Wide-scope target screening characterization of legacy and emerging contaminants in the Danube River Basin by liquid and gas chromatography coupled with high-resolution mass spectrometry.

A state-of-the-art wide-scope target screening of 2,362 chemicals and their transformation products (TPs) was performed in samples collected within the Joint Danube Survey 4 (JDS4) performed in 2019. The analysed contaminants of emerging concern (CECs) included three major categories: plant protection products (PPPs), industrial chemicals and pharmaceuticals and personal care products (PPCPs). In total, 586 CECs were detected in the samples including 158 PPPs, 71 industrial chemicals, 348 PPCPs, and 9 other chemicals. A wide-variety of sample matrices were collected including influent and effluent wastewater, groundwater, river water, sediment and biota. Forty-five CECs (19 PPPs, 8 industrial chemicals, 18 PPCPs) were detected at levels above their ecotoxicological thresholds (lowest predicted no-effect concentration (PNEC) values) in one or more of the investigated environmental compartments, indicating potential adverse effects on the impacted ecosystems. Among them 12 are legacy substances; 33 are emerging and qualify as potential Danube River Basin Specific Pollutants (RBSPs). Moreover, the efficiency of the wastewater treatment plants (WWTPs) was evaluated using 20 selected performance indicator chemicals. WWTPs showed effective removal (removal rate ≥80%) and medium removal (removal rate 25-80%) for 6 and 8 of the indicator chemicals, respectively. However, numerous contaminants passed the WWTPs with a lower removal rate. Further investigation on performance of WWTPs is suggested at catchment level to improve their removal efficiency. WWTP effluents are proven to be one of the major sources of contaminants in the Danube River Basin (DRB). Other sources include sewage discharges, industrial and agricultural activities. Continuous monitoring of the detected CECs is suggested to ensure water quality of the studied area.

New RT-PCR Assay for the Detection of Current and Future SARS-CoV-2 Variants.

Multiple lineages of SARS-CoV-2 have been identified featuring distinct sets of genetic changes that confer to the virus higher transmissibility and ability to evade existing immunity. The continuous evolution of SARS-CoV-2 may pose challenges for current treatment options and diagnostic tools. In this study, we have first evaluated the performance of the 14 WHO-recommended real-time reverse transcription (RT)-PCR assays currently in use for the detection of SARS-CoV-2 and found that only one assay has reduced performance against Omicron. We then developed a new duplex real-time RT-PCR assay based on the amplification of two ultra-conserved elements present within the SARS-CoV-2 genome. The new duplex assay successfully detects all of the tested SARS-CoV-2 variants of concern (including Omicron sub-lineages BA.4 and BA.5) from both clinical and wastewater samples with high sensitivity and specificity. The assay also functions as a one-step droplet digital RT-PCR assay. This new assay, in addition to clinical testing, could be adopted in surveillance programs for the routine monitoring of SARS-CoV-2's presence in a population in wastewater samples. Positive results with our assay in conjunction with negative results from an Omicron-specific assay may provide timely indication of the emergence of a novel SARS-CoV-2 variant in a certain community and thereby aid public health interventions.

Lagrangian profiles of riverine autotrophy, organic matter transformation, and micropollutants at extreme drought.

On their way from inland to the ocean, flowing water bodies, their constituents and their biotic communities are exposed to complex transport and transformation processes. However, detailed process knowledge as revealed by Lagrangian measurements adjusted to travel time is rare in large rivers, in particular at hydrological extremes. To fill this gap, we investigated autotrophic processes, heterotrophic carbon utilization, and micropollutant concentrations applying a Lagrangian sampling design in a 600 km section of the River Elbe (Germany) at historically low discharge. Under base flow conditions, we expect the maximum intensity of instream processes and of point source impacts. Phytoplankton biomass and photosynthesis increased from upstream to downstream sites but maximum chlorophyll concentration was lower than at mean discharge. Concentrations of dissolved macronutrients decreased to almost complete phosphate depletion and low nitrate values. The longitudinal increase of bacterial abundance and production was less pronounced than in wetter years and bacterial community composition changed downstream. Molecular analyses revealed a longitudinal increase of many DOM components due to microbial production, whereas saturated lipid-like DOM, unsaturated aromatics and polyphenols, and some CHOS surfactants declined. In decomposition experiments, DOM components with high O/C ratios and high masses decreased whereas those with low O/C ratios, low masses, and high nitrogen content increased at all sites. Radiocarbon age analyses showed that DOC was relatively old (890-1870 years B.P.), whereas the mineralized fraction was much younger suggesting predominant oxidation of algal lysis products and exudates particularly at downstream sites. Micropollutants determining toxicity for algae (terbuthylazine, terbutryn, isoproturon and lenacil), hexachlorocyclohexanes and DDTs showed higher concentrations from the middle towards the downstream part but calculated toxicity was not negatively correlated to phytoplankton. Overall, autotrophic and heterotrophic process rates and micropollutant concentrations increased from up- to downstream reaches, but their magnitudes were not distinctly different to conditions at medium discharges.

