European demonstration program on the effect-based and chemical identification and monitoring of organic pollutants in European surface waters.

Growing concern about the adverse environmental and human health effects of a wide range of micropollutants requires the development of novel tools and approaches to enable holistic monitoring of their occurrence, fate and effects in the aquatic environment. A European-wide demonstration program (EDP) for effect-based monitoring of micropollutants in surface waters was carried out within the Marie Curie Initial Training Network EDA-EMERGE. The main objectives of the EDP were to apply a simplified protocol for effect-directed analysis, to link biological effects to target compounds and to estimate their risk to aquatic biota. Onsite large volume solid phase extraction of 50 L of surface water was performed at 18 sampling sites in four European river basins. Extracts were subjected to effect-based analysis (toxicity to algae, fish embryo toxicity, neurotoxicity, (anti-)estrogenicity, (anti-)androgenicity, glucocorticoid activity and thyroid activity), to target analysis (151 organic micropollutants) and to nontarget screening. The most pronounced effects were estrogenicity, toxicity to algae and fish embryo toxicity. In most bioassays, major portions of the observed effects could not be explained by target compounds, especially in case of androgenicity, glucocorticoid activity and fish embryo toxicity. Estrone and nonylphenoxyacetic acid were identified as the strongest contributors to estrogenicity, while herbicides, with a minor contribution from other micropollutants, were linked to the observed toxicity to algae. Fipronil and nonylphenol were partially responsible for the fish embryo toxicity. Within the EDP, 21 target compounds were prioritized on the basis of their frequency and extent of exceedance of predicted no effect concentrations. The EDP priority list included 6 compounds, which are already addressed by European legislation, and 15 micropollutants that may be important for future monitoring of surface waters. The study presents a novel simplified protocol for effect-based monitoring and draws a comprehensive picture of the surface water status across Europe.

Miniaturization of a transthyretin binding assay using a fluorescent probe for high throughput screening of thyroid hormone disruption in environmental samples.

Thyroid hormone (TH) disrupting compounds are potentially important environmental contaminants due to their possible adverse neurological and developmental effects on both humans and wildlife. Currently, the most successful bio-analytical method to detect and evaluate TH disruptors, which target the plasma transport of TH in environmental samples, is the radio-ligand thyroxine-transthyretin (T4-TTR) binding assay. Yet, costly materials and tedious handling procedures prevent the use of this assay in high throughput analysis that is nowadays urgently demanded in environmental quality assessment. For the first time a miniaturized fluorescence T4-TTR binding assay was developed in a 96 well microplate and tested with eight TH disrupting compounds. For most of the compounds, the sensitivity of the newly developed assay was slightly lower than the radio-ligand binding assay, however, throughput was enhanced at least 100-fold, while using much cheaper materials. The TH disrupting potency of 22 herring gull (Larus argentatus) egg extracts, collected from two different locations (Musvær and Reiaren) in Norway, was evaluated to demonstrate the applicability of the assay for environmental samples.

Non-target analysis of household dust and laundry dryer lint using comprehensive two-dimensional liquid chromatography coupled with time-of-flight mass spectrometry.

Household dust and laundry dryer lint are important indoor environmental matrices that may have notable health effects on humans due to chronic exposure. However, due to the sample complexity the studies conducted on these sample matrices until now were almost exclusively on the basis of target analysis. In this study, comprehensive two-dimensional liquid chromatography coupled with time-of-flight mass spectrometry (LC × LC-ToF MS) was applied, to enable non-target analysis of household dust as well as laundry dryer lint for the first time. The higher peak capacity and good orthogonality of LC × LC, together with reduced ion suppression in the MS enabled rapid identification of environmental contaminants in these complex sample matrices. A number of environmental contaminants were tentatively identified based on their accurate masses and isotopic patterns, including plasticizers, flame retardants, pesticides, drug metabolites, etc. The identity of seven compounds: tris(2-butoxyethyl) phosphate, tris(2-chloropropyl) phosphate, n-benzyl butyl phthalate, dibutyl phthalate, tributyl phosphate, triethyl phosphate and N, N-diethyl-meta-toluamide was confirmed using two-dimensional retention alignment and their concentrations in the samples were semi-quantitatively determined.

Rapid Screening of Acetylcholinesterase Inhibitors by Effect-Directed Analysis Using LC × LC Fractionation, a High Throughput in Vitro Assay, and Parallel Identification by Time of Flight Mass Spectrometry.

Effect-directed analysis (EDA) is a useful tool to identify bioactive compounds in complex samples. However, identification in EDA is usually challenging, mainly due to limited separation power of the liquid chromatography based fractionation. In this study, comprehensive two-dimensional liquid chromatography (LC × LC) based microfractionation combined with parallel high resolution time of flight (HR-ToF) mass spectrometric detection and a high throughput acetylcholinesterase (AChE) assay was developed. The LC × LC fractionation method was validated using analytical standards and a C18 and pentafluorophenyl (PFP) stationary phase combination was selected for the two-dimensional separation and fractionation in four 96-well plates. The method was successfully applied to identify AChE inhibitors in a wastewater treatment plant (WWTP) effluent. Good orthogonality (>0.9) separation was achieved and three AChE inhibitors (tiapride, amisulpride, and lamotrigine), used as antipsychotic medicines, were identified and confirmed by two-dimensional retention alignment as well as their AChE inhibition activity.

Comprehensive two-dimensional liquid chromatography coupled to high resolution time of flight mass spectrometry for chemical characterization of sewage treatment plant effluents.

For the first time a comprehensive two-dimensional liquid chromatography (LC×LC) system coupled with a high resolution time-of-flight mass spectrometer (HR-ToF MS) was developed and applied for analysis of emerging toxicants in wastewater effluent. The system was optimized and validated using environmental standard compound mixtures of e.g. carbamate pesticides and polycyclic aromatic hydrocarbons (PAHs), to characterize the chromatographic system, to test the stability of the retention times and orthogonality. Various stationary phases in the second dimension were compared for the LC×LC analysis of silicon rubber passive sampler extracts of a wastewater effluent. A combination of C18 and Pentafluorophenyl (PFP) was found to be most effective. Finally, the hyphenation of LC×LC with HR-ToF MS was optimized, including splitter settings, transfer of data files between the different software packages and background subtraction using instrument software tools, after which tentative identification of 20 environmental contaminants was achieved, including pesticides, pharmaceuticals and food additives. As examples, three pesticides (isoproturon, terbutryn and diazinon) were confirmed by two-dimensional retention alignment.