Fluoxetine chronic exposure affects growth, behavior and tissue structure of zebrafish.

Fluoxetine (FLX) is among the top 100 pharmaceutical prescribed annually worldwide and consequently is often detected in wastewater treatment plant effluent and surface waters, in concentrations up to 2.7 and 0.33 µg/L, respectively. Despite the presence of FLX in surface waters, little is known about its chronic effects in fish. Thus, this study aimed at investigating the chronic toxicity of FLX to Danio rerio adults. Rate of weight gain, behavior (feeding and swimming activity) and tissue organization (liver and intestine) were evaluated, after 30 days exposure. A lower rate of weight gain was observed at 100 µg/L FLX. The food intake time decreased, showing a decrease in fish appetite. The preference for the upper aquarium layer was observed at 10 and 100 µg/L of FLX, indicating an inhibition of the stress level (anxiolytic effect). Mild to moderate damage of hepatic tissue and a decrease epithelium height and increase in villus height of intestine were observed in fish exposed to concentrations as low as 0.01 µg/L. Based on obtained results, chronic exposure of fish to FLX could affect swimming and feeding behavior and alter morphological structure of liver and intestine tissues at environmental levels.

Contribution of priority PAHs and POPs to Ah receptor-mediated activities in sediment samples from the River Elbe Estuary, Germany.

The estuary of the River Elbe between Hamburg and the North Sea (Germany) is a sink for contaminated sediment and suspended particulate matter (SPM). One major concern is the effect of human activities on the hydrodynamics, particularly the intensive dredging activities in this area that may result in remobilization of sediment-bound pollutants. The aim of this study was to identify pollutants contributing to the toxicological risk associated with re-suspension of sediments in the Elbe Estuary by use of an effect-directed analysis that combines chemical and biological analyses in with specific fractionation techniques. Sediments were collected from sites along the Elbe Estuary and a site from a small harbor basin of the Elbe Estuary that is known to be polluted. The sixteen priority EPA-PAHs were quantified in organic extracts of sediments. In addition, dioxin equivalents of sediments were investigated by use of the 7-ethoxyresorufin O-deethylase assay with RTL-W1 cells and the Ah receptor-mediated luciferase transactivation assay with H4IIE-luc cells. Quantification of the 16 priority PAHs revealed that sediments were moderately contaminated at all of the sites in the Elbe River Estuary (<0.02-0.906 µg/g dw). Sediments contained relatively small concentrations of dioxin equivalents (Bio-TEQ) with concentrations ranging from 15.5 to 322 pg/g dw, which were significantly correlated with dioxin equivalents calculated based on toxicity reference values and concentrations of PAH. The concentration of Bio-TEQ at the reference site exceeded 200,000 pg/g dw. In a potency balance the 16 PAHs explained between 47 and 118% of the Bio-TEQ in the luciferase assay, which can be explained by the constant input of PAHs bound to SPM from the upper course of the Elbe River into its estuary. Successful identification of a significant portion of dioxin-like activity to priority PAHs in complex environmental samples such as sediments has rarely been reported.

Sediment-contact fish embryo toxicity assay with Danio rerio to assess particle-bound pollutants in the Tietê River Basin (São Paulo, Brazil).

The Tietê River and its tributary Pinheiros River receive a highly complex organic and inorganic pollutants load from sanitary sewage and industrial sources, as well as agricultural and agroindustrial activities. The aim of the present study was to evaluate the embryotoxic and teratogenic effects of sediments from selected locations in the Tietê River Basin by means of the sediment contact embryo toxicity assay with Danio rerio, in order to provide a comprehensive and realistic insight into the bioavailable hazard potential of these sediment samples. Lethal and sub-lethal effects were recorded, and high embryo toxicity could be found in the samples not only in the vicinity of the megacity São Paulo (Billings reservoir and Pinheiros River samples), but also downstream (in the reservoirs Barra Bonita, Promissão and Três Irmãos). Results confirm that most toxicity is due to the discharges of the metropolitan area of São Paulo. However, they also indicate additional sources of pollutants along the river course, probably from industrial, agricultural and agroindustrial residues, which contribute to the degradation of each area. The sediment contact fish embryo test showed to be powerful tool to detect embryo toxicity in sediments, not only by being a sensitive method, but also for taking into account bioavailability. This test provides an ecological highly realistic and relevant exposure scenario, and should therefore be added in ecotoxicological sediment quality assessments.