Prevalence and circulation patterns of SARS-CoV-2 variants in European sewage mirror clinical data of 54 European cities.

For community-level monitoring, the European Commission under the EU Sewage Sentinel System recommends wastewater-based SARS-CoV-2 surveillance. Tracking SARS-CoV-2 variants in a community is pivotal for appropriate public health response. Genome sequencing of SARS-CoV-2 in wastewater samples for tracking variants is challenging, often resulting in low coverage genome sequences, thereby impeding the detection of the SARS-CoV-2 mutations. Therefore, we aimed at high-coverage SARS-CoV-2 genome sequences from sewage samples which we successfully accomplished. This first pan-European surveillance compared the mutation profiles associated with the variants of concerns: B.1.1.7, P.1, B.1.351 and B.1.617.2 across 20 European countries, including 54 municipalities. The results highlight that SARS-CoV-2 variants detected in the wastewater samples mirror the variants profiles reported in clinical data. This study demonstrated that >98% coverage of SARS-CoV-2 genomic sequences is possible and can be used to track SARS-CoV-2 mutations in wastewater to support identifying variants circulating in a city at the community level.

Genome Sequencing of Wastewater Confirms the Arrival of the SARS-CoV-2 Omicron Variant at Frankfurt Airport but Limited Spread in the City of Frankfurt, Germany, in November 2021.

Wastewater-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surveillance of Frankfurt Airport by genome sequencing was used to detect SARS-CoV-2 variants entering the region. In November 2021, we found all characteristic mutations of Omicron in wastewater originating from Frankfurt Airport before the first confirmed clinical report from an arriving passenger on 26 November 2021.

Chemical mixtures and fluorescence in situ hybridization analysis of natural microbial community in the Tiber river.

The Water Framework Directive (WFD) regulates freshwater and coastal water quality assessment in Europe. Chemical and ecological water quality status is based on measurements of chemical pollutants in water and biota together with other indicators such as temperature, nutrients, species compositions (phytoplankton, microalgae, benthos and fish) and hydromorphological conditions. However, in the current strategy a link between the chemical and the ecological status is missing. In the present WFD, no microbiological indicators are foreseen for integrating the different anthropogenic pressures, including mixtures of chemicals, nutrients and temperature changes, to provide a holistic view of the freshwater ecosystem water quality. The main aim of this work was to evaluate if natural microbial populations can be valuable indicators of multiple stressors (e.g. chemical pollutants, temperature, nutrients etc.) to guide preventive and remediation actions by water authorities. A preliminary survey was conducted to identify four sites reflecting a contamination gradient from the source to the mouth of a river suitable to the objectives of the European Marie Curie project, MicroCoKit. The River Tiber (Italy) was selected as a pilot case study to investigate the correlation between bacteria taxa and the chemical status of the river. The main physicochemical parameters, inorganic elements, organic pollutants and natural microbial community composition were assessed at four selected sites corresponding to pristine, agricultural, industrial and urban areas for three consecutive years. The overall chemical results indicated a correspondence between different groups of contaminants and the main contamination sources at the selected sampling points. Phylogenetic analysis of the microbial community analyzed by Fluorescence In Situ Hybridization method (FISH) revealed differences among the four sampling sites which could reflect an adaptive bacterial response to the different anthropogenic pressures.

Analysis of emerging organic contaminants in water, fish and suspended particulate matter (SPM) in the Joint Danube Survey using solid-phase extraction followed by UHPLC-MS-MS and GC-MS analysis.

