Izonete Cristina Guiloski

Effects of the herbicide atrazine in neotropical catfish (Rhamdia quelen)

The exposure to a world-wide used herbicide atrazine (ATZ) (96h exposure to 2, 10, and 100μgL(-1)), was investigated on the freshwater fish Rhamdia quelen through a multi biomarker approach. Liver histopathology revealed leukocyte infiltration, hepatocyte vacuolization like steatosis and necrosis areas, leading to raised lesion index levels in all tested concentrations. The increase of free melanomacrophage numbers was observed. Gill filaments revealed considerable loss of the microridges on pavement cells at 10 and 100μgL(-1) of ATZ, and a significantly increased of chloride cell (CC) number and density on apical surface area at 100μgL(-1) of ATZ. CAT, GST, GPx, and GR activities were inhibited by all tested concentrations. GSH levels were reduced in individuals exposed to 100µgL(-1). Osmoregulatory function was also disturbed. We observed an increase of plasma magnesium concentrations at 10µgL(-1). Additionally the inhibition of branchial carbonic anhydrase activity was observed at 100µgL(-1). In the kidney, carbonic anhydrase activity decreased only in the group exposed to 2µgL(-1). These results suggest that ATZ, represents a potential ecotoxicological hazard and can be hepatotoxic and nephrotoxic even low concentrations. The current study was the first to show the nephrotoxic effect of ATZ in fish. Besides, in Brazil, the environmental protection agency (CONAMA) establishes that the maximum allowed level of dissolved ATZ in water is 2μgL(-1), but the present results showed that this concentration may cause histopathological, biochemical and physiological changes in R. quelen.

Effects of trophic exposure to dexamethasone and diclofenac in freshwater fish

Steroidal and non-steroidalanti-inflammatories are pharmaceutical prescribed in human medicine and have the potential to contaminate water and sediments via inputs from sewage treatment plants. Their impacts on humans and ecosystems are emerging issues in environmental health. The aim of the present work was to evaluate the effects of diclofenac and dexamethasone in male fish Hoplias malabaricus after trophic exposure. Fish were fed twice every week with Astyanax sp. submitted to intraperitoneal inoculation with diclofenac (0; 0.2; 2.0 or 20.0 μg/kg) or dexamethasone (0; 0.03; 0.3 or 3.0 μg/kg). After 12 doses, blood was collected for testosterone dosage. The gonad and liver were collected to calculate gonadosomatic (GSI) and hepatosomatic index (HSI). Antioxidants enzymes activity and biotransformation were also evaluated in liver and gonads. In liver, diclofenac caused oxidative stress with increased superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities and lipoperoxidation (LPO). The GST activity was reduced by diclofenac in liver. Trophic exposure of H. malabaricus to dexamethasone caused an increase in antioxidant system (GPx, CAT, GST, and GSH) and LPO in liver. However, it reduced antioxidant system (GPX and GST activities and GSH) in gonads. Both diclofenac and dexamethasone reduced the levels of testosterone, causing impairment to reproduction. Diclofenac reduced HSI at the 0.2 μg/kg, but not GSI. Our results suggest that the anti-inflammatory drugs diclofenac and dexamethasone caused oxidative stress and reduced testosterone levels that can have a negative impact in aquatic organisms.

Risks of waterborne copper exposure to a cultivated freshwater Neotropical catfish (Rhamdia quelen)

As it is the case in all animal food production systems, it is often necessary to treat farmed fish for diseases and parasites. Quite frequently, fish farmers still rely on the aggressive use of copper to control bacterial infections and infestations by ecto-parasites, and to manage the spread of diseases. The susceptibility of the neotropical fish Rhamdia quelen to copper was here evaluated at different waterborne copper concentrations (2, 7 or 11 μg Cu L(-1)) for 96 h, through a multi biomarkers approach. Liver histopathological findings revealed leukocyte infiltration, hepatocyte vacuolization and areas of necrosis, causing raised levels of lesions upon exposure to 7 and 11 μg Cu L(-1). Decreased occurrence of free melano-macrophages and increased densities of melano-macrophage centers were noted upon exposure to 11 μg Cu L(-1). Gills showed damages on their secondary lamellae already at 2 μg Cu L(-1); hypertrophy and loss of the microridges of pavement cells at 7 and 11 μg L(-1), and increased in chloride cell (CC) apical surface area (4.9-fold) and in CC density (1.5-fold) at 11 μg Cu L(-1). In the liver, catalase (CAT), glutathione peroxidase activities (GPx) and glutathione concentration (GSH) remained unchanged, compared to the control group. However, there was inhibition of 7-ethoxyresorufin-O-deethylase (EROD) at all copper concentrations tested. Glutathione reductase activity (GR) was reduced and levels of lipid peroxidation (LPO) were increased at 11 μg Cu L(-1). Glutathione S-transferase activity (GST) at 7 μg Cu L(-1) and superoxide dismutase activity (SOD) at both 7 and 11 μg Cu L(-1) were reduced. However, copper exposure did not alter brain and muscle acetylcholinesterase (AChE) activity. Osmoregulatory function was also disturbed, in agreement with the above-mentioned changes noted in the gills, as detected by plasma osmolality reduction in the group exposed to 11 μg Cu L(-1), and plasma chloride reduction at 2 μg Cu L(-1). These concentrations also, coherently, lead to inhibition of branchial carbonic anhydrase activity. In the kidney, increased carbonic anhydrase activity was measured in the groups exposed to 2 and 7 μg Cu L(-1). When these effects are compared to data available in the literature for other freshwater fish, also for 96 h of exposure, R. quelen appears as a relatively sensitive species. In addition, the concentrations employed here were quite low in comparison to levels used for disease control in real culture practices (ranging from 4 μg Cu L(-1) used against bacteria to 6000 μg Cu L(-1) against fungal infections). We can conclude that the concentrations frequently employed in aquaculture are in fact not safe enough for this species. Such data are essential for the questioning and establishment of new policies to the sector.

