First Novel Workflow for Semiquantification of Emerging Contaminants in Environmental Samples Analyzed by Gas Chromatography-Atmospheric Pressure Chemical Ionization-Quadrupole Time of Flight-Mass Spectrometry.

The ionization efficiency of emerging contaminants was modeled for the first time in gas chromatography-high-resolution mass spectrometry (GC-HRMS) which is coupled to an atmospheric pressure chemical ionization source (APCI). The recent chemical space has been expanded in environmental samples such as soil, indoor dust, and sediments thanks to recent use of high-resolution mass spectrometric techniques; however, many of these chemicals have remained unquantified. Chemical exposure in dust can pose potential risk to human health, and semiquantitative analysis is potentially of need to semiquantify these newly identified substances and assist with their risk assessment and environmental fate. In this study, a rigorously tested semiquantification workflow was proposed based on GC-APCI-HRMS ionization efficiency measurements of 78 emerging contaminants. The mechanism of ionization of compounds in the APCI source was discussed via a simple connectivity index and topological structure. The quantitative structure-property relationship (QSPR)-based model was also built to predict the APCI ionization efficiencies of unknowns and later use it for their quantification analyses. The proposed semiquantification method could be transferred into the household indoor dust sample matrix, and it could include the effect of recovery and matrix in the predictions of actual concentrations of analytes. A suspect compound, which falls inside the application domain of the tool, can be semiquantified by an online web application, free of access at http://trams.chem.uoa.gr/semiquantification/.

Development and Application of Liquid Chromatographic Retention Time Indices in HRMS-Based Suspect and Nontarget Screening.

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/.

Assessment of the chemical pollution status of the Dniester River Basin by wide-scope target and suspect screening using mass spectrometric techniques.

The quality of the Dniester River Basin has been seriously impacted by the chemicals released by agriculture, industry, and wastewater discharges. To assess its current chemical pollution status, a transboundary monitoring campaign was conducted in May 2019. Thirteen surface water, 13 sediment, and three biota samples were collected and analyzed using generic sample preparation methods for the determination of organic substances by liquid chromatography high-resolution mass spectrometry (LC-HRMS) and metals by inductively coupled plasma mass spectrometry (ICP-MS). Wide-scope target and suspect screening resulted in detection of Water Framework Directive (WFD) priority substances and emerging contaminants, whereas the raw data were stored in NORMAN Digital Sample Freezing Platform (DSFP) for future retrospective screening. Furthermore, risk assessment was performed to prioritize detected substances and propose a draft list of river basin-specific pollutants. All studied metals (As, Hg, Zn, Cu, Cr, Cd, Pb, Ni) were detected in the surface water and sediments. In total, 139 organic contaminants belonging to various chemical classes (pesticides, pharmaceuticals, drugs of abuse, stimulants, sweeteners, industrial chemicals, and their transformation products) were detected. The highest cumulative concentration of contaminants was observed in surface water from the Byk River, a tributary of the Dniester (Moldova). Concentrations of WFD priority substances diuron and mercury and EU Watch List neonicotinoid compounds imidacloprid and thiamethoxam exceeded their environmental quality standards (EQS), whereas concentrations of 23 emerging substances exceeded their predicted no-effect concentration (PNEC) at minimum one site. Emerging contaminants telmisartan, metolachlor, terbuthylazine, and 4-acetamidoantipyrine were prioritized as potential river basin-specific pollutants. Graphical abstract.

Exploring the Potential of a Global Emerging Contaminant Early Warning Network through the Use of Retrospective Suspect Screening with High-Resolution Mass Spectrometry.

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.

Reflection of Socioeconomic Changes in Wastewater: Licit and Illicit Drug Use Patterns.