In the third Joint Danube Survey (JDS3), emerging organic contaminants were analysed in the dissolved water phase of samples from the Danube River and its major tributaries. Analyses were performed using solid-phase extraction (SPE) followed by ultra-high-pressure liquid chromatography triple-quadrupole mass spectrometry (UHPLC-MS-MS) and gas chromatography-mass spectrometry (GC-MS). The polar organic compounds analysed by UHPLC-MS-MS were 1H-benzotriazole, methylbenzotriazoles, carbamazepine, 10,11-dihydro-10,11-dihydroxy-carbamazepine, diclofenac, sulfamethox-azole, 2,4-D (2,4-dichlorophenoxyacetic acid), MCPA (2-methyl-4-chlorophenoxyacetic acid), metolachlor, cybutryne (irgarol), terbutryn, DEET (N,N-diethyl-m-toluamide), and several perfluoroalkyl acids (C6-C9; C8=perfluorooctanoic acid (PFOA)) and perfluorooctansulfonic acid (PFOS). In addition, several organophosphorus flame retardants were analysed by GC-MS. The most relevant compounds identified in the 71 water samples, in terms of highest median and maximum concentrations, were 1H-benzotriazole, tris(1-chloro-2-propyl)phosphate (TCPP), methylbenzotriazoles, carbama-zepine and its metabolite, DEET, sulfamethoxazole, tris(isobutyl)phosphate (TiBP), tris(2-chloroethyl)phosphate (TCEP), PFOA, PFOS and diclofenac. The concentrations of these compounds in the samples were generally below the environmental quality standard (EQS) threshold values, with the exception of PFOS, the concentration of which exceeded the annual average water EQS limit of 0.65ng/L along the whole river, and also exceeded the fish biota EQS of 9.1µg/kg. In addition, the proposed EQS for diclofenac, of 0.1µg/L, was exceeded in the Arges River in Romania (255ng/L).

Comparing measured and modelled PFOS concentrations in a UK freshwater catchment and estimating emission rates.

The lifecycle, sources and fate of perfluorooctane sulfonate (PFOS) continue to generate scientific and political interest, particularly since PFOS was listed by the Stockholm Convention and largely restricted in Europe. It continues to be detected in aquatic environments, with only limited studies into the on-going sources. This paper explores PFOS emissions discharged by the general population into a small catchment comprising two rivers in the UK. A sampling campaign was undertaken to improve our understanding of population-derived PFOS sources from sewage treatment plants (STPs) and in rivers. A corresponding modelling exercise allowed an emission estimate of 13µg/day/per capita to be derived for the Aire and Calder rivers. PFOS emission was linked to STP discharges bylinear regression of measured and modelled concntrations (R(2)=0.49-0.85). The model was able to accurately estimate the spatial trends of PFOS in the rivers, while predicted concentrations were within a factor of three based on per capita emission values taken from the literature. Measured PFOS concentrations in rivers suggested that emissions from STPs are partially dependent on treatment type, where plants with secondary or tertiary treatment such as activated sludge processes emit less PFOS, possibly due to increased partitioning and retention. With refinements based on the type of treatment at each STP, predictions were further improved. The total PFOS mass discharged annually via rivers from the UK has been estimated to be between 215 and 310kg, based on the per capita emission range derived in this study.

Looking for protein expression signatures in European eel peripheral blood mononuclear cells after in vivo exposure to perfluorooctane sulfonate and a real world field study.

The decline of European eel population can be attributed to many factors such as pollution by xenobiotics present in domestic and industrial effluents. Perfluorooctane sulfonate (PFOS) is a ubiquitous compound of a particular concern in Europe. PFOS can reach high concentrations in tissues of organisms and many toxic effects have been reported in fish. This study aimed at evaluating the toxicological effects of PFOS in European eel peripheral blood mononuclear cells (PBMCs) at the protein expression level. To identify proteins whose expression was modified by PFOS, we performed a proteomic analysis on the post-nuclear fraction of PBMCs after a chronic exposure (28 days) of yellow eels to zero, 1 or 10 µg/L PFOS. This in vivo study was completed by a proteomic field study on eels sampled in Belgian rivers presenting different PFOS pollution degrees. Proteins were separated by two-dimensional in-gel electrophoresis (2D-DIGE) to compare the post-nuclear fraction of PBMCs from the reference group with cells from fish exposed to the pollutant of interest. On the 28 spots that were significantly (p < 0.05; ANOVA followed by a Dunnett post-hoc test) affected by PFOS in the in vivo experiment, a total of 17 different proteins were identified using nano-LC ESI-MS/MS and the Peptide and Protein Prophet of Scaffold software. In the field experiment, 18 significantly (p < 0.05; ANOVA followed by Dunnett's test) affected spots conducted to the identification of 16 different proteins. Interestingly, only three proteins were found in common between in vivo and in situ experiments: plastin-2, alpha-enolase and glyceraldehyde 3-phosphate dehydrogenase. Comparing the results with a previous study, plastin-2 and alpha-enolase were also been found to be affected after in vitro exposure of PBMCs during 48 h to either 10 µg or 1 mg PFOS/L. Potential use of these proteins as biomarkers of PFOS exposure in European eel could indicate early warning signals.

