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There is an increasing need for comparable and harmonized retention times (tR) in liquid chromatography (LC) among different laboratories, to provide supplementary evidence for the identity of compounds in high-resolution mass spectrometry (HRMS)-based suspect and nontarget screening investigations. In this study, a rigorously tested, flexible, and less system-dependent unified retention time index (RTI) approach for LC is presented, based on the calibration of the elution pattern. Two sets of 18 calibrants were selected for each of ESI+ and ESI-based on the maximum overlap with the retention times and chemical similarity indices from a total set of 2123 compounds. The resulting calibration set, with RTI set to range between 1 and 1000, was proposed as the most appropriate RTI system after rigorous evaluation, coordinated by the NORMAN network. The validation of the proposed RTI system was done externally on different instrumentation and LC conditions. The RTI can also be used to check the reproducibility and quality of LC conditions. Two quantitative structure-retention relationship (QSRR)-based models were built based on the developed RTI systems, which assist in the removal of false-positive annotations. The applicability domains of the QSRR models allowed completing the identification process with higher confidence for substances within the domain, while indicating those substances for which results should be treated with caution. The proposed RTI system was used to improve confidence in suspect and nontarget screening and increase the comparability between laboratories as demonstrated for two examples. All RTI-related calculations can be performed online at http://rti.chem.uoa.gr/. ©

Non-target analysis (NTA) employing high-resolution mass spectrometry is a commonly applied approach for the detection of novel chemicals of emerging concern in complex environmental samples. NTA typically results in large and information-rich datasets that require computer aided (ideally automated) strategies for their processing and interpretation. Such strategies do however raise the challenge of reproducibility between and within different processing workflows. An effective strategy to mitigate such problems is the implementation of inter-laboratory studies (ILS) with the aim to evaluate different workflows and agree on harmonized/standardized quality control procedures. Here we present the data generated during such an ILS. This study was organized through the Norman Network and included 21 participants from 11 countries. A set of samples based on the passive sampling of drinking water pre and post treatment was shipped to all the participating laboratories for analysis, using one pre-defined method and one locally (i.e. in-house) developed method. The data generated represents a valuable resource (i.e. benchmark) for future developments of algorithms and workflows for NTA experiments. © 2021, The Author(s).

The Partnership for Chemicals Risk Assessment (PARC) is currently under development as a joint research and innovation programme to strengthen the scientific basis for chemical risk assessment in the EU. The plan is to bring chemical risk assessors and managers together with scientists to accelerate method development and the production of necessary data and knowledge, and to facilitate the transition to next-generation evidence-based risk assessment, a non-toxic environment and the European Green Deal. The NORMAN Network is an independent, well-established and competent network of more than 80 organisations in the field of emerging substances and has enormous potential to contribute to the implementation of the PARC partnership. NORMAN stands ready to provide expert advice to PARC, drawing on its long experience in the development, harmonisation and testing of advanced tools in relation to chemicals of emerging concern and in support of a European Early Warning System to unravel the risks of contaminants of emerging concern (CECs) and close the gap between research and innovation and regulatory processes. In this commentary we highlight the tools developed by NORMAN that we consider most relevant to supporting the PARC initiative: (i) joint data space and cutting-edge research tools for risk assessment of contaminants of emerging concern; (ii) collaborative European framework to improve data quality and comparability; (iii) advanced data analysis tools for a European early warning system and (iv) support to national and European chemical risk assessment thanks to harnessing, combining and sharing evidence and expertise on CECs. By combining the extensive knowledge and experience of the NORMAN network with the financial and policy-related strengths of the PARC initiative, a large step towards the goal of a non-toxic environment can be taken. © 2020, The Author(s).