The economic crisis plaguing Greece was expected to impact consumption of pharmaceuticals and illicit drugs - a priori to an unknown extent. We quantified the change of use for various classes of licit and illicit drugs by monitoring Athens' wastewater from 2010 to 2014. A high increase in the use of psychoactive drugs was detected between 2010 and 2014, especially for antipsychotics (35-fold), benzodiazepines (19-fold), and antidepressants (11-fold). This directly reflects the perceived increase of incidences associated with mental illnesses in the population, as a consequence of severe socioeconomic changes. Other therapeutic classes, like antiepileptics, hypertensives, and gastric and ulcer drugs also showed an increase in use (from 2-fold increase for antiepileptics to 13-fold for hypertensives). In contrast, the overall use of antibiotics and NSAIDs decreased. For mefenamic acid, an almost 28-fold decrease was observed. This finding is likely related to the reduction in drug expenditure applied in public health. A 2-fold increase of methamphetamine use was detected, associated with a cheap street drug called ″sisa″ (related to marginal conducts), which is a health concern. MDMA (5-fold) and methadone (7-fold) use showed also an increase, while cocaine and cannabis estimates did not show a clear trend.

Olive oil authenticity studies by target and nontarget LC-QTOF-MS combined with advanced chemometric techniques.

Food analysis is continuously requiring the development of more robust, efficient, and cost-effective food authentication analytical methods to guarantee the safety, quality, and traceability of food commodities with respect to legislation and consumer demands. Hence, a novel reversed-phase ultra high performance liquid chromatography-electrospray ionization quadrupole time of flight tandem mass spectrometry analytical method was developed that uses target, suspect, and nontarget screening strategies coupled with advanced chemometric tools for the investigation of the authenticity of extra virgin olive oil. The proposed method was successfully applied in real olive oil samples for the identification of markers responsible for the sensory profile. The proposed target analytical method includes the determination of 14 phenolic compounds and demonstrated low limits of detection ranging from 0.015 µg mL-1 (apigenin) to 0.039 µg mL-1 (vanillin) and adequate recoveries (96-107 %). A suspect list of 60 relevant compounds was compiled, and suspect screening was then applied to all the samples. Semiquantitation of the suspect compounds was performed with the calibration curves of target compounds having similar structures. Then, a nontarget screening workflow was applied with the aim to identify additional compounds so as to differentiate extra virgin olive oils from defective olive oils. Robust classification-based models were built with the use of supervised discrimination techniques, partial least squares-discriminant analysis and counterpropagation artificial neural networks, for the classification of olive oils into extra virgin olive oils or defective olive oils. Variable importance in projection scores were calculated to select the most significant features that affect the discrimination. Overall, 51 compounds were identified and suggested as markers, among which 14, 26, and 11 compounds were identified by target, suspect, and nontarget screening respectively. Retrospective analysis was also performed and identified 19 free fatty acids. Graphical Abstract Development of a novel RP-LC-ESI-QTOFMS analytical method employing target, suspect and non-target screening strategies coupled to advanced chemometric tools for the investigation of markers responsible for the sensory profile of extra virgin olive oil and guarantee authenticity.

Wide-Scope Target and Suspect Screening of Antibiotics in Effluent Wastewater from Wastewater Treatment Plants in Europe.

The occurrence of antibiotics in the environment could result in the development of antibiotic-resistant bacteria, which could result in a public health crisis. The occurrence of 676 antibiotics and the main transformation products (TPs) was investigated in the 48 wastewater treatment plants (WWTPs) from 11 countries (Germany, Romania, Serbia, Croatia, Slovenia, Hungary, Slovakia, Czechia, Austria, Cyprus, and Greece) by target and suspect screening. Target screening involved the investigation of antibiotics with reference standards (40 antibiotics). Suspect screening covered 676 antibiotics retrieved from the NORMAN Substance Database (antibiotic list on NORMAN network). Forty-seven antibiotics were detected in effluent wastewater samples: thirty-two by target screening and fifteen additional ones by suspect screening. An ecotoxicological risk assessment was performed based on occurrence data and predicted no effect concentration (PNEC), which involved the derivation of frequency of appearance (FoA), frequency of PNEC exceedance (FoE), and extent of PNEC exceedance (EoE). Azithromycin, erythromycin, clarithromycin, ofloxacin, and ciprofloxacin were prioritized as the calculated risk score was above 1. The median of antibiotics' load to freshwater ecosystems was 0.59 g/day/WWTP. The detection of antibiotics across countries indicates the presence of antibiotics in the ecosystems of Europe, which may trigger unwanted responses from the ecosystem, including antibiotic resistance.