EU-wide monitoring survey on emerging polar organic contaminants in wastewater treatment plant effluents.

In the year 2010, effluents from 90 European wastewater treatment plants (WWTPs) were analyzed for 156 polar organic chemical contaminants. The analyses were complemented by effect-based monitoring approaches aiming at estrogenicity and dioxin-like toxicity analyzed by in vitro reporter gene bioassays, and yeast and diatom culture acute toxicity optical bioassays. Analyses of organic substances were performed by solid-phase extraction (SPE) or liquid-liquid extraction (LLE) followed by liquid chromatography tandem mass spectrometry (LC-MS-MS) or gas chromatography high-resolution mass spectrometry (GC-HRMS). Target microcontaminants were pharmaceuticals and personal care products (PPCPs), veterinary (antibiotic) drugs, perfluoroalkyl substances (PFASs), organophosphate ester flame retardants, pesticides (and some metabolites), industrial chemicals such as benzotriazoles (corrosion inhibitors), iodinated x-ray contrast agents, and gadolinium magnetic resonance imaging agents; in addition biological endpoints were measured. The obtained results show the presence of 125 substances (80% of the target compounds) in European wastewater effluents, in concentrations ranging from low nanograms to milligrams per liter. These results allow for an estimation to be made of a European median level for the chemicals investigated in WWTP effluents. The most relevant compounds in the effluent waters with the highest median concentration levels were the artificial sweeteners acesulfame and sucralose, benzotriazoles (corrosion inhibitors), several organophosphate ester flame retardants and plasticizers (e.g. tris(2-chloroisopropyl)phosphate; TCPP), pharmaceutical compounds such as carbamazepine, tramadol, telmisartan, venlafaxine, irbesartan, fluconazole, oxazepam, fexofenadine, diclofenac, citalopram, codeine, bisoprolol, eprosartan, the antibiotics trimethoprim, ciprofloxacine, sulfamethoxazole, and clindamycine, the insect repellent N,N'-diethyltoluamide (DEET), the pesticides MCPA and mecoprop, perfluoroalkyl substances (such as PFOS and PFOA), caffeine, and gadolinium.

Analysis of polar organic contaminants in surface water of the northern Adriatic Sea by solid-phase extraction followed by ultrahigh-pressure liquid chromatography-QTRAP® MS using a hybrid triple-quadrupole linear ion trap instrument.

Water-soluble polar organic contaminants are discharged by rivers, cities, and ships into the oceans. Little is known on the fate, pollution effects, and thresholds of toxic chemical mixtures in the marine environment. A new trace analytical method was developed for the multi-compound analysis of polar organic chemical contaminants in marine waters. The method is based on automated solid-phase extraction (SPE) of one-liter water samples followed by ultrahigh-pressure liquid chromatography triple-quadrupole linear ion-trap mass spectrometry (UHPLC-QTRAP(®) MS). Marine water samples from the open Adriatic Sea taken 16 km offshore from Venice (Italy) were analyzed. Method limits of quantification (LOQs) in the low picogram per liter (pg/l) concentration range were achieved. Among the 67 target chemicals analyzed, 45 substances could be detected above the LOQ. The chemicals detected at the highest concentrations were caffeine (up to 367 ng/l), nitrophenol (36 ng/l), 2,4-dinitrophenol (34 ng/l), 5-methyl-1H-benzotriazole (18.5 ng/l), sucralose (11 ng/l), 1H-benzotriazole (9.2 ng/l), terbuthylazine (9 ng/l), alachlor (7.7 ng/l), atrazine-desisopropyl (6.6 ng/l), diethyltoluamide (DEET) (5.0 ng/l), terbuthylazine-desethyl (4.3 ng/l), metolachlor (2.8 ng/l), perfluorooctanoic acid (PFOA) (2.5 ng/l), perfluoropentanoic acid (PFPeA) (2.3 ng/l), linuron (2.3 ng/l), perfluorohexanoic acid (PFHxA) (2.2 ng/l), diuron (2.0 ng/l), perfluorohexane sulfonate (PFHxS) (1.6 ng/l), simazine (1.6 ng/l), atrazine (1.5 ng/l), and perfluorooctane sulfonate (PFOS) (1.3 ng/l). Higher concentrations were detected during summer due to increased levels of tourist activity during this period.

Pharmacokinetics and pharmacodynamics of ritodrine hydrochloride administered orally and intramuscularly to female healthy volunteers.