This study presents the development and validation of a comprehensive quantitative target methodology for the analysis of 2316 emerging pollutants in water based on Ultra-Performance Liquid Chromatography Quadrupole-Time-Of-Flight Mass Spectrometry (UPLC-Q-ToF-HRMS/MS). Target compounds include pesticides, pharmaceuticals, drugs of abuse, industrial chemicals, doping compounds, surfactants and transformation products, among others. The method was validated for 195 analytes, chosen to be representative of the chemical space of the target list, enabling the assessment of the performance of the method. The method involves a generic sample preparation based on mixed mode solid phase extraction, a UPLC-QTOF-MS/MS screening method using Data Independent Acquisition (DIA) mode, which provides MS and MS/MS spectra simultaneously and an elaborate strong post-acquisition evaluation of the data. The processing method was optimized to provide a successful identification rate >95 % and to minimize the number of false positive results (< 5 %). Decision limit (CCα) and detection capability (CCβ) were also introduced in the validation scheme to provide more realistic metrics on the performance of a HRMS-based wide-scope screening method. A new system of identification points (IPs) based on the one described in the Commission Decision 2002/657/EC was applied to communicate the confidence level in the identification of the analytes. This system considers retention time, mass accuracy, isotopic fit and fragmentation; taking full advantage of the capacities of the HRMS instruments. Finally, 398 contaminants were detected and quantified in real wastewater. © 2019 Elsevier B.V.

Non-target screening (NTS) including suspect screening with high resolution mass spectrometry has already shown its feasibility in detecting and identifying emerging contaminants, which subsequently triggered exposure mitigating measures. NTS has a large potential for tasks such as effective evaluation of regulations for safe marketing of substances and products, prioritization of substances for monitoring programmes and assessment of environmental quality. To achieve this, a further development of NTS methodology is required, including: (i) harmonized protocols and quality requirements, (ii) infrastructures for efficient data management, data evaluation and data sharing and (iii) sufficient resources and appropriately trained personnel in the research and regulatory communities in Europe. Recommendations for achieving these three requirements are outlined in the following discussion paper. In particular, in order to facilitate compound identification it is recommended that the relevant information for interpretation of mass spectra, as well as about the compounds usage and production tonnages, should be made accessible to the scientific community (via open-access databases). For many purposes, NTS should be implemented in combination with effect-based methods to focus on toxic chemicals. © 2019, The Author(s).

Peak prioritization plays a key role in non-target analysis of complex samples in order to focus the elucidation efforts on potentially relevant substances. The present work shows the development of a computational workflow capable of detecting compounds that exhibit large variation in intensity over time. The developed approach is based on three open-source R packages (xcms, CAMERA and TIMECOURSE) and includes the use of the statistical test Multivariate Empirical Bayes Approach to rank the compounds based on the Hotelling T2 coefficient, which is an indicator of large concentration variations of unknown components. The approach was applied to replicate series of 24 h composite flow-proportional influent wastewater samples collected during 8 consecutive days. 60 events involving unknown substances with high fluctuation over time were successfully prioritized. 14 of those compounds were tentatively identified using HRMS/MS libraries, chemical databases, in-silico fragmentation tools, and retention time prediction models. Four compounds were confirmed with standards from which two never reported before in wastewater. © 2018 Elsevier B.V.

Urban regions of the world are expanding rapidly, placing additional stress on water resources. Urban water bodies serve many purposes, from washing and sources of drinking water to transport and conduits for storm drainage and effluent discharge. These water bodies receive chemical emissions arising from either single or multiple point sources, diffuse sources which can be continuous, intermittent, or seasonal. Thus, aquatic organisms in these water bodies are exposed to temporally and compositionally variable mixtures. We have delineated source-specific signatures of these mixtures for diffuse urban runoff and urban point source exposure scenarios to support risk assessment and management of these mixtures. The first step in a tiered approach to assessing chemical exposure has been developed based on the event mean concentration concept, with chemical concentrations in runoff defined by volumes of water leaving each surface and the chemical exposure mixture profiles for different urban scenarios. Although generalizations can be made about the chemical composition of urban sources and event mean exposure predictions for initial prioritization, such modeling needs to be complemented with biological monitoring data. It is highly unlikely that the current paradigm of routine regulatory chemical monitoring alone will provide a realistic appraisal of urban aquatic chemical mixture exposures. Future consideration is also needed of the role of nonchemical stressors in such highly modified urban water bodies.