Hematologic and hepatic responses of the freshwater fish Hoplias malabaricus after saxitoxin exposure

The bioaccumulation of saxitoxins (STX) in the trophic chain, mainly in freshwater, are not completely known. This work aimed to elucidate the effects of STX on Hoplias malabaricus through trophic bioassay. The fish were fed once every five days with Astyanax sp. before being subjected to an intraperitoneal inoculation with the lysate of Cylindrospermopsis raciborskii culture containing 97% STX and 3% by neosaxitoxin and gonyautoxin during 20 days. The animals liver was assessed using biomarkers as activities of superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), and glutathione peroxidase (GPx), and concentrations of reduced glutathione (GSH) and lipoperoxidation (LPO) and protein carbonylation (PCO). In the blood was analyzed the genotoxic and hematological parameters. The hepatosomatic index and the relative condition factor did not show a significant difference between the exposed and control groups. The values of mean corpuscular hemoglobin concentration and mean corpuscular hemoglobin increased in the STX group. The hepatic tissue from both groups exhibited a typical pattern that have been already described for most teleost fish. The results suggested the generation of reactive oxygen species, with increased activity of GPx and concentrations of LPO and GSH; whereas the specific activity of SOD decreased. However, no changes were observed in the CAT, PCO, and DNA damage. Although the STX effects are known as neurotoxic, this cyanotoxin caused liver biochemical alterations that can be considered ecologically relevant.

Multibiomarker in fish to evaluate a river used to water public supply.

We aimed to evaluate the ecological integrity of a large river, which receives agricultural and urban effluents and is used to water public supply. The fish species Astyanax bifasciatus was used as bioindicator during winter and spring 2012, and summer 2013 at the Middle Iguaçu River basin in Paraná state, Brazil. Water chemical and physical measures and ecotoxicological tests were carried out as well biochemical and genetic biomarkers in sampled fish in each period. The studied area was divided in three sample points: SP1, located where the water is collected to public supply; SP2, located in an urbanized area, and SP3, located at an urbanized area with the discharge of the sewage treatment. Although water chemical and physical analyzes were range of the Brazilian law to hydric bodies, anticholinesterasic effects were found in winter, oxidative stress in summer and spring. The higher genotoxic effect was in winter to all sample points. The temporal variation in biomarkers and the detection of caffeine in the water call attention to the water quality in this river mainly to be used to public supply.

Biomarkers of waterborne copper exposure in the Neotropical fish Prochilodus lineatus

The main goal of the present study was to investigate the effects of acute exposure to copper (Cu) using a Neotropical freshwater fish as sentinel species through multi biomarkers analysis at different biological levels. Juveniles of Prochilodus lineatus were kept under control condition (no Cu addition in the water) or exposed to environmentally relevant concentrations of waterborne Cu (5, 9 and 20μgL(-1)) for 96h. These concentrations were selected to bracket the current Brazilian water quality criteria for Cu in fresh water (9 and 13μgL(-1) dissolved copper). Endpoints analyzed included ethoxyresorufin-O-deethylase (EROD), glutathione-S-transferase (GST), catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity, reduced glutathione (GSH) and metallothionein-like protein (MT) concentration, lipid peroxidation (LPO) level, tissue damage index, and incidence of free melano-macrophages (FMM) and melano-macrophage centers (MMC) in the liver. They also included DNA damage (frequency of nucleoids per comet class, number of damaged nucleoids per fish and DNA damage score) in erythrocytes, as well as muscle and brain acetylcholinesterase (AChE) activity and behavioral parameters (swimming distance and velocity, time spent swimming and swimming activity in the upper and lower layers of the water column). Fish exposed to any of the Cu concentrations tested showed increased liver MT concentration and LPO level, higher number of damaged nucleoids in erythrocytes per fish, and inhibited muscle AChE activity. Also, increased liver SOD activity was observed in fish exposed to 9 and 20μgL(-1) Cu. Fish exposed to 5 and 9μgL(-1) Cu spent lower amount of time swimming. Fish exposed to 9μgL(-1) Cu showed increased swimming distance and velocity while those exposed to 20μgL(-1) Cu had lower swimming distance and velocity, as well as, spent less time swimming in the lower layer of the water column when compared to those kept under control condition. These findings indicate that Cu exposure at environmentally relevant concentrations (below or close to the current Brazilian water quality criteria) induced significant biological (histological, biochemical and genetic) and ecological (swimming and exploratory abilities) damages in the Neotropical fish P. lineatus. They also suggest that MT concentration, DNA damage (comet assay), LPO (TBARS method), SOD and AChE activity, together with swimming behavior analyses are potential biomarkers to assess and monitor areas impacted by Cu in fresh water.