Comparison of in vitro and in situ genotoxicity in the Danube River by means of the comet assay and the micronucleus test.

Genotoxicity can be correlated with adverse reproductive effects or may even result in elevated extinction risk for particular species of an ecosystem. It may thus be a valuable tool for screening of pollution and potential environmental harm. Since many genotoxicants tend to adsorb onto particulate matter, sediments and suspended materials are of particular interest for genotoxicity screening under field conditions. In order to correlate the genotoxic potential of sediments with genetic damage in fish, rainbow-trout liver (RTL-W1) cells were exposed in vitro to acetone extracts of sediments collected at 10 selected sites along the upper Danube River and analyzed in the comet and micronucleus assays. These in vitro results were compared with micronucleus formation in erythrocytes of the European barbel (Barbus barbus) caught in the field. The two in vitro bioassays showed excellent correlation, indicating comparability of genotoxic potentials in vitro. Sampling sites could be clearly differentiated with respect to severity of effects, with Rottenacker as the most heavily contaminated site, Ehingen and Schwarzach as moderately genotoxic, and with the weakest effects in the tributary Lauchert. All other sediment extracts showed intermediate genotoxic or clastogenic effects. In situ, micronucleus formation in barbel erythrocytes indicated severe genotoxicity at Rottenacker, moderate effects at Ehingen, but minor contamination at Riedlingen and Sigmaringen. In situ observations thus showed excellent correlation with corresponding in vitro tests and document the ecological relevance of in vitro studies with sediment extracts. With respect to the ecological status of the Danube River, the results overall indicate a moderate to severe genotoxic potential with a highly differential localization.

Sediment genotoxicity in the Tietê River (São Paulo, Brazil): in vitro comet assay versus in situ micronucleus assay studies.

The in vitro comet assay with the permanent fish cell line RTL-W1 and the in situ micronucleus assay using erythrocytes from indigenous tilapia (Oreochromis niloticus) were used to detect genotoxicity in Tietê River sediments (São Paulo, Brazil). Either test was successful in identifying site-specific differences in genotoxicity, with a high correlation between in situ and in vitro results indicating the relevance of the latter even for environmental studies. Discharges from São Paulo city have major impact on genotoxic effects by sediment-bound contaminants; however, overall genotoxicity decreases downstream. The high genotoxic burden of the Tietê River warrants measures to reduce the input of toxic effluents.

Perfluorooctane sulfonate increases the genotoxicity of cyclophosphamide in the micronucleus assay with V79 cells. Further proof of alterations in cell membrane properties caused by PFOS.

Perfluorooctane sulfonate (PFOS; C8F17SO3-) is a fully fluorinated organic compound which has been manufactured for decades and was used widely in industrial and commercial products. The recent toxicological knowledge of PFOS mainly concerns mono-substance exposures of PFOS to biological systems, leaving the potential interactive effects of PFOS with other compounds as an area where understanding is significantly lacking. However, a recent study, reported the potential of PFOS to enhance the toxicity of two compounds by increasing cell membrane permeability. This is of particular concern since PFOS has been reported to be widely distributed in the environment where contaminants are known to occur in complex mixtures. In this study, PFOS was evaluated alone and in combination with cyclophosphamide (CPP) to investigate whether a presence of PFOS leads to an increased genotoxic potential of CPP towards hamster lung V79 cells. Genotoxicity was investigated using the micronucleus (MN) assay according to the recent draft ISO/DIS 21427-2 method. PFOS alone demonstrated no genotoxicity up to a concentration of 12.5 microg/ml. However, PFOS combined with two different concentrations of CPP, with metabolic activation, caused a significant increase in the number of micronucleated cells compared to treatments with CPP alone. These results provide a first indication that PFOS has the potential to enhance the genotoxic action of CPP towards V79 cells, suggesting, together with the alterations in cell membrane properties shown previously, that genotoxicity of complex mixtures may be increased significantly by changes in chemical uptake. Together with an earlier study performed by the own working group, it can be concluded that PFOS alone is not genotoxic in this bioassay using V79 cells up to 12.5 microg/ml, but that further investigations are needed to assess the potential interaction between PFOS and other substances, in particular regarding the impact of membrane alterations on the uptake of toxic substances.