Alterations in neurotransmitters, steroid hormones, vitellogenin, and antioxidant system induced by di-n-butyl phthalate and di-isopentyl phthalate on catfish Rhamdia quelen.

Phthalates, such as di-n-butyl phthalate (DBP) and di-isopentyl phthalate (DiPeP), are pollutants with a high potential for endocrine disruption. This study aimed to evaluate parameters of endocrine disruption in specimens of the Neotropical fish Rhamdia quelen exposed to DBP and DiPeP through their food. After 30 days of exposure, the fish were anesthetized and then euthanized, and blood, hypothalamus, liver, and gonads were collected. DBP caused statistically significant alterations in the serotoninergic system of males (5 and 25 ng/g) and females (5 ng/g) of R. quelen and it increased testosterone levels in females (25 ng/g). DiPeP significantly altered the dopaminergic system in females, reduced plasma estradiol levels (125 ng/g) and hepatic vitellogenin expression (25 ng/g), and changed the antioxidant system in gonads (125 ng/g). The results suggest that DBP and DiPeP may have different response patterns in females, with the former being androgenic and the latter being anti-estrogenic. These findings provide additional evidence regarding the molecular events involving DBP and DiPeP in the endocrine disruption potential in juvenile specimens of Rhamdia quelen.

Evidence of genotoxicity, neurotoxicity, and antioxidant imbalance in silver catfish Rhamdia quelen after subchronic exposure to diisopentyl phthalate.

Diisopentyl phthalate (DiPeP) is a plasticizer with significant offer and application in Brazilian industries. This is attributed to its origin, which is closely linked to the refining process of sugarcane for ethanol production in the country. In this work, we developed a model for trophic exposure to environmentally relevant doses (5, 25, and 125 ng/g of DiPeP) to identify possible target tissues and toxic effects promoted by subchronic exposure to DiPeP in a Neotropical catfish species (Rhamdia quelen). After thirty days of exposure, blood, liver, kidney, brain, and muscle were collected and studied regarding DNA damage in blood cells and biochemical analyses. The kidney was the most affected organ, as in the head kidney, genotoxicity was evidenced in all groups exposed to DiPeP. Besides, the caudal kidney showed a reduction in the superoxide dismutase and glutathione peroxidase activities as well as a reduced glutathione concentration. In the liver, exposure to 125 ng/g of DiPeP increased glutathione S-transferase activity and reduced glutathione levels. In muscle, acetylcholinesterase (AChE) was reduced. However, in the brain, an increase in AChE activity was observed after the exposure to lowest doses. In contrast, a significant reduction of brain AChE activity after exposure to the highest dose was detected. The pronounced genotoxicity observed in head kidney cells is of concern, as it may compromise different functions performed by this organ (e.g., hematopoiesis, immune and endocrine functions). In our study, DiPeP proved to be a compound of environmental concern since we have evidenced its nephrotoxic and neurotoxic potential even in low doses.

Salvinia molesta phytoremediation capacity as a nature-based solution to prevent harmful effects and accumulation of ciprofloxacin in Neotropical catfish.

Phytoremediation has been a potential solution for the removal of pharmaceuticals from water. Here, we evaluated the toxicological safety of ciprofloxacin-contaminated water treated by 96 h with Salvinia molesta. The Neotropical catfish Rhamdia quelen was used as a model, and the potential of the phytoremediation technique for mitigating the drug accumulation in the fishes was also studied. Fish exposed to Cipro (1 and 10 µg·L-1) in untreated water showed toxic responses (alteration of hematological, genotoxicity, biochemical, and histopathological biomarkers) and accumulated Cipro in their muscles at concentrations high for human consumption (target hazardous quotient > 1). Fish exposed to water treated with S. molesta showed no toxic effect and no accumulation of Cipro in their tissues. This must be related to the fact that S. molesta removed up to 97% of Cipro from the water. The decrease in Cipro concentrations after water treatment with S. molesta not only prevented the toxic effects of Cipro on R. quelen fish but also prevented the antimicrobial accumulation in fish flesh, favouring safe consumption by humans. For the very first time, we showed the potential of phytoremediation as an efficiently nature-based solution to prevent environmental toxicological effects of antimicrobials to nontarget organisms such as fish and humans. The use of S. molesta for Cipro-removal from water is a green technology to be considered in the combat against antimicrobial resistance.

Alkylphenols cause cytotoxicity and genotoxicity induced by oxidative stress in RTG-2 cell line.

