H.C. Silva de Assis

Toxic effects of DDT and methyl mercury on the hepatocytes from Hoplias malabaricus

Here, we examined the impact of dichlorodiphenyltrichloroethane (DDT) and monomethyl mercury (MeHg) on the redox milieu and survival of hepatocytes from Hoplias malabaricus (traíra). After isolation and attachment of cells, we established one control and four treatments: DDT (50nM of DDT), MeHg I (0.25microM of MeHg), MeHg II (2.5microM of MeHg) and DDT * MeHg I (combination of 50nM of DDT and 0.25microM of MeHg). After four days the exposed hepatocytes presented significantly increased damage in lipids (all treatments), proteins (DDT * MeHg I and MeHg II) and reduced cell viability (all treatments). Also the antioxidant enzymes catalase, glucose-6-phosphate dehydrogenase (G6PDH), glutathione reductase and superoxide dismutase were affected. The current data showed that despite of some protective responses, the increased disturbs on membrane lipids and proteins, increased hydrogen peroxide levels, and decreased glutathione concentration and cell viability strongly indicate oxidative stress as the reason of hepatotoxicity due to DDT and MeHg exposure. In addition, DDT and MeHg together had greater effect than alone when G6PDH and glutathione-S-transferase activities and lipids damage were considered. These findings are indicative of hepatotoxicity occurring at realistic concentrations of DDT and MeHg found in Amazonian fish tissues.

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.

An assessment of acute biomarker responses in the demersal catfish Cathorops spixii after the Vicuña Oil Spill in a harbour estuarine area in Southern Brazil

The Vicuña oil tanker exploded in Paranaguá Bay (South of Brazil), during methanol unloading operations in front of Paranaguá Harbour, on November 15th, 2004, releasing a large amount of bunker oil and methanol. Two weeks after the accident, the acute effects of the Vicuña Oil Spill (VOS) were evaluated in the demersal catfish Cathorops spixii, comparing a contaminated (at the spill site) and a reference site inside the Bay. Data were compared to previous measurements, taken before the accident, in the same species, from the same sites. The physiological biomarkers were the ones that best reflected acute effects of the spill: plasma osmolality, chloride, calcium, magnesium, and potassium. Morphological (liver and gill histopathology) and genetic (piscine micronucleus and DNA strand breaks) biomarkers revealed that damage was already present in fishes from both reference and contaminated sites inside the Bay. Thus, the reference site is not devoid of contamination, as water circulation tends to spread the contaminants released into other areas of the Bay. Acute field surveys of oil spill effects in harbour areas with a long history of contamination should thus be viewed with caution, and whenever possible previous evaluations should be considered for proper appraisal of biomarker sensitivity, especially in mobile bioindicators such as fish.

Evaluation of waterborne exposure to oil spill 5 years after an accident in Southern Brazil

Fish (Astyanax sp.) were exposed to water samples from the Arroio Saldanha, locality of an oil spill, and the Barigui River (upstream and downstream of the confluence of the river and Arroio Saldanha), Brazil. Histopathological and biochemical biomarkers, as acetylcholinesterase (AchE), ethoxyresorufin-O-deethylase (EROD), catalase (CAT), glutathione-S-transferase (GST) activities, lipid peroxidation (LPO) levels, and amounts of polynuclear aromatic hydrocarbon residues (PAHs) in bile were evaluated. PAHs, metals, and surfactants were investigated in the water samples. CAT and GST activities increased in the Arroio Saldanha sample, whereas CAT activity and LPO levels were increased in the upstream and downstream sites. The results suggested a toxic action of the free radicals and disturbance of the antioxidant defense mechanisms. However, the EROD and AchE activities were not affected. Histopathology studies showed severe lesions. The oil is still bioavailable to biota, moreover, other pollution sources continue to affect the water in the Barigui River.

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.

Subchronic effects of dipyrone on the fish species Rhamdia quelen

The use of the non-steroidal anti-inflammatory drugs such as dipyrone is so widespread that this drug and its metabolites have been detected in effluents and surface water. This study aimed to evaluate the potential toxic effects of dipyrone on the aquatic environment, using a native fish species, Rhamdia quelen. Fish were exposed to three concentrations of dipyrone, 0.5, 5 and 50 μg/L, in the water for 15 days, and hematological, biochemical, genetic and morphological biomarkers were evaluated. The glutathione S-transferase activity decreased in the highest concentration in relation to the control group. In addition, hematocrit, red blood cells and thrombocyte counts were decreased in all three exposed groups in relation to the control group. The comet assay showed DNA damage at the lowest concentration of dipyrone and significant kidney damage. Those results suggest that a constant exposure of aquatic organisms to dipyrone presents potential toxic effects.

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.

Effects of ecologically relevant concentrations of cadmium in a freshwater fish

Sub-chronic effects of ecologically relevant concentrations of cadmium (Cd) were evaluated in the catfish Rhamdia quelen. The fish were exposed to Cd (0, 0.1, 1, 10 and 100μgL(-1)) for 15 days. Bioconcentration was observed in the liver of fish exposed to 10 and 100μgL(-1) of cadmium. The liver glutathione S-transferase activity decreased at 0.1 and 1μgL(-1) and increased at 100μgL(-1) and lipoperoxidation increased in all tested concentrations. Fish exposed to 0.1, 1 and 100µgL(-1) Cd presented increase in hepatic lesion index. In the kidney, the catalase activity and LPO reduced in all exposed groups. The gluthatione peroxidase, etoxiresorufin-O-deethylase activities and metallothionein increased at the highest concentration of Cd, but the level of reduced glutathione decreased. The genotoxicity was observed at 0.1 and 100μgL(-1). Neurotoxicity was not observed. The results showed that low concentrations (range of μgL(-1)) of Cd caused hepato-, nephro- and hematological alterations in this freshwater fish species.

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.

Biochemical changes in the liver and gill of Cathorops spixii collected seasonally in two Brazilian estuaries under varying influences of anthropogenic activities

In order to understand environmental health by the use of a bioindicator species in estuaries, biochemical responses observed in the catfish Cathorops spixii such as catalase (CAT), glutathione-S-transferase (GST), lipid peroxidation (LPO) and acetylcholinesterase (AChE) were evaluated in liver and muscle. Furthermore, histological changes were also verified in liver and gills preparations. Fish were collected in three sites of the Santos-São Vicente estuary located at São Paulo (Brazil), subjected to varying levels of inputs of pollutants. For a reference site, specimens were sampled at Cananéia estuary at southern coast of São Paulo, a region with low anthropogenic influence. In general, no significant seasonal differences in antioxidant enzymes and lipid peroxidation responses were found in the organisms from the Cananéia estuary. However, in the polluted estuary (Santos-São Vicente), biochemical responses were observed by increases in GST hydroperoxides and decreases in AChE activities in the summer. Inhibition of AChE expression in fish from different areas of the Santos-São Vicente estuary in the summer was also found and can indicate neurotoxic effects in these organisms. Histopathological observation of gill and liver showed severe lesions, such as lamellar fusion and necrosis.