An analytical method was developed for determining macrolide antibiotics in treated wastewater effluents and in ambient water based on solid-phase extraction and LC/MS analysis as well as on LC/MS/MS for structural confirmation. In wastewater treatment plants (WWTPs) macrolides are only partly eliminated and can therefore reach the aquatic environment. In treated effluents from three WWTPs in Switzerland, clarithromycin, roxithromycin, and erythromycin-H2O, the main degradation product of erythromycin, were found. The most abundant, clarithromycin, reflects the consumption pattern of macrolide antibiotics. Summer concentrations of clarithromycin varied between 57 and 330 ng/L in treated WWTP effluents. In the WWTP Kloten-Opfikon seasonal differences revealed a load two times higher in winter than in summer. The higher abundance of erythromycin-H2O in the effluent of WWTP Kloten-Opfikon can be explained by distinct consumption patterns due to the main international airport of Switzerland in the catchment area. In the Glatt River clarithromycin reached concentrations of up to 75 ng/L. Mass flux determinations in treated effluents and in river water in the Glatt Valley watershed showed that elimination of clarithromycin along the river stretch of 36 km is insignificant (<20%). Investigations in the Glatt River before and after the diversion of the largest WWTP revealed an observable decrease in clarithromycin loads.

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An analytical method has been developed and validated for the simultaneous trace determination of four macrolide antibiotics, six sulfonamides, the human metabolite N4-acetylsulfamethoxazole, and trimethoprim in wastewater. The method was validated for tertiary, secondary, and-unlike in previously published methods-also for primary effluents of municipal wastewater treatment plants. This wide range of application is necessary to thoroughly investigate the occurrence and fate of chemicals in wastewater treatment. Wastewater samples were enriched by solid-phase extraction, followed by reversed-phase liquid chromatography coupled to tandem mass spectrometry using positive electrospray ionization. Recoveries from all sample matrixes were generally above 80%, and the combined measurement uncertainty varied between 2 and 18%. Concentrations measured in tertiary effluents ranged between 10 ng/L for roxithromycin and 423 ng/L for sulfamethoxazole. Corresponding levels in primary effluents varied from 22 to 1450 ng/L, respectively. Trace amounts of these emerging contaminants reach ambient waters, since all analytes were not fully eliminated during conventional activated sludge treatment followed by sand filtration. In the case of sulfamethoxazole, the amount present as human metabolite N4-acetylsulfamethoxazole had to be taken into account in order to correctly assess the fate of sulfamethoxazole in wastewater treatment.

Here we describe results from a proteomic study of protein-nanoparticle interactions to further the understanding of the ecotoxicological impact of silver nanoparticles (AgNPs) in the environment. We identified a number of proteins from Escherichia coli that bind specifically to bare or carbonatecoated AgNPs. Of these proteins, tryptophanase (TNase) was observed to have an especially high affinity for both surface modifications despite its low abundance in E. coli. Purified TNase loses enzymatic activity upon associating with AgNPs, suggesting that the active site may be in the vicinity of the binding site(s). TNase fragments with high affinities for both types of AgNPs were identified using matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectrometry. Differences in peptide abundance/presence in mass spectra for the two types of AgNPs suggest preferential binding of some protein fragments based on surface coating. One highbinding protein fragment contained a residue (Arg103) that is part of the active site. Ag adducts were identified for some fragments and found to be characteristic of strong binding to AgNPs rather than association of the fragments with ionic silver. These results suggest a probable mechanism for adhesion of proteins to the most commonly used commercial nanoparticles and highlight the potential effect of nanoparticle surface coating on bioavailability.

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Significance Comprehending the responses of organisms to pollutants by a systems-based approach allows characterization of molecular events and the cellular pathways that have been perturbed. However, mapping only adverse outcomes of a toxicant in an organism falls short of describing the defense response that is mounted to maintain homeostasis and resistance to the toxic insult. Our study provides the understanding of molecular mechanisms of algae in response to silver, which in turn indicates how the algae might behave in a silver contamination scenario. We have used complementary information obtained from the transcriptome, proteome, and physiology to gain mechanistic insights into the responses of Chlamydomonas reinhardtii . We show here the importance of stress and adaptive responses, especially at sublethal concentrations of pollutant.

One central goal of evolutionary biology is to explain how biological diversity emerges and is maintained in nature. Given the complexity of the phenotype and the multifaceted nature of inheritance, modern evolutionary ecological studies rely heavily on the use of molecular tools. Here, we show how molecular tools help to gain insight into the role of egg coats (i.e. the extracellular structures surrounding eggs and embryos) in evolutionary diversification. Egg coats are maternally derived structures that have many biological functions from mediating fertilization to protecting the embryo from environmental hazards. They show great molecular, structural and functional diversity across species, but intraspecific variability and the role of ecology in egg coat evolution have largely been overlooked. Given that much of the variation that influences egg coat function is ultimately determined by their molecular phenotype, cutting-edge molecular tools (e.g. proteomics, glycomics and transcriptomics), combined with functional assays, are needed for rigorous inferences on their evolutionary ecology. Here, we identify key research areas and highlight emerging molecular techniques that can increase our understanding of the role of egg coats in the evolution of biological diversity, from adaptation to speciation.

A facultative anaerobic bacterium, Pseudomonas sp. strain Chol1, degrading cholate and other bile acids was isolated from soil. We investigated how strain Chol1 grew with cholate and whether growth was affected by the toxicity of this compound. Under anoxic conditions with nitrate as electron acceptor, strain Chol1 grew by transformation of cholate to 7,12-dihydroxy-1,4-androstadiene-3,17-dione (DHADD) as end product. Under oxic conditions, strain Chol1 grew by transformation of cholate to 3,7,12-trihydroxy-9, 10-seco-1,3,5(10)-androstatriene-9,17-dione (THSATD), which accumulated in the culture supernatant before its further oxidation to CO 2 . Strain Chol1 converted DHADD into THSATD by an oxygenase-dependent reaction. Addition of cholate ( ‡10 mM) to cell suspensions of strain Chol1 caused a decrease of optical density and viable counts but aerobic growth with these toxic cholate concentrations was possible. Addition of CCCP or EDTA strongly increased the sensitivity of the cells to 10 mM cholate. EDTA also increased the sensitivity of the cells to DHADD and THSATD ( £ 1.7 mM). The toxicity of cholate and its degradation intermediates with a steroid structure indicates that strain Chol1 requires a strategy to minimize these toxic effects during growth with cholate. Apparently, the proton motive force and the outer membrane are necessary for protection against these toxic effects.