Alkylphenols ethoxylates are industrial surfactants, and the release in the environmental matrices produces degraded products, of which nonylphenol (NP) and octylphenol (OP) were the most common. They can be classified as endocrine disruptors since the estrogenic potential is widely recognized, but some others toxic aspects are in discussion. This study aimed to evaluate the toxicity of NP, OP, and mixtures of both through cellular, biochemical and genetic biomarkers in fish gonadal cell line RTG-2 exposed to nominal concentrations of 0.05; 0.5; 5; 50, and 100 µg mL-1 of each chemical and their mixtures of 0.05, 0.5; 5 µg mL-1 concentrations. After 24 h, the cells were collected for cytotoxic (neutral red - NR; crystal violet - CV, resazurin assay - RA and lactate-dehydrogenase - LDH), antioxidant system (glutathione-s-transferase - GST; superoxide-dismutase - SOD; glutathione-peroxidase - GPx and malondialdehyde - MDA) and genotoxic assays (alkaline comet assay and Fpg-modified alkaline comet assay). The chemicals and their mixtures were cytotoxic at 50 and 100 µg mL-1, in general aspect, but LDH showed cytotoxicity since 0.05 µg mL-1. The GST and SOD showed an activity increase trend in most tested groups, while GPx decreased at 5 µg mL-1 of the mixture. The MDA increase in all groups resulted in lipid peroxidation. The reactive oxygen species caused DNA damage for all groups. The tested chemicals and concentrations have been found in the freshwater systems. They can induce cell toxicity in several parameters that could impair the gonadal tissues considering the RTG-2 responses.

Development of 3D cultures of zebrafish liver and embryo cell lines: a comparison of different spheroid formation methods.

Fish cell spheroids are promising 3D culture models for vertebrate replacement in ecotoxicology. However, new alternative ecotoxicological methods must be adapted for applications in industry and for regulatory purposes; such methods must be cost-effective, simple to manipulate and provide rapid results. Therefore, we compared the effectiveness of the traditional hanging drop (HD), orbital shaking (OS), and HD combined with OS (HD+OS) methods on the formation of zebrafish cell line spheroids (ZFL and ZEM2S). Time in HD (3-5 days) and different 96-well plates [flat-bottom or ultra-low attachment of round-bottom (ULA-plates)] in OS were evaluated. Easy handling, rapid spheroid formation, uniform-sized spheroids, and circularity were assessed to identify the best spheroid protocol. Traditional HD alone did not result in ZFL spheroid formation, whereas HD (5 days)+OS did. When using the OS, spheroids only formed on the ULA-plate. Both HD+OS and OS were reproducible in size (177.50 ± 2.81 µm and 225.62 ± 19.20 µm, respectively) and circularity (0.83 ± 0.02 and 0.80 ± 0.01, respectively) of ZFL spheroids. Nevertheless, HD+OS required a considerable time to completely form spheroids (10 days) and intensive handling, whereas the OS was fast (5 days of incubation) and simple. OS also yielded reproducible ZEM2S spheroids in 1 day (226.23 ± 0.57 µm diameter and 0.80 ± 0.01 circularity). In conclusion, OS in ULA-plate is an effective and simple spheroid protocol for high-throughput ecotoxicity testing. This study contributes to identify a fast, reproducible, and simple protocol of single piscine spheroid formation in 96-well plates and supports the application of fish 3D model in industry and academia.

Tissue-specific genotoxicity and antioxidant imbalance of titanium dioxide nanoparticles (NPTiO2) and inorganic lead (PbII) in a neotropical fish species.

The aquatic environment is the major recipient of wastes containing nanoparticles and other contaminants. Titanium dioxide nanoparticles (NPTiO2) are one of the most produced and used nanoparticle worldwide. This study investigated the toxicity of NPTiO2, as well as the toxicity interaction between NPTiO2 and lead (Pb), in response to genetic and biochemical biomarkers using freshwater fish Rhamdia quelen, as an animal model. The results showed genotoxicity in blood and kidney tissues. No effect of NPTiO2 alone or in co-exposure with Pb on liver genotoxicity were observed. Alterations in the antioxidant hepatic enzymes activities, as well as alterations in glutathione levels indicated that NPTiO2 alone or in co-exposure with Pb can cause antioxidant imbalance. The lipid peroxidation was also raised after exposure to NPTiO2. In general, the results of this study indicated that both NPTiO2 alone and their co-exposure with Pb are capable of producing significant toxic effects in short-term exposure.

