Bioavailability of pollutants sets risk of exposure to biota and human population in reservoirs from Iguaçu River (Southern Brazil).

The Iguaçu River, located at the Southern part of Brazil, has a great socioeconomic and environmental importance due to its high endemic fish fauna and its potential to generate hydroelectric power. However, Iguaçu River suffers intense discharge of pollutants in the origin of the river. In a previous report, the local environmental agency described water quality to improve along the river course. However, no study with integrated evaluation of chemical analysis and biological responses has been reported so far for the Iguaçu River. In the current study, three different Brazilian fish species (Astyanax bifasciatus, Chrenicicla iguassuensis, and Geophagus brasiliensis) were captured in the five cascading reservoirs of Iguaçu River for a multi-biomarker study. Chemical analysis in water, sediment, and muscle indicated high levels of bioavailable metals in all reservoirs. Polycyclic aromatic hydrocarbons (PAHs) were detected in the bile of the three fish species. Integration of the data through a FA/PCA analysis demonstrated the poorest environmental quality of the reservoir farthest from river's source, which is the opposite of what has been reported by the environmental agency. The presence of hazardous chemicals in the five reservoirs of Iguaçu River, their bioaccumulation in the muscle of fish, and the biological responses showed the impacts of human activities to this area and did not confirm a gradient of pollution between the five reservoirs, from the source toward Iguaçu River's mouth. Therefore, diffuse source of pollutants present along the river course are increasing the risk of exposure to biota and human populations.

Effects of realistic concentrations of TiO2 and ZnO nanoparticles in Prochilodus lineatus juvenile fish.

The impact of nanoparticles on fish health is still a matter of debate, since nanotechnology is quite recent. In this study, freshwater benthonic juvenile fish Prochilodus lineatus were exposed through water to three concentrations of TiO2 (0.1, 1, and 10 µg l(-1)) and ZnO (7, 70, and 700 µg l(-1)) nanoparticles, as well as to a mixture of both (TiO2 1 µg l(-1) + ZnO 70 µg l(-1)) for 5 and 30 days. Nanoparticle characterization revealed an increase of aggregate size in the function of concentration, but suspensions were generally stable. Fish mortality was high at subchronic exposure to 70 and 700 µg l(-1) of ZnO. Nanoparticle exposure led to decreased acetylcholinesterase activity either in the muscle or in the brain, depending on particle composition (muscle-TiO2 10 µg l(-1); brain-ZnO 7 and 700 µg l(-1)), and protein oxidative damage increased in the brain (ZnO 70 µg l(-1)) and gills (ZnO 70 µg l(-1) and mixture) but not in the liver. Exposed fish had more frequent alterations in the liver (necrosis, vascular congestion, leukocyte infiltration, and basophilic foci) and gills (hyperplasia and epithelial damages, e.g., epithelial disorganization and epithelial loss) than the control fish. Thus, predicted concentrations of TiO2 and ZnO nanoparticles caused detectable effects on P. lineatus that may have important consequences to fish health. But, these effects are much more subtle than those usually reported in the scientific literature for high concentrations or doses of metal nanoparticles.

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.

Alterations in morphometric and organosomatic indices and histopathological analyses indicative of environmental contamination in Mullet, Mugil liza, from Southeastern Brazil.

Mullet (Mugil liza) were sampled in five different areas along the Guanabara Bay, southeastern Brazil, classified as non-contaminated, moderately contaminated and contaminated. Morphometric (Fulton condition factor, relative condition factor and weight to length scaling coefficient) and organosomatic (hepatosomatic index) indices of environmental stress were analysed. Fish from the differentially contaminated areas show statistically different Fulton and relative condition factors and hepatosomatic indices, but not the weight to length scaling coefficient. The Kn and the FCF followed the same trend, with fish from São Gonçalo (1.07 ± 0.04 and 0.89 ± 0.03), Itaipu (0.84 ± 0.01 and 0.86 ± 0.01) and the Rodrigo de Freitas Lagoon (1.03 ± 0.01 and 0.87 ± 0.20) showing higher FCFs than fish from Magé (0.96 ± 0.01 and 0.81 ± 0.01). Fish from Itaipu showed significantly higher HSI values than the other sampling sites (1.68 ± 0.07), with fish from Olaria and Ipiranga showing the lowest (1.56 ± 0.12 and 1.60 ± 0.07, respectively).

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.

Responses of hepatocytes to DDT and methyl mercury exposure.

The aim of the current work was to investigate the effects of dichlorodiphenyltrichloroethane (DDT) and monomethyl mercury (MeHg) on hepatocytes from tropical fish Hypostomus commersoni (cascudo). In order to verify DDT and MeHg impacts on the redox milieu, cells were exposed for 4 days to 50 nM of DDT, 0.25 and 2.5 microM of MeHg and to a combination of 50 nM of DDT and 0.25 microM of MeHg. These concentrations were compared with those previously published (Filipak Neto et al., 2008) for the predator fish Hoplias malabaricus (traíra). The effects were mostly noticeable on reduced glutathione concentration and delta-aminolevulinic acid dehydratase and glutathione S-transferase activity. Catalase activity increased in the group exposed to 2.5 microM of MeHg and hydrogen peroxide levels decreased in all exposed groups. Also, superoxide anion levels decreased in the groups exposed to 2.5 microM of MeHg and DDT *MeHg group. Cell viability decreased only in the DDT exposed group, demonstrating that the antioxidant defense mechanism of H. commersoni hepatocytes is more efficient than H. malabaricus. These results corroborate the resistance of H. commersoni to polluted areas and support the hypothesis that this species is more resistant to DDT and MeHg than H. malabaricus species.