Effects of environmentally relevant concentrations of the anti-inflammatory drug diclofenac in freshwater fish Rhamdia quelen

The presence of pharmaceuticals in the aquatic environment and its impact on humans and the ecosystem are emerging issues in environmental health. This study evaluated the potential biochemical, genetic and reproductive effects of the diclofenac by waterborne exposure, in a semi-static bioassay for 21 days. The fish Rhamdia quelen were exposed to environmental concentrations of diclofenac (0, 0.2, 2 and 20µg/L). The results showed that in the liver, diclofenac reduced the catalase and ethoxyresorufin- O- deethylase activities in fish exposed to 2µg/L, and superoxide dismutase in all exposed groups. The levels of reduced glutathione and glutathione S-transferase (GST) activity increased at all tested concentrations. Lipid peroxidation (LPO) was reduced in the groups exposed to 0.2 and 20µg/L of diclofenac, but there was no protein oxidation. In the testis, the concentration of 0.2µg/L caused major changes as inhibition of SOD, glutathione peroxidase and GST activities and also LPO decrease. Diclofenac was not genotoxic and not altered plasma testosterone and estradiol levels and testicular morphology. In brain, there was a reduction of dopamine and its metabolite DOPAC (3, 4-dihydroxyphenylacetic acid) in exposure to diclofenac, but this not disrupted the hypothalamic-pituitary-gonadal axis.

Saxitoxins induce cytotoxicity, genotoxicity and oxidative stress in teleost neurons in vitro.

The aim of this study was establish a protocol for isolation and primary culture of neurons from tropical freshwater fish species Hoplias malabaricus for assessment of the effects of neurotoxic substances as saxitoxins (STXs). Cells from brain of H. malabaricus were treated with different concentrations of trypsin, dispase and papain for tissue dissociation. Cells type was separated by cellular gradient and basic fibroblast growth factor (bFGF) supplement nutrition media were added. The dissociated cells were plated with medium and different STXs concentrations and the toxic cellular effects such as oxidative stress, neurotoxicity, and genotoxicity and apoptosis process were evaluated. Cultures treated with bFGF showed the greatest adherence, survival and cellular development. STXs increased specific activity of glutathione peroxidase and lipoperoxidation levels, were cytotoxic and genotoxic indicated by the comet assay. Although the STXs effects due the blockage of sodium channels is reported to be reversible, the time exposure and concentration of STXs suggested cellular injuries which can lead to neuropathology. The establishment of primary neuronal culture protocol enables new applications for neurotoxicological assessments.

Mesotrione herbicide promotes biochemical changes and DNA damage in two fish species

Highlights • Our results are novel, never before the toxicity of mesotrione was tested with biomarkers in fish• We assess the acute effects of mesotrione using 9 different biomarkers.• DNA damage was assessed in three different cell types in two species exposed to mesotrione.• We found a positive ROS response in O. niloticus and G. brasiliensis induced by mesotrione.• Adverse effects were found on concentrations lower than the applied on crop fields.

Low malathion concentrations influence metabolism in Chironomus sancticaroli (Diptera, Chironomidae) in acute and chronic toxicity tests

Low malathion concentrations influence metabolism in Chironomus sancticaroli (Diptera, Chironomidae) in acute and chronic toxicity tests. Organophosphate compounds are used in agro-systems, and in programs to control pathogen vectors. Because they are continuously applied, organophosphates often reach water sources and may have an impact on aquatic life. The effects of acute and chronic exposure to the organophosphate insecticide malathion on the midge Chironomus sancticaroli are evaluated. To that end, three biochemical biomarkers, acetylcholinesterase (AChE), alpha (EST-α) and beta (EST-β) esterase were used. Acute bioassays with five concentrations of malathion, and chronic bioassays with two concentrations of malathion were carried out. In the acute exposure test, AChE, EST-α and EST-β activities declined by 66, 40 and 37%, respectively, at 0.251 µg L-1 and more than 80% at 1.37, 1.96 and 2.51 µg L-1. In chronic exposure tests, AChE and EST-α activities declined by 28 and 15% at 0.251 µg L-1. Results of the present study show that low concentrations of malathion can influence larval metabolism, indicating high toxicity for Chironomus sancticaroli and environmental risk associated with the use of organophosphates.