A key challenge in the environmental and exposure sciences is to establish experimental evidence of the role of chemical exposure in human and environmental systems. High resolution and accurate tandem mass spectrometry (HRMS) is increasingly being used for the analysis of environmental samples. One lauded benefit of HRMS is the possibility to retrospectively process data for (previously omitted) compounds that has led to the archiving of HRMS data. Archived HRMS data affords the possibility of exploiting historical data to rapidly and effectively establish the temporal and spatial occurrence of newly identified contaminants through retrospective suspect screening. We propose to establish a global emerging contaminant early warning network to rapidly assess the spatial and temporal distribution of contaminants of emerging concern in environmental samples through performing retrospective analysis on HRMS data. The effectiveness of such a network is demonstrated through a pilot study, where eight reference laboratories with available archived HRMS data retrospectively screened data acquired from aqueous environmental samples collected in 14 countries on 3 different continents. The widespread spatial occurrence of several surfactants (e.g., polyethylene glycols (PEGs) and C12AEO-PEGs), transformation products of selected drugs (e.g., gabapentin-lactam, metoprolol-acid, carbamazepine-10-hydroxy, omeprazole-4-hydroxy-sulfide, and 2-benzothiazole-sulfonic-acid), and industrial chemicals (3-nitrobenzenesulfonate and bisphenol-S) was revealed. Obtaining identifications of increased reliability through retrospective suspect screening is challenging, and recommendations for dealing with issues such as broad chromatographic peaks, data acquisition, and sensitivity are provided. © 2018 American Chemical Society.

High-resolution tandem mass spectrometry (HRMS2) with electrospray ionization is frequently applied to study polar organic molecules such as micropollutants. Fragmentation provides structural information to confirm structures of known compounds or propose structures of unknown compounds. Similarity of HRMS2 spectra between structurally related compounds has been suggested to facilitate identification of unknown compounds. To test this hypothesis, the similarity of reference standard HRMS2 spectra was calculated for 243 pairs of micropollutants and their structurally related transformation products (TPs); for comparison, spectral similarity was also calculated for 219 pairs of unrelated compounds. Spectra were measured on Orbitrap and QTOF mass spectrometers and similarity was calculated with the dot product. The influence of different factors on spectral similarity [e.g., normalized collision energy (NCE), merging fragments from all NCEs, and shifting fragments by the mass difference of the pair] was considered. Spectral similarity increased at higher NCEs and highest similarity scores for related pairs were obtained with merged spectra including measured fragments and shifted fragments. Removal of the monoisotopic peak was critical to reduce false positives. Using a spectral similarity score threshold of 0.52, 40% of related pairs and 0% of unrelated pairs were above this value. Structural similarity was estimated with the Tanimoto coefficient and pairs with higher structural similarity generally had higher spectral similarity. Pairs where one or both compounds contained heteroatoms such as sulfur often resulted in dissimilar spectra. This work demonstrates that HRMS2 spectral similarity may indicate structural similarity and that spectral similarity can be used in the future to screen complex samples for related compounds such as micropollutants and TPs, assisting in the prioritization of non-target compounds. [Figure not available: see fulltext.]. © 2017, American Society for Mass Spectrometry.

The existing target analytical methods described in the literature only cover a tiny fraction of the large number of compounds present in environmental samples. As a result, many potential chemical stressors are systematically omitted and there is an urgent need of analytical methodologies capable of detecting and identifying compounds using nontarget methods. In this regard, high resolution mass spectrometry coupled to liquid chromatography (LC-HRMS) has opened up new windows of opportunity for the detection of polar organic contaminants in complex samples. The objective of this chapter is to provide a critical overview of the state-of-the-art of the application of LC-HRMS to the nontarget analysis of organic contaminants in environmental samples. The existing methodologies and the different prioritisation strategies as well as workflows to characterise unknown organic pollutants are evaluated. © 2016 Elsevier B.V.