What is in the fish? Collaborative trial in suspect and non-target screening of organic micropollutants using LC- and GC-HRMS.

A collaborative trial involving 16 participants from nine European countries was conducted within the NORMAN network in efforts to harmonise suspect and non-target screening of environmental contaminants in whole fish samples of bream (Abramis brama). Participants were provided with freeze-dried, homogenised fish samples from a contaminated and a reference site, extracts (spiked and non-spiked) and reference sample preparation protocols for liquid chromatography (LC) and gas chromatography (GC) coupled to high resolution mass spectrometry (HRMS). Participants extracted fish samples using their in-house sample preparation method and/or the protocol provided. Participants correctly identified 9-69 % of spiked compounds using LC-HRMS and 20-60 % of spiked compounds using GC-HRMS. From the contaminated site, suspect screening with participants' own suspect lists led to putative identification of on average ∼145 and ∼20 unique features per participant using LC-HRMS and GC-HRMS, respectively, while non-target screening identified on average ∼42 and ∼56 unique features per participant using LC-HRMS and GC-HRMS, respectively. Within the same sub-group of sample preparation method, only a few features were identified by at least two participants in suspect screening (16 features using LC-HRMS, 0 features using GC-HRMS) and non-target screening (0 features using LC-HRMS, 2 features using GC-HRMS). The compounds identified had log octanol/water partition coefficient (KOW) values from -9.9 to 16 and mass-to-charge ratios (m/z) of 68 to 761 (LC-HRMS and GC-HRMS). A significant linear trend was found between log KOW and m/z for the GC-HRMS data. Overall, these findings indicate that differences in screening results are mainly due to the data analysis workflows used by different participants. Further work is needed to harmonise the results obtained when applying suspect and non-target screening approaches to environmental biota samples.

Application of a novel prioritisation strategy using non-target screening for evaluation of temporal trends (1969-2017) of contaminants of emerging concern (CECs) in archived lynx muscle tissue samples.

Most environmental monitoring studies of contaminants of emerging concern (CECs) focus on aquatic species and target specific classes of CECs. Even with wide-scope target screening methods, relevant CECs may be missed. In this study, non-target screening (NTS) was used for tentative identification of potential CECs in muscle tissue of the terrestrial top predator Eurasian lynx (Lynx lynx). Temporal trend analysis was applied as a prioritisation tool for archived samples, using univariate statistical tests (Mann-Kendall and Spearman rank). Pooled lynx muscle tissue collected from 1969 to 2017 was analysed with an eight-point time series using a previously validated screening workflow. Following peak detection, peak alignment, and blank subtraction, 12,941 features were considered for statistical analysis. Prioritisation by time-trend analysis detected 104 and 61 features with statistically significant increasing and decreasing trends, respectively. Following probable molecular formula assignment and elucidation with MetFrag, two compounds with increasing trends, and one with a decreasing trend, were tentatively identified. These results show that, despite low expected concentration levels and high matrix effects in terrestrial species, it is possible to prioritise CECs in archived lynx samples using NTS and univariate statistical approaches.

Novel prioritisation strategies for evaluation of temporal trends in archived white-tailed sea eagle muscle tissue in non-target screening.

