecotoxicological risks posed by these pollutants.

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.

Behavioral changes in Japanese quails exposed to predicted environmentally relevant abamectin concentrations.

Abamectin (ABA) toxicity in fish, amphibian and mammals was already proven, but its effect on birds is almost unknown. Thus, the aim of our study is to assess the impact of exposure to water with ABA for 40 days at predicted environmentally relevant concentrations on the behavior of female Japanese quails (Coturnix coturnix japonica). The three following experimental groups (n = 10 each) were set: "control", quails exposed to drinking water, without ABA, "EC1x" and "EC1000x" (0.31 mg a.i./L and 310.0 a.i./L, respectively; via commercial formulation Kraft® 36EC). The open field test showed anxiolytic response in birds exposed to ABA. These birds did not show locomotor changes or aggressive behavior in the aggressiveness test. Quails exposed to the pesticide did not react to the introduction of an object in the experimental box during the object recognition test, and it suggested perception deficit due to ABA. Moreover, these birds did not recognize the cat (Felix catus) and the vocalization of a hawk (Rupornis magnirostris) as potential predatory threats. These responses also suggest anti-predatory behavior deficit due to the pesticide. Thus, our study is pioneer in showing that water with ABA, at tested concentrations, influences the behavior of C. coturnix japonica, as well as in highlighting the potential impacts of this pesticide on this group of birds.

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.

Insights about the toxic effects of tannery effluent on Lithobates catesbeianus tadpoles.

Tannery industries are considered highly polluting due to the large production of polluted wastewater [untreated tannery effluent (UTE)]. Although previous studies have already shown the consequences from fish, birds and mammals' exposure to this pollutant, little is known about its toxicological effect on representatives of class amphibian. Thus, we aim at assessing whether short-term UTE exposure, even at low concentrations (0.2% UTE diluted in water), would be able to induce behavioral, morphological and cyto-genotoxic changes in L. catesbeianus tadpoles. In order to do so, two experimental groups were set (control and tannery effluent) and exposed, or not, to UTE for seven days. A positive control group (cyclophosphamide) was included in the experimental design in order to assess cyto-genotoxicity. Our behavioral results showed that tadpoles exposed to the contaminant presented abnormal responses in the predator-response test; therefore, it evidenced losses in their capacity to recognize chemical olfactory cues of a potential predator. We also searched for changes in mouth length, in dentition, in body length before and after the eyes of animals exposed to UTE. Besides, we observed higher nuclear abnormality frequency in the circulating erythrocytes of tadpoles exposed to the contaminant, as well as in animals belonging to the positive control group. Some of the observed abnormalities were micronuclei, binucleated, notched, kidney-shaped and blebbed cells, multilobulated nuclei, as well as lower mitotic index. Therefore, our data confirm the hypothesis that UTE causes behavioral, morphological and cyto-genotoxic changes in L. catesbeianus tadpoles, fact that opens new perspectives to other investigations about how and which UTE constituents were responsible for the observed effects.

Zinc oxide nanoparticles in predicted environmentally relevant concentrations leading to behavioral impairments in male swiss mice.

Although the potential neurotoxic effects from the exposure to zinc oxide nanoparticles (ZnO NPs) on humans and on experimental models have been reported in previous studies, the effects from the exposure to environmentally relevant concentrations of them remain unclear. Thus, the aim of the present study is to investigate the effects from the exposure to environmentally relevant concentrations of ZnO NPs on the behavior of male Swiss mice. The animals were daily exposed to environmentally relevant concentrations of ZnO NPs (5.625×10-5mgkg-1) at toxic level (300mgkg-1) through intraperitoneal injection for five days; a control group was set for comparison purposes. Positive control groups (clonazepam and fluoxetine) and a baseline group were included in the experimental design to help analyzing the behavioral tests (open field, elevated plus maze and forced swim tests). Although we did not observe any behavioral change in the animals subjected to the elevated plus maze and forced swim tests, our data evidence the anxiogenic behavior of animals exposed to the two herein tested ZnO NPs concentrations in the open field test. The animals stayed in the central part of the apparatus and presented lower locomotion ratio in the central quadrants/total of locomotion during this test. It indicates that the anxiogenic behavior was induced by ZnO NP exposure, because it leads to Zn accumulation in the brain. Thus, the current study is the first to demonstrate that the predicted environmentally relevant ZnO NPs concentration induces behavioral changes in mammalian experimental models. Our results corroborate previous studies that have indicated the biological risks related to the water surface contamination by metal-based nanomaterials.