Assessing the risk posed by complex environmental samples is a challenging task that requires reliable tools for detection and evaluation of effects. Among hazardous compounds, endocrine disrupting chemicals (EDC) in mixtures need to be identified given their potential to interfere with wild life reproduction. In this context, the aim of our work is to apply a set of effect-based tools, both in vivo and in vitro, to environmental samples in order to gain a wide overview of adverse outcomes related to estrogenic pathways in fish. As part of the FP7-SOLUTIONS project, we screened surface water extracts of Danube River (22 sites) and extracts of Rhine tributaries (water extracts of 3 STP effluents, upstream, and downstream sites in Switzerland) using an in vitro human ER reporter system (MELN cell line). Additionally, Rhine tributaries extracts were assessed in vivo for induction of CYP19a1b gene, which is strictly ER-regulated, in transgenic zebrafish embryos (EASZY test). Results obtained so far with the MELN assay show very low estradiol-equivalents (E2-EQ) levels at Danube sites (only 3 sites displaying E2-EQ above 0.1ng/L), while slightly higher estrogenic activity was measured at Swiss sites (E2-EQ ranged from 2.9-5.5 ng/L in effluents, and 0.1-0.3 and 0.5-1.5 at upstream and downstream sites, respectively). The estrogenic activity detected in vitro in effluents was correlated to estrogenicity in vivo for one site, based on CYP19a1b induction; the other effluents displayed developmental toxicity which impeded reporter gene activation detection. To gather further information on estrogenicity of these samples, work is under progress to assess their effects on zebrafish (zf) cell lines stably transfected with zebrafish estrogen receptors (i.e. zfERalpha, zfERbeta1 and zfERbeta2). Results will be interpreted in relation to targeted chemical analyses done at these sites. Eventually, the study should provide a comprehensive assessment of estrogenicity of given environmental samples using a combination of mammalian and zebrafish cell lines, as well as an in vivo fish embryo bioassay. This study is supported by INERIS and by SOLUTIONS Project from the European Union Seventh Framework Programme (FP7-ENV-2013-two-stage Collaborative project) under grant agreement 603437.

Identification of transformation products (TPs) of emerging pollutants is challenging, due to the vast number of compounds, mostly unknown, the complexity of the matrices and their often low concentrations, requiring highly selective, highly sensitive techniques. We compile background information on biotic and abiotic formation of TPs and analytical developments over the past five years. We present a database of biotic or abiotic TPs compiled from those identified in recent years. We discuss mass spectrometric (MS) techniques and workflows for target, suspect and non-target screening of TPs with emphasis on liquid chromatography coupled to MS (LC-MS). Both low- and high-resolution (HR) mass analyzers have been applied, but HR-MS is the technique of choice, due to its high confirmatory capabilities, derived from the high resolving power and the mass accuracy in MS and MS/MS modes, and the sophisticated software developed. © 2014 Elsevier B.V.

In this article, a dataset from a collaborative nontarget screening trial organised by the NORMAN Association is used to review the state-of-the-art and discuss future perspectives of non-target creening using high-resolution mass spectrometry in water analysis. A total of 18 institutes from 12 European countries analysed an extract of the same water sample collected from the River Danube with either one or both of liquid and gas chromatography coupled with mass spectrometry detection. This article focuses mainly on the use of high resolution screening techniques with target, suspect, and non-target workflows to identify substances in environmental samples. Specific examples are given to emphasise major challenges including isobaric and co-eluting substances, dependence on target and suspect lists, formula assignment, the use of retention information, and the confidence of identification. Approaches andmethods applicable to unit resolution data are also discussed. Although most substances were identified using high resolution data with target and suspect-screening approaches, some participants proposed tentative non-target identifications. This comprehensive dataset revealed that non-target analytical techniques are already substantially harmonised between the participants, but the data processing remains time-consuming. Although the objective of a "fully-automated identification workflow" remains elusive in the short term, important steps in this direction have been taken, exemplified by the growing popularity of suspect screening approaches. Major recommendations to improve non-target screening include better integration and connection of desired features into software packages, the exchange of target and suspect lists, and the contribution ofmore spectra from standard substances into (openly accessible) databases. © Springer-Verlag Berlin Heidelberg 2015.