Environmental monitoring studies based on target analysis capture only a small fraction of contaminants of emerging concern (CECs) and miss pollutants potentially harmful to wildlife. Environmental specimen banks, with their archived samples, provide opportunities to identify new CECs by temporal trend analysis and non-target screening. In this study, archived white-tailed sea eagle (Haliaeetus albicilla) muscle tissue was analysed by non-targeted high-resolution mass spectrometry. Univariate statistical tests (Mann-Kendall and Spearman rank) for temporal trend analysis were applied as prioritisation methods. A workflow for non-target data was developed and validated using an artificial time series spiked at five levels with gradient concentrations of selected CECs (n = 243). Pooled eagle muscle tissues collected 1965-2017 were then investigated with an eight-point time series using the validated screening workflow. Following peak detection, peak alignment, and blank subtraction, 14 409 features were considered for statistical analysis. Prioritisation by time-trend analysis detected 207 features with increasing trends. Following unequivocal molecular formula assignment to prioritised features and further elucidation with MetFrag and EU Massbank, 13 compounds were tentatively identified, of which four were of anthropogenic origin. These results show that it is possible to prioritise new CECs in archived biological samples using univariate statistical approaches.

Screening of legacy and emerging substances in surface water, sediment, biota and groundwater samples collected in the Siverskyi Donets River Basin employing wide-scope target and suspect screening.

Siverskyi Donets is the fourth longest river in Ukraine and its ecosystem is heavily affected by numerous agricultural and industrial activities. An impact of the on-going armed military conflicts in the Eastern Ukraine to the overall pollution by the chemicals has been studied. Considering the uncontrolled activities in the catchment due to the conflict, there is a high demand to assess the contamination status of the Siverskyi Donets basin. In this study, the occurrence of the EU Water Framework Directive priority substances, selected physicochemical parameters and wide-range emerging contaminants were investigated in surface water, groundwater, biota and river sediments samples from 13 sampling sites in the river basin. The study included metals, inorganic, non-polar and polar organic contaminants. The wide-scope target screening of 2316 substances and suspect screening of 2219 substances revealed occurrence of 83 compounds in the studied samples. A few industrial chemicals such as plasticizers bisphenol A and DEHP, as well as flame retardant brominated diphenylethers were found to be potentially hazardous to the ecosystem, exceeding the established legacy environmental quality standards (EQS) or the provisional no-effect concentration (PNEC) values. River sediment samples contained traces of long-term banned chemicals such as polychlorinated biphenyls (PCBs) and degradation products of DDT (p,p'-DDD and p,p'-DDE). A simplified risk assessment based on comparison of measured concentration of the detected compounds against their (eco)toxicity threshold values from the NORMAN Ecotoxicology Database has been performed to aid their prioritization in future monitoring and, eventually, establishing the list of Siverskyi Donets River Basin Specific Pollutants. A comparison with the recent similar studies in the Dniester and Dnieper river basins in Ukraine has shown that the overall pollution by chemicals in the Siverskyi Donets basin is significantly lower.

European scale assessment of the potential of ozonation and activated carbon treatment to reduce micropollutant emissions with wastewater.

Micropollutants (MPs) in wastewater pose a growing concern for their potential adverse effects on the receiving aquatic environment, and some countries have started requiring that wastewater treatment plants remove them to a certain extent. Broad spectrum advanced treatment processes, such as ozonation, activated carbon or their combination, are expected to yield a significant reduction in the toxicity of effluents. Here we quantify the reduction of effluent toxicity potentially achieved by implementing these advanced treatment solutions in a selection of European wastewater treatment plants. To this end, we refer to a list of "total pollution proxy substances" (TPPS) composed of 1337 chemicals commonly found in wastewater effluents according to a compilation of datasets of measured concentrations. We consider these substances as an approximation of the "chemical universe" impinging on the European wastewater system. We evaluate the fate of the TPPS in conventional and advanced treatment plants using a compilation of experimental physicochemical properties that describe their sorption, volatilization and biodegradation during activated sludge treatment, as well as known removal efficiency in ozonation and activated carbon treatment, while filling the gaps through in silico prediction models. We estimate that the discharge of micropollutants with wastewater effluents in the European Union has a cumulative MP toxicity to the environment equal to the discharge of untreated wastewater of ca. 160 million population equivalents (PE), i.e. about 30 % of the generated wastewater in the EU. If all plants above a capacity of 100,000 PE were equipped with advanced treatment, we show that this load would be reduced to about 95 million PE. In addition, implementing advanced treatment in wastewater plants above 10,000 PE discharging to water bodies with an average dilution ratio smaller than 10 would yield a widespread improvement in terms of exposure of freshwater ecosystems to micropollutants, almost halving the part of the stream network exposed to the highest toxic risks. Our analysis provides background for a cost-effectiveness appraisal of advanced treatment "at the end of the pipe", which could lead to optimized interventions. This should not be regarded as a stand-alone solution, but as a complement to policies for the control of emissions at the source for the most problematic MPs.