Ritodrine hydrochloride ((R,S)-4-hydroxy-alpha-[1-[2-((4-hydroxyphenyl)ethyl]amino)ethyl]benzenemethanol, CAS 26652-09-5) is a direct-acting sympathomimetic agent with a predominant beta-adrenergic activity and a selective action on beta2-receptors. A clinical trial was carried out to investigate the pharmacokinetics, pharmacodynamics and safety of ritodrine hydrochloride administered at the doses of 10, 20 and 30 mg p.o. and 10 mg by i. m. route. A four-way randomised crossover design was adopted on 12 healthy female volunteers with a wash-out of at least 14 days. Concentrations of ritodrine and of the pool of ritodrine in plasma and concentrations of the pool of ritodrine in urine of volunteers were bioassayed with tandem mass spectrometry. The following pharmacokinetic parameters were calculated, using the non-compartmental model: Cmax, AUC0-t, AUC0-INF, t1/2, Vd/f, and Aet after each administration. The distribution volume of ritodrine proved to be about 3 times higher than that of the pool of ritodrine after i. m. injection, confirming the good permeability of ritodrine that massively enters tissue compartments. Statistical analyses of pharmacokinetic parameters ascertained that the p. o. absorption of ritodrine hydrochloride was linearly related with the doses administered in the 10-30 mg range. The pharmacodynamic parameters evaluated complied with the mechanism of action of this drug.

Pharmacokinetics of isoxsuprine hydrochloride administered orally and intramuscularly to female healthy volunteers.

Isoxsuprine (1-(4-hydroxyphenyl)-2-(1-methyl-2-phenoxyethylamino)-1-propanol, CAS 395-28-8) is a peripheral vasodilator that also stimulates beta-adrenergic receptors. It causes a direct relaxation of vascular and uterine smooth muscles and produces positive inotropic and chronotropic effects. It is widely used to arrest premature labour and miscarriage. The aim of this trial was to investigate the pharmacokinetics of isoxsuprine hydrochloride administered orally to healthy young female volunteers as an extended-release formulation at the doses of 30, 60 and 90 mg compared to 10 mg by i.m. route. A randomised, crossover, four-period, multisequence, single-dose design was adopted. Plasma and urine concentrations of free and total isoxsuprine were evaluated by tandem mass spectrometry that reached a low quantification limit of 1 ng/ml. From plasma concentrations Cmax, tmax, AUC(0-t), AUC(0-infinity), t1/2 and Vd and from urine concentration CUE(0-24h) were evaluated by the non-compartmental model. The free drug was present only in plasma after i.m. route, whereas total isoxsuprine, namely the drug after an enzymatic hydrolysis of the conjugate form, was detected in all plasma and urine samples. The distribution volume of the free drug proved to be 2.5 times higher than that of total isoxsuprine, which indicates a good penetration of the free drug into tissue compartments. Oral absorption was evaluated from the p.o./i.m. percentualized ratio of AUC and CUE and proved to be on average around 51%, being linearly correlated with the three doses administered. The oral absorption proved to be sustained as expected from the zero-order kinetics of the drug release from the core of the extended-release formulation. This has justified different values of half-life that was on average 2.2 h after the i.m. route and around 10 h after the three oral doses. After isoxsuprine administration, both oral and i.m. routes, the heart rate increased from baseline during the 9 h monitoring period. This was an expected finding attributable to the stimulating activity of beta-adrenergic receptors. The tolerability of isoxsuprine proved to be very good with all the four administrations performed.

Column-switching system with restricted access pre-column packing for an integrated sample cleanup and liquid chromatographic-mass spectrometric analysis of alkylphenolic compounds and steroid sex hormones in sediment.

A novel methodology based on column switching LC-MS, using restricted access material (RAM), for an integrated sample clean-up and analysis of endocrine disrupting compounds in sediment samples is described. The use of RAM precolumns, that combines size exclusion and reversed-phase retention mechanisms, enables fast on-line clean-up of sediment extracts and sensitive determination of alkylphenolic compounds, bisphenol A and steroid sex hormones at low ppb level (LODs=0.5-5 ng/g). Different LiChrospher ADS RAM precolumns (Merck, Germany) with C4, C8 and C18, respectively, modification of inner pore surface were tested. ADS C4 precolumns gave the best results in terms of recovery, selectivity and sensitivity, eliminating efficiently matrix components and consequently reducing ion suppression effects. Except for the most polar compounds, all compounds exhibit complete recovery with RSD from 0.87 to 15%. A complete analysis, including efficient pressurized liquid extraction (PLE) of target compounds, on-line clean-up, chromatographic separation and MS detection takes approximately 2 h, which is a significant improvement in comparison to the methods reported previously.