Genotoxicity of titanium dioxide nanoparticles and triggering of defense mechanisms in Allium cepa.

Titanium dioxide nanoparticles (TiO2NPs) are widely used and may impact the environment. Thus, this study used a high concentration of TiO2NP (1000 mg/L) to verify the defense mechanisms triggered by a plant system - an indicator of toxicity. Furthermore, this study aimed at completely characterizing TiO2NP suspensions to elucidate their toxic behavior. TiO2NPs were taken up by meristematic cells of Allium cepa, leading to slight inhibition of seed germination and root growth. However, severe cellular and DNA damages were observed in a concentration-dependent manner (10, 100, and 1000 mg/L). For this reason, we used the highest tested concentration (1000 mg/L) to verify if the plant cells developed defense mechanisms against the TiO2NPs and evaluated other evidences of TiO2NP genotoxicity. Nucleolar alterations and plant defense responses (i.e., increased lytic vacuoles, oil bodies and NP phase change) were observed in meristematic cells exposed to TiO2NP at 1000 mg/L. In summary, TiO2NPs can damage the genetic material of plants; however, plants displayed defense mechanisms against the deleterious effects of these NPs. In addition, A. cepa was found to be a suitable test system to evaluate the cyto- and genotoxicity of NPs.

The evaluation of the potential ecotoxicity of pyroligneous acid obtained from fast pyrolysis.

Pyroligneous acid (PA) is a by-product of bio-oil, which is obtained by pyrolysis of the wood. This product has been tested for use in several areas, such as agriculture, as a promising green herbicide; however, there are few scientific data regarding its environmental impacts. For this study, an ecotoxicity testing battery, composed of Daphnia magna acute toxicity test, Allium cepa test and in vitro Comet assay with the rainbow trout gonad-2 cell fish line (RTG-2) were used to evaluate the acute toxicity and genotoxicity of PA obtained from fast pyrolysis of eucalyptus wood fines. The PA presented acute toxicity to D. magna (microcrustacea) with EC50 of 26.12 mg/L, and inhibited the seed germination (EC50 5.556 g/L) and root development (EC50 3.436 g/L) of A. cepa (higher plant). No signs of genotoxicity (chromosomal aberrations and micronuclei in A. cepa and primary DNA lesions in RTG-2 cells) were detected to this product. The acute toxicity and absence of genotoxicity may relate to the molecules found in the PA, being the phenolic fraction the key chemical candidate responsible for the toxicity observed. In addition, daphnids seem to be more sensitivity to the toxicity of PA than higher plants based on their EC50 values. This first ecotoxicological evaluation of PA from fast pyrolysis pointed out the need of determining environmental exposure limits to promote the safer agriculture use of this product, avoiding impacts to living organisms.

Antioxidant imbalance and genotoxicity detected in fish induced by titanium dioxide nanoparticles (NpTiO2) and inorganic lead (PbII).

Titanium dioxide nanoparticles (NpTiO2) are the most widely-used nanoparticle type and the adsorption of metals such as lead (PbII) onto their surface is a major source of concern to scientists. This study evaluated the effects of the associated exposure to both types of contaminant, i.e., lead (a known genotoxic metal) and NpTiO2, in a freshwater fish (Astyanax serratus) through intraperitoneal injection for an acute assay of 96 h. The effects of this exposure were evaluated using the comet assay, DNA diffusion assay and piscine micronucleus test, as well as the quantification of antioxidant enzymes (SOD, CAT, and GST) and metallothioneins. Our findings indicate that co-exposure of PbII with NpTiO2 can provoke ROS imbalances, leading to DNA damage in the blood and liver tissue of A. serratus, as well as modifying erythropoiesis in this species, inducing necrosis and changing the nuclear morphology of the erythrocytes.

Toxicological evaluation of nail polish waste discarded in the environment.

Nail polish has been widely used around the world. However, the hazards of nail polishes discarded in the environment are still poorly investigated. Thus, the toxicogenetic effects of solubilized (SE) and leached (LE) extracts from nail polishes were investigated, simulating their disposal on water and landfill, respectively, and identifying their physicochemical properties and chemical constituents. Organic compounds and metals were detected in both extracts. SE and LE only induced mutagenic effects in TA98 Salmonella strain in the presence and absence of exogenous metabolic activation. Although both extracts did not significantly increase the frequency of micronucleated HepG2 cells, the cell viability was affected by 24-h exposure. No DNA damage was observed in gonad fish cells (RTG-2) exposed to both extracts; however, the highest SE and LE concentrations induced significant lethal and sublethal effects on zebrafish early-life stages during 96-h exposure. Based on our findings, it can be concluded that if nail polishes enter aquatic systems, it may cause negative impacts to the environment.

