Monitoring estrogenic activities of waste and surface waters using a novel in vivo zebrafish embryonic (EASZY) assay: Comparison with in vitro cell-based assays and determination of effect-based trigger values.

This study reports the use of the recently developed EASZY assay that uses transgenic cyp19a1b-GFP zebrafish (Danio rerio) embryos to assess in vivo estrogenic activity of 33 surface (SW) and waste water (WW) samples collected across Europe that were previously well-characterized for estrogen hormones and in vitro estrogenic activity. We showed that 18 out of the 33 SW and WW samples induced estrogenic responses in the EASZY assay leading to a significant and concentration-dependent up-regulation of the ER-regulated cyp19a1b gene expression in the developing brain. The in vivo 17ß-estradiol-equivalents (EEQs) were highly correlated with, both, the chemical analytical risk quotient (RQ) based on steroidal estrogen concentrations and EEQs reported from five different in vitro reporter gene assays. Regression analyses between the vitro and in vivo effect concentrations allowed us to determine an optimal cut-off value for each in vitro assay, above which in vivo responses were observed. These in vitro assay-specific effect-based trigger values (EBTs), ranging from 0.28 to 0.58 ng EEQ/L define the sensitivity and specificity of the individual in vitro assays for predicting a risk associated with substances acting through the same mode of action in water samples. Altogether, this study demonstrates the toxicological relevance of in vitro-based assessment of estrogenic activity and recommends the use of such in vitro/in vivo comparative approach to refine and validate EBTs for mechanism-based bioassays.

Chemical and microbial hypotheses explaining the effect of wastewater treatment plant discharges on the nitrifying communities in freshwater sediment.

Nitrification is a microbial key step of the nitrogen cycle, which performs the oxidation of ammonium to nitrate, via nitrite. In aquatic environments, it mainly takes place in the sediment or is associated with suspended particles. Wastewater treatment plant (WTP) discharges in rivers may disrupt sediment nitrification: this impact is related to nitrogen inputs (mainly NH(4)(+) and organic nitrogen) but could also depend on the nitrifying bacteria inputs which have been proved to survive downstream WTP discharge points. The aim of the present study was to assess the effect of NH(4)(+) and nitrifying bacteria inputs on the two steps of nitrification in freshwater sediments. To avoid natural sites constraints and to control the main environmental parameters, we used microcosms to simulate a river receiving different types of WTP discharges. These microcosms were composed of five glass dual-walls reactors (6 l) containing sediment and continuously filled (controlled flow) with river water and WTP effluent. Two types of effluents were tested: a non-nitrified one (high NH(4)(+) input, very few nitrifying bacteria) and a nitrified one (low NH(4)(+) input, more nitrifying bacteria), at different effluent/freshwater ratios (0/100, 20/80, 40/60 and 80/20). Changes in the ammonium- and nitrite-oxidizing communities were assessed by the Most Probable Number method, and changes in potential ammonium-oxidizing activity and potential nitrite-oxidizing activity were determined by incubations with specific inhibitors (sodium chlorate and allylthiourea). In most of the cases the presence of effluent induced significant changes of the nitrifying bacteria densities and potential activities in the sediment. This effect indicates generally a loss of specific potential activity and in most of the time is significant for a high effluent/river water ratio (40% to 80%). In our experimental conditions and in the case of a large WTP discharge, the nitrifying potential in freshwater sediments could thus be significantly modified.

Competition between two nitrite-oxidizing bacterial populations: a model for studying the impact of wastewater treatment plant discharge on nitrification in sediment.

Abstract Nitrobacter, a ubiquitous nitrite oxidizer in natural and anthropized environments, is commonly studied as the model genus performing the second stage of nitrification. In rivers, wastewater treatment plant discharges may affect the nitrite-oxidizing activity and the responsible genera that are largely associated with sediment. We used a laboratory batch culture approach with Nitrobacter wynogradskyi ssp. agilis strain AG and Nitrobacter hamburgensis strain X(14) to characterize the possible stress effect of wastewater effluent on these populations and to study the possible competition between an effluent strain (X(14)) and a sediment strain (AG) over a 42-day incubation time. Immunofluorescence enumerations of each strain showed that they both survived and settled in the sediment, indicating that there was no significant stress effect due to chemical changes caused by the effluent. The development of the strains' density and activity was directly correlated with the available nitrite concentration. Nevertheless, the potential specific activity was not constant along the so-called mixotrophic (non-limiting nitrite concentration) and heterotrophic (nitrite depletion) conditions. This illustrates the inducibility of the nitrite oxidoreductase and indicates the metabolic versatility of the strains. In our experimental conditions, the preferentially autotrophic AG strain appeared more competitive than the preferentially mixo- or heterotrophic X(14) strain, including in heterotrophic environment.