Vitellogenesis and other physiological responses induced by 17-beta-estradiol in males of freshwater fish Rhamdia quelen.

This study investigated the effects of different doses of 17-beta-estradiol (E(2)) in Rhamdia quelen. Groups of males exposed to different doses of E(2) (0.1 mg kg(-)(1), 1 mg kg(-)(1) and 10 mg kg(-)(1)) were compared with non-exposed male and female fish groups. Among the considered biomarkers, no significant differences were observed for micronuclei test, reduced glutathione concentration and lipid peroxidation. All E(2)-treated individuals had decreased glutathione S-transferase activity. Increased catalase and superoxide dismutase activities, increased vitellogenin expression and decreased metallothionein concentration were observed in males treated with the highest dose. Liver of all test groups showed necrotic areas, but cytoplasm vacuolization was again found only in the individuals exposed to highest dose. E(2) causes deleterious hepatic effects to R. quelen, and vitellogenin expression, catalase and superoxide dismutase activity and metallothionein concentration represent appropriate biomarkers for studying E(2) effects. Additionally, the response of some biomarkers was similar in males exposed to E(2) and unexposed females, and therefore exposure to endocrine disruptors may cause consequences for fish populations.

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.

Bioaccumulation of chlorinated pesticides and PCBs in the tropical freshwater fish Hoplias malabaricus: histopathological, physiological, and immunological findings.

For assessing the impact of chlorinated compounds, such as organochlorine pesticides, polychlorinated biphenyls, chlorotriazines (atrazine, simazine), and chlorinated phenylureas (diuron), on the Ponta Grossa lake South of Brazil, ten freshwater trahira fish (Hoplias malabaricus) were collected in October 2005. The contamination status was evaluated by the energy budget and various histopathological markers. The results showed detectable amounts of persistent organic pollutants (POPs) in the liver and muscle; the bioaccumulation was higher in the liver than in the muscle. The presence of some banned pesticides, such as hexachlorobenzene and dichlorodiphenyltrichloroethane, in the liver suggests an acute exposure to these compounds. Some physiological disturbances and morphological damages found in the liver of H. malabaricus were associated with chlorinated-compound bioaccumulation. The most important alterations in the liver were lesions such as fibrosis, large necrosis area, leukocyte infiltration, and the absence of melanomacrophages (MM). Individuals containing higher concentrations of pesticides, such as aldrin, alachlor, and dichloroaniline (a metabolite of diuron), showed the nonoccurrence of MM in the liver. These data suggest an immunosuppression in the individuals from Ponta Grossa Lake after exposure to POPs. According to the present data, the POPs found in the studied site are bioavailable, induce severe damages in target organs such as the liver, and can disturb the immune system of the trahira. This is the first study of POPs in the Paraná state, and one among the few studies in the south of Brazil. The present data suggest and motivate further chemical and biomonitoring studies in freshwater ecosystems in the south of Brazil.

Histopathological evidence of antagonistic effects of tributyltin on benzo[a]pyrene toxicity in the Arctic charr (Salvelinus alpinus).

We previously reported that long-term (54 days), repeated intraperitoneal exposure to low doses of tributyltin (TBT; 0.3 mg/kg) inhibited the metabolic activation of co-administered benzo[a]pyrene (BaP; 3 mg/kg) in the Arctic charr (Salvelinus alpinus); BaP, in turn, stimulated the metabolism and/or excretion of TBT. Here, we report the results of histopathological examinations of liver, kidney and pseudobranch tissue samples originating from these same fish. The results revealed higher lesion incidences at all sampling time points (Days 8, 32 and 56) among BaP-exposed fish compared with fish exposed to either TBT alone or combined with BaP. The severity of lesions like necrosis was also higher in BaP-exposed fish. Moreover, hepatic basophilic foci were observed exclusively in fish exposed to BaP alone. Together, these results provide new evidences that TBT can antagonize BaP toxicity in fish exposed to both pollutants under controlled laboratory conditions. In contrast, BaP does not appear to provide protection against TBT toxicity.

Losses of immunoreactive parvalbumin amacrine and immunoreactive alphaprotein kinase C bipolar cells caused by methylmercury chloride intoxication in the retina of the tropical fish Hoplias malabaricus.