Wide-scope target screening of >2000 emerging contaminants in wastewater samples with UPLC-Q-ToF-HRMS/MS and smart evaluation of its performance through the validation of 195 selected representative analytes.

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 (CCß) 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.

Evaluation of chemical and biological contaminants of emerging concern in treated wastewater intended for agricultural reuse.

The occurrence of chemical and biological contaminants of emerging concern (CECs) was investigated in treated wastewater intended for reuse in agriculture. An agarose hydrogel diffusion-based passive sampler was exposed to the outlet of a wastewater treatment plant (WWTP) located in Cyprus, which is equipped with membrane bioreactor (MBR). Passive samplers in triplicate were exposed according to a time-series exposure plan with maximum exposure duration of 28 days. Composite flow-proportional wastewater samples were collected in parallel with the passive sampling exposure plan and were processed by solid phase extraction using HORIZON SPE-DEX 4790 and the same sorbent material (Oasis HLB) as in the passive sampler. The analysis of passive samplers and wastewater samples enabled (i) the field-scale calibration of the passive sampler prototype by the calculation of in situ sampling rates of target substances, and (ii) the investigation of in silico predicted transformation products of the four most ecotoxicologically hazardous antibiotics (azithromycin, clarithromycin, erythromycin, ofloxacin). Additionally, the wastewater samples were subjected to the analysis of seven preselected antibiotic resistant genes (ARGs) and one mobile resistant element (int1). All extracts were analyzed for chemicals in a single batch using a highly sensitive method for pharmaceuticals, antibiotics and illicit drugs by liquid chromatography tandem MS/MS (LC-QQQ) and for various other target compounds (2316 compounds in total) by liquid chromatography high-resolution mass spectrometry (LC-HRMS). 279 CECs and all investigated ARGs (except for blaCTX-M-32) were detected, highlighting potential chemical and biological hazards related to wastewater reuse practices. 16 CECs were prioritized following ecotoxicological risk assessment, whereas sul1 and the mobile resistant element (int1) showed the highest abundance. Comprehensive monitoring efforts using novel sampling methods such as passive sampling, wide-scope target screening and molecular analysis are required to assure safe application of wastewater reuse and avoid spread and crop uptake of potentially hazardous chemicals.

Spatio-temporal assessment of illicit drug use at large scale: evidence from 7 years of international wastewater monitoring.

BACKGROUND AND AIMS: Wastewater-based epidemiology is an additional indicator of drug use that is gaining reliability to complement the current established panel of indicators. The aims of this study were to: (i) assess spatial and temporal trends of population-normalized mass loads of benzoylecgonine, amphetamine, methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA) in raw wastewater over 7 years (2011-17); (ii) address overall drug use by estimating the average number of combined doses consumed per day in each city; and (iii) compare these with existing prevalence and seizure data. DESIGN: Analysis of daily raw wastewater composite samples collected over 1 week per year from 2011 to 2017. SETTING AND PARTICIPANTS: Catchment areas of 143 wastewater treatment plants in 120 cities in 37 countries. MEASUREMENTS: Parent substances (amphetamine, methamphetamine and MDMA) and the metabolites of cocaine (benzoylecgonine) and of Δ9 -tetrahydrocannabinol (11-nor-9-carboxy-Δ9 -tetrahydrocannabinol) were measured in wastewater using liquid chromatography-tandem mass spectrometry. Daily mass loads (mg/day) were normalized to catchment population (mg/1000 people/day) and converted to the number of combined doses consumed per day. Spatial differences were assessed world-wide, and temporal trends were discerned at European level by comparing 2011-13 drug loads versus 2014-17 loads. FINDINGS: Benzoylecgonine was the stimulant metabolite detected at higher loads in southern and western Europe, and amphetamine, MDMA and methamphetamine in East and North-Central Europe. In other continents, methamphetamine showed the highest levels in the United States and Australia and benzoylecgonine in South America. During the reporting period, benzoylecgonine loads increased in general across Europe, amphetamine and methamphetamine levels fluctuated and MDMA underwent an intermittent upsurge. CONCLUSIONS: The analysis of wastewater to quantify drug loads provides near real-time drug use estimates that globally correspond to prevalence and seizure data.