An integrated workflow based on liquid chromatography coupled to a quadrupole-time-of-flight mass spectrometer (LC-QTOF-MS) was developed and applied to detect and identify suspect and unknown contaminants in Greek wastewater. Tentative identifications were initially based on mass accuracy, isotopic pattern, plausibility of the chromatographic retention time and MS/MS spectral interpretation (comparison with spectral libraries, in silico fragmentation). Moreover, new specific strategies for the identification of metabolites were applied to obtain extra confidence including the comparison of diurnal and/or weekly concentration trends of the metabolite and parent compounds and the complementary use of HILIC. Thirteen of 284 predicted and literature metabolites of selected pharmaceuticals and nicotine were tentatively identified in influent samples from Athens and seven were finally confirmed with reference standards. Thirty four nontarget compounds were tentatively identified, four were also confirmed. The sulfonated surfactant diglycol ether sulfate was identified along with others in the homologous series (SO4C2H4(OC2H4)xOH), which have not been previously reported in wastewater. As many surfactants were originally found as nontargets, these compounds were studied in detail through retrospective analysis. © 2015 American Chemical Society.

EDA-EMERGE aims to train a new generation of young scientists in the interdisciplinary techniques required to meet the major challenges in the monitoring, assessment and management of toxic pollution in European river basins considering the enormous complexity of contamination, effects and cause-effect relationships. By integrating innovative mode-of-action based biodiagnostic tools including in vitro tests, transgenic organisms and 'omics'techniques with powerful fractionation and cutting edge analytical and computational structure elucidation tools, a new generation of effect directed analysis (EDA) approaches will be developed for the identification of toxicants in European surface and drinking waters. Innovative method development by young researchers at major universities, research centres and private companies will be closely interlinked with a joint European demonstration program and higher tier EDA and extensive training courses. EDA-EMERGE ESRs will learn to organise and run international and interdisciplinary sampling and monitoring campaigns and benefit from the expertise of one of the most experienced private companies in this field. Strong networking between academia, the private sector and leading regulators in the field of river basin management and pollution management ensures the relevance of the research for practice and excellent employment opportunities for EDA-EMERGE ESRs.

The degradation of radiolabeled 4(3',5'-dimethyl-3'-heptyl)-phenol [nonylphenol (NP)] was tested with resting cells of Sphingomonas sp. strain TTNP3. Concomitantly to the degradation of NP, a metabolite identified as hydroquinone transiently accumulated and short-chain organic acids were then produced at the expense of hydroquinone. Two other radiolabeled isomers of NP, 4(2',6'-dimethyl-2'-heptyl)-phenol and 4(3',6'-dimethyl-3'-heptyl)-phenol, were synthesized. In parallel experiments, the 4(2',6'-dimethyl-2'-heptyl)-phenol was degraded more slowly than the other isomers of NP by strain TTNP3, possibly because of effects of the side-chain structure on the kinetics of degradation. Alkylbenzenediol and alkoxyphenol derivatives identified as metabolites during previous studies were synthesized and tested as substrates. The derivatives were not degraded, which indicated that the mineralization of NP does not proceed via alkoxyphenol as the principal intermediate. The results obtained led to the elucidation of the degradation pathway of NP isomers with a quaternary alpha-carbon. The proposed mechanism is a type II ipso substitution, leading to hydroquinone and nonanol as the main metabolites and to the dead-end metabolites alkylbenzenediol or alkoxyphenol, depending on the substitution at the alpha-carbon of the carbocationic intermediate formed.