To quantify the effects of methylmercury (MeHg) on amacrine and on ON-bipolar cells in the retina, experiments were performed in MeHg-exposed groups of adult trahiras (Hoplias malabaricus) at two dose levels (2 and 6 microg/g, ip). The retinas of test and control groups were processed by mouse anti-parvalbumin and rabbit anti-alphaprotein kinase C (alphaPKC) immunocytochemistry. Morphology and soma location in the inner nuclear layer were used to identify immunoreactive parvalbumin (PV-IR) and alphaPKC (alphaPKC-IR) in wholemount preparations. Cell density, topography and isodensity maps were estimated using confocal images. PV-IR was detected in amacrine cells in the inner nuclear layer and in displaced amacrine cells from the ganglion cell layer, and alphaPKC-IR was detected in ON-bipolar cells. The MeHg-treated group (6 microg/g) showed significant reduction of the ON-bipolar alphaPKC-IR cell density (mean density = 1306 +/- 393 cells/mm2) compared to control (1886 +/- 892 cells/mm2; P < 0.001). The mean densities found for amacrine PV-IR cells in MeHg-treated retinas were 1040 +/- 56 cells/mm2 (2 microg/g) and 845 +/- 82 cells/mm2 (6 microg/g), also lower than control (1312 +/- 31 cells/mm2; P < 0.05), differently from the data observed in displaced PV-IR amacrine cells. These results show that MeHg changed the PV-IR amacrine cell density in a dose-dependent way, and reduced the density of alphaKC-IR bipolar cells at the dose of 6 microg/g. Further studies are needed to identify the physiological impact of these findings on visual function.

Bioaccumulation and the effects of organochlorine pesticides, PAH and heavy metals in the Eel (Anguilla anguilla) at the Camargue Nature Reserve, France.

Pesticides (organochlorines-OC), polycyclic aromatic hydrocarbons (PAH) and heavy metals are toxic to fish and may be taken in through gills, skin and contaminated foods. Here we measure concentrations of OC, PAH and heavy metals, and their effects in the eel Anguilla anguilla from three locations in the Camargue Reserve in southern France. The Camargue Biosphere Reserve is the largest coastal wetland in Western Europe, and A. anguilla is a common predator at the top of the food chain. Livers and spleens were analyzed for histopathological, chemical and organo-somatic (HSI and SSI) effects. Gill, liver and spleen samples were collected for histopathological studies. Livers and muscles were sampled for metabolic parameters and persistent organic pollutant analysis. Total lipids were estimated by spectrophotometry and lipid-free residues were used in protein and glycogen analysis. OC pesticides were extracted from lipids of muscles and livers, analyzed by gas chromatography, and PAH from bile were analyzed by fixed wavelength fluorescence spectrofluorimetry. Heavy metals were measured by inductively coupled plasma with optical or with mass spectrometers. High concentrations of contaminants were found in eel tissues. La Capelière had the greatest OC and PAH concentrations; unexpected lesions in gills, livers and spleens were more common at the other sites. Liver and spleen tumors and lipidosis in livers were associated with chronic, and gill lesions with acute exposure. High pesticide and PAH concentrations and lesions in eels from the Camargue reserve demonstrate the contamination of the area. A more complete study in the Camargue reserve is necessary to better understand the impact on wildlife and humans. Also, this study suggests that eel biology must be better understood before continued use of this species as a biomonitor of polluted areas.

Morphological and neurotoxicological findings in tropical freshwater fish (Astyanax sp.) after waterborne and acute exposure to water soluble fraction (WSF) of crude oil.

The water-soluble fraction (WSF) of crude oil is a complex highly volatile and toxic mixture of hydrocarbon chains (polyaromatics, heterocyclics), phenols, and heterocyclic compounds containing nitrogen and sulfur. To evaluate the toxic effects of WSF in tropical freshwater teleosts and to develop methodologies that could investigate the toxic mechanisms of WSF in tropical organisms, an acute toxicity experiment was conducted with Astyanax sp. Three dilutions (15%, 33%, and 50%) of WSF obtained from Campos Bay's crude oil (Brazil) were used to study morphological and biochemical responses of the fish. Prior to exposure, the distribution and rate of volatilization of the WSF into each aquarium for the same exposure period was quantified by spectrofluorimetry. Five individuals of Astyvanax sp. were exposed to duplicate WSF of 0, 15, 33, and 50% for each of 12-, 24-, and 96-h exposures for a total of 120 individuals. Liver and gills were sampled from five fish from each treatment and were analyzed by histology, scanning and transmission electron microscopy. A fragment of muscle was also collected from each fish to measure acetylcholinesterase activity. Water analysis showed that only 4 h after dilution, an important loss of hydrocarbons in 33% and 50% of WSF was observed. In addition, 50% of hydrocarbon mass was lost in all tested dilutions after 24 h with significant difference for the 50% WSF at all measured times, demonstrating the high volatility of WSF in freshwater. Damage in the liver and the gills included the presence of necrosis, loss of hepatocytes limit, inflammation areas, cellular proliferation, aneurysms, and disorganization of the second lamellae. The 33% WSF significantly reduced acetylcholinesterase activity in fish. Our study demonstrated that the WSF of crude oil caused damage in organs and tissues of tropical freshwater Astyanax sp. and provided also the basis for a better understanding of the toxic mechanisms of WSF in freshwater fishes.