Untargeted time-pattern analysis of LC-HRMS data to detect spills and compounds with high fluctuation in influent wastewater.

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.

Characterization of wastewater effluents in the Danube River Basin with chemical screening, in vitro bioassays and antibiotic resistant genes analysis.

Averaged 7-day composite effluent wastewater samples from twelve wastewater treatment plants (WWTPs) in nine countries (Romania, Serbia, Hungary, Slovenia, Croatia, Slovakia, Czechia, Austria, Germany) in the Danube River Basin were collected. WWTPs' selection was based on countries' dominant technology and a number of served population with the aim to get a representative holistic view of the pollution status. Samples were analyzed for 2248 chemicals of emerging concern (CECs) by wide-scope target screening employing LC-ESI-QTOF-MS. 280 compounds were detected at least in one sample and quantified. Spatial differences in the concentrations and distribution of the compounds classes were discussed. Additionally, samples were analyzed for the possible agonistic/antagonistic potencies using a panel of in vitro transactivation reporter gene CALUX® bioassays including ERα (estrogenics), anti-AR (anti-androgens), GR (glucocorticoids), anti-PR (anti-progestins), PPARα and PPARγ (peroxisome proliferators) and PAH assays. The potency of the wastewater samples to cause oxidative stress and induce xenobiotic metabolism was determined using the Nrf2 and PXR CALUX® bioassays, respectively. The signals from each of the bioassays were compared with the recently developed effect-based trigger values (EBTs) and thus allowed for allocating the wastewater effluents into four categories based on their measured toxicity, proposing a putative action plan for wastewater operators. Moreover, samples were analyzed for antibiotics and 13 antibiotic-resistant genes (ARGs) and one mobile genetic element (intl1) with the aim to assess the potential for antibiotic resistance. All data collected from these various types of analysis were stored in an on-line database and can be viewed via interactive map at https://norman-data.eu/EWW_DANUBE.

Occurrence and potential environmental risk of surfactants and their transformation products discharged by wastewater treatment plants.

Seven-day composite effluent samples from a German monitoring campaign including 33 conventional wastewater treatment plants (WWTP) were analyzed for linear alkylbenzene sulfonates (LAS) and alkyl ethoxysulfates (AES) and were screened by wide-scope suspect screening for 1564 surfactants and their transformation products (TPs) by UHPLC-ESI-QTOF-MS. Corresponding seven-day composite influent samples of selected WWTPs showed high influent concentrations as well as very high removal rates for LAS and AES. However, average total LAS and AES effluent concentrations were still 14.4 µg/L and 0.57 µg/L, respectively. The LAS-byproducts di-alkyl tetralin sulfonates (DATSs), the TPs sulfophenyl alkyl carboxylic acids (SPACs) and sulfo-tetralin alkyl carboxylic acids (STACs) reached maximum effluent concentrations of 19 µg/L, 17 µg/L and 5.3 µg/L, respectively. In many cases the sum of the concentration of all LAS-related byproducts and TPs surpassed the concentration of the precursors. High concentrations of up to 7.4 µg/L were found for 41 polyethylenoglycol homologs. Quantified surfactants and their TPs and by-products together accounted for concentrations up to 82 µg/L in WWTP effluents. To determine the risk of individual surfactants and their mixtures, single homologs were grouped by a "weighted carbon number approach" to derive normalized Predicted No-Effect Concentrations (PNEC), based on experimental ecotoxicity data from existing risk assessments, complemented by suitable Quantitative Structure-Activity Relationships (QSAR) predictions. Predicted Environmental Concentrations (PEC) were derived by dividing effluent concentrations of surfactants by local dilution factors. Risks for all analyzed surfactants were below the commonly accepted PEC/PNEC ratio of 1 for single compounds, while contributions to mixture toxicity effects from background levels of LAS and DATS cannot be excluded. Maximum LAS concentrations exceeded half of its PNEC, which may trigger country-wide screening to investigate potential environmental risks.