Co-exposure to titanium dioxide nanoparticles (NpTiO2) and lead at environmentally relevant concentrations in the Neotropical fish species Hoplias intermedius.

Growing production and utilization of titanium dioxide nanoparticles (NpTiO2) invariably lead to their accumulation in oceans, rivers and other water bodies, thus increasing the risk to the welfare of this ecosystem. The progressive launch of these nanoparticles in the environment has been accompanied by concern in understanding the dynamics and the toxic effect of these xenobiotic in different ecosystems, either on their own or in tandem with different contaminants (such as organic compounds and heavy metals), possibly altering their toxicity. Nevertheless, it remains unknown if these combined effects may induce damage in freshwater organisms. Therefore, this study aimed to analyze the consequences caused by NpTiO2, after a waterborne exposure of 96 h to a Neotropical fish species Hoplias intermedius, as well as after a co-exposure with lead, whose effects for fish have already been well described in the literature. The characterization of NpTiO2 stock suspension was carried out in order to provide additional information and revealed a stable colloidal suspension. As a result, NpTiO2 showed some genotoxic effects which were observed by comet assay in gill, kidney and brain cells. Also, the activity of brain acetylcholinesterase (AChE) has not changed, but the activity of muscle AChE decreased in the group exposed only to PbII. Regarding the hepatic antioxidant system, catalase (CAT) did not show any change in its activity, whereas that of superoxide dismutase (SOD) intensified in the groups submitted only to PbII and NpTiO2 alone. As for lipid peroxidation, there was a decrease in the group exposed to the NpTiO2 alone and to the co-exposed group (NpTiO2+PbII). As far as metallothionein is concerned, its concentration rose for the co-exposed group (NpTiO2+PbII) and for the group exposed to PbII alone. Overall, we may conclude that NpTiO2 alone caused DNA damage to vital tissues. Also, some impairment related to the antioxidant mechanism was described but it is probably not related to the DNA damage observed, suggesting that the genotoxic effect observed may be due to a different mechanism instead of ROS production.

Genetic and Hematologic Endpoints in Astyanax altiparanae (Characidae) After Exposure and Recovery to Water-Soluble Fraction of Gasoline (WSFG).

The sublethal effects of water-soluble fraction of gasoline (WSFG, 1.5 % v/v) were evaluated in the freshwater fish, Astynax altiparanae, after acute exposure (96 h) under a semi-static system. In addition, the recovery process was assessed in the fish following contaminant depuration. Recovery treatments were carried out with gradual depuration (GD), consisting of 7 days in the WSFG, followed by 8 days in clean water; and treatments with total depuration in clean water for 15 (DEP 15) and 30 days (DEP 30). The effects were evaluated through the piscine micronucleus test and by differential counting of organic defense cells. Acute exposure increased the frequency of neutrophils. In the GD treatment, the thrombocyte count and erythrocytic nuclear abnormalities (ENA) increased. In the DEP 15 treatment, there was a reduction of ENA; and following 30 days of depuration (i.e., DEP 30), the number of lymphocytes increased and the thrombocyte count remained high. These results indicate a long-term response to a condition of stress from WSFG.

Handling of Astyanax sp. for biomonitoring in Cangüiri Farm within a fountainhead (Iraí River Environment Preservation Area) through the use of genetic biomarkers.

Aquatic environmental pollution may cause biodiversity loss. Thus, monitoring studies are very important because fish health reflects both quality and sustainability of the environment, as well as of the individuals that live there. In the present report, genetic biomarkers (piscine micronucleus test; comet assay with blood, liver, and kidney cells) were used in specimens of Astyanax sp. to analyze the contamination level of the Cangüiri Farm through biomonitoring. The Cangüiri Farm, the old school farm of the Federal University of Paraná, is inside the Iraí River Environment Preservation Area, created in 1996 to preserve the sources of public water supply in Curitiba and metropolitan area. We verified that the fishes collected within the Cangüiri Farm area presented high damage levels, showing more environment contamination when compared to the specimens collected in the Costa Ecologic Park, used as reference in the present report. The results indicate that the Cangüiri Farm, which is inside an environment protection area, created especially for the protection of the fountainhead for water supply, may be contaminated. These toxic residues, which were remarkably persistent in the environment, are possibly derived from agricultural activities in the wider area. Thus, we suggest the analysis of the area with other biomarkers and for a longer time period.