Two stage algorithm vs commonly used approaches for the suspect screening of complex environmental samples analyzed via liquid chromatography high resolution time of flight mass spectroscopy: A test study.

LC-HR-QTOF-MS recently has become a commonly used approach for the analysis of complex samples. However, identification of small organic molecules in complex samples with the highest level of confidence is a challenging task. Here we report on the implementation of a two stage algorithm for LC-HR-QTOF-MS datasets. We compared the performances of the two stage algorithm, implemented via NIVA_MZ_Analyzer™, with two commonly used approaches (i.e. feature detection and XIC peak picking, implemented via UNIFI by Waters and TASQ by Bruker, respectively) for the suspect analysis of four influent wastewater samples. We first evaluated the cross platform compatibility of LC-HR-QTOF-MS datasets generated via instruments from two different manufacturers (i.e. Waters and Bruker). Our data showed that with an appropriate spectral weighting function the spectra recorded by the two tested instruments are comparable for our analytes. As a consequence, we were able to perform full spectral comparison between the data generated via the two studied instruments. Four extracts of wastewater influent were analyzed for 89 analytes, thus 356 detection cases. The analytes were divided into 158 detection cases of artificial suspect analytes (i.e. verified by target analysis) and 198 true suspects. The two stage algorithm resulted in a zero rate of false positive detection, based on the artificial suspect analytes while producing a rate of false negative detection of 0.12. For the conventional approaches, the rates of false positive detection varied between 0.06 for UNIFI and 0.15 for TASQ. The rates of false negative detection for these methods ranged between 0.07 for TASQ and 0.09 for UNIFI. The effect of background signal complexity on the two stage algorithm was evaluated through the generation of a synthetic signal. We further discuss the boundaries of applicability of the two stage algorithm. The importance of background knowledge and experience in evaluating the reliability of results during the suspect screening was evaluated.

Occurrence and spatial distribution of 158 pharmaceuticals, drugs of abuse and related metabolites in offshore seawater.

The occurrence and spatial distribution of 158 pharmaceuticals and drugs of abuse were studied in seawater of the Eastern Mediterranean Sea (Saronikos Gulf and Elefsis Bay in central Aegean Sea). This area is affected by various anthropogenic pressures as it receives the treated wastewater of the greatest Athens area and off-shore input fluxes. This study constitutes the largest one in terms of number of analytes in this environmental compartment. It provides the first evidence on the occurrence of several pharmaceuticals in marine environment including amoxicillin, lidocaine, citalopram or tramadol, among others. 22 samples were collected at three different depths in 9 sampling stations in order to assess the presence and the spatial distribution of the target compounds. A multi-residue method based on solid phase extraction and liquid chromatography coupled to tandem mass spectrometry was developed for the determination of the 158 target substances and validated for seawater sample analysis. 38 out of the 158 target compounds were detected, 15 of them with frequencies of detection equal to or higher than 50%. The highest detected values corresponded to amoxicillin, caffeine and salicylic acid, with concentrations in the range of < 5.0-127.8 ng L(-1); 5.2-78.2 ng L(-1) and < 0.4-53.3 ng L(-1), respectively. Inputs from the wastewater treatment plant (WWTP) of Athens revealed to be the main source of pollution in the Inner Saronikos Gulf, whereas, other anthropogenic pressures such as contamination from shipping activity, industrial effluents, dredging and/or inputs from land proved to be also relevant. Τhe concentrations of some compounds varied significantly with depth suggesting that currents play an important role in the dilution of the target compounds.