Genotoxic evaluation of different doses of methylmercury (CH3Hg⁺) in Hoplias malabaricus.

The survey of the effects of toxic metals and its organic compounds are of critical importance because these compounds tend to accumulate in aquatic environments. In the present work, the genotoxic potential of methylmercury, an organomercurial compound with high toxicity and present in large amounts in fish of the Amazon due to the mining process, was evaluated using the piscine micronucleus test and comet assay in fish. Specimens of Hoplias malabaricus (popularly known as traira), a neotropical specie, was exposed to methylmercury via food web, over 70 days, in two different concentrations: two groups of fifteen fish were tested with 0.075 µg CH(3)Hg(+)/g and 0.75 µg CH(3)Hg(+)/g of methylmercury per gram of fish, at 5-day intervals and over 14 successive intervals whereas control group, composed by nine fish, was fed by uncontaminated prey fish (Astyanax sp). One of the aims of this study was to reproduce the contamination found in nature in an attempt to increase our biological knowledge on the neotropical species. Hoplias malabaricus specimens were then anesthetized for removal of blood samples and dissected, for cephalic kidney removal. As a result, it was observed that the piscine micronucleus test showed no significant differences between exposed groups and control group. The comet assay performed on erythrocyte system cells, showed a significant difference between controls and contaminated, but there was no difference between doses. In contrast, the kidney cells comet assay showed no significant difference between groups, probably due to the type of damage caused by xenobiotics to be related to the tissues of most contact with it, as well as its mode of action and the chain of bioaccumulation within bodies.

Chronic genetic damages in Geophagus brasiliensis exposed to anthropic impact in estuarine lakes at Santa Catarina coast--southern of Brazil.

Biological monitoring through animals exposed to pollutants using biomarkers provides a promising tool for the identification of pollutants that may cause damage to human health and/or to sustainability of ecosystems. The effects of pollutants in fish tissues are important tools to understand the impact of human activities in natural ecosystems. The aim of this work was to study the water quality of two estuarine lakes in Santa Catarina, Brazil (Camacho Lake and Santa Marta Lake). Geophagus brasiliensis is a species widely distributed in Brazil and was used in this work. Comet assays in peripheral red blood and kidney cells, micronucleus tests in peripheral red blood cells, measurements of acetylcholinesterase activity in axial muscle and histopathological analysis of liver were used as biomarkers. Three sampling campaigns were undertaken in November 2004, June 2005 and November 2005. Thirty adult animals were sampled from each of three different sites (P1--Santa Marta Lake, P2 and P3--Camacho Lake). A negative control was sampled in a non-polluted site at Costa Ecological Park, Paraná. The positive control for genotoxicity was obtained by treating animals with copper sulphate. The results showed that both studied lakes are impacted by potential genotoxic substances. Severe lesions in liver of G. brasiliensis were also observed. The inhibition of acetylcholinesterase activity suggests the presence of pesticides or metals in the studied sites. This work shows that the water quality of Santa Marta and Camacho Lakes have been compromised and further control source of pollutants into these ecosystems is required.

Toxicity evaluation of water samples collected near a hospital waste landfill through bioassays of genotoxicity piscine micronucleus test and comet assay in fish Astyanax and ecotoxicity Vibrio fischeri and Daphnia magna.

In this study, we analyzed samples of water from a river and a lake located near a hospital waste landfill with respect to physico-chemical parameters and conducted bioassays of ecotoxicity using Vibrio fischeri and Daphnia magna, which are species commonly used to evaluate the water toxicity. We also evaluated damage to the genetic material of fish (Astyanax sp. B) that were exposed (96 h) to water from these two sites that were located near the tank ditch, using the alkaline comet assay and the piscine micronucleus test. Parameters including aluminum, manganese, biochemical oxygen demand, sulfide, conductivity, phenol, total coliforms and Escherichia coli counts, were above acceptable levels that have been established in environmental legislation. However, the toxicity bioassays that we carried out in Vibrio fischeri and Daphnia magna and the piscine micronucleus test in fish showed no immediate risk due to acute effects. Based on the results of the comet assay, however, it was possible to detect damage to genetic material in fish that were acutely exposed in the laboratory to water samples from the river and lake that are located near the trench septic tank. Thus, our results suggest that tests beyond those usually employed to test water toxicity, such as the comet assay we used in the fish, are required to assess the toxicity of water with greater accuracy.