HRMS-based suspect screening of pharmaceuticals and their transformation products in multiple environmental compartments: An alternative to target analysis?

The comprehensive monitoring of pharmaceutically active compounds (PhACs) in the environment is challenging given the myriad of substances continuously discharged, the increasing number of new compounds being produced (and released), or the variety of the associated human metabolites and transformation products (TPs). Approaches such as high-resolution mass spectrometry (HRMS)-based suspect analysis have emerged to overcome the drawbacks of classical target analytical methods, e.g., restricted chemical coverage. In this study, we assess the readiness of HRMS-based suspect screening to replace or rather complement target methodologies by comparing the performance of both approaches in terms of i) detection of PhACs in various environmental samples (water, sediments, biofilm, fish plasma, muscle and liver) in a field study; ii) PhACs (semi)quantification and iii) prediction of their environmental risks. Our findings revealed that target strategies alone significantly underestimate the variety of PhACs potentially impacting the environment. However, relying solely on suspect strategies can misjudge the presence and risk of low-level but potentially risky PhACs. Additionally, semiquantitative approaches, despite slightly overestimating concentrations, can provide a realistic overview of PhACs concentrations. Hence, it is recommended to adopt a combined strategy that first evaluates suspected threats and subsequently includes the relevant ones in the established target methodologies.

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.

Contaminants of emerging concern fate and fluvial biofilm status as pollution markers in an urban river.

Contaminants of emerging concern (CEC) are still under research given the vast diversity of compounds reaching freshwater ecosystems and adverse effects they might cause. In this study, the environmental fate of 73 CEC, comprising sweeteners, stimulants and several pharmaceutical therapeutic classes, and changes in fluvial biofilm photosynthetic parameters were evaluated in a semi-arid urban river receiving diffuse and point sources of pollution (Suquía river, Argentina). Out of the 37 CEC detected, 30 were quantified in surface water (n.d. - 9826 ng/L), 10 in biofilm (n.d. - 204 ng/gd.w.) and 9 in the clay fraction of sediments (n.d. - 64 ng/gd.w.). CEC distribute differently among the 3 matrices: water phase presents the biggest diversity of compounds (14 CEC families), being analgesic/anti-inflammatories the most abundant family. Antibiotics largely predominated in biofilms (7 CEC families), while the stimulant caffeine and some antibiotics where the most abundant in sediments (6 CEC families). Different CEC accumulated in biofilms and sediments upstream and downstream the city, and big shifts of biofilm community occurred downstream WWTP. The shift of biofilm community upstream (F0 > 0) and downstream the WWTP (F0 = 0) shows a sensitive response of F0 to the impact of WWTP. Biofilm photosynthetic parameters responded in less impacted urban sites (sites 1, 2 and 3), where significant correlations were found between ketoprofen and some antibiotics and biofilm parameters. The diversity and amount of CEC found in the urban section of Suquía river alert to the magnitude of point and non-point sources of pollution.

International Comparison, Risk Assessment, and Prioritisation of 26 Endocrine Disrupting Compounds in Three European River Catchments in the UK, Ireland, and Spain.

Endocrine-disrupting compounds (EDCs) constitute a wide variety of chemistries with diverse properties that may/can pose risks to both humans and the environment. Herein, a total of 26 compounds, including steroids, flame retardants, and plasticizers, were monitored in three major and heavily urbanized river catchments: the R. Liffey (Ireland), the R. Thames (UK), and the R. Ter (Spain), by using a single solid-phase extraction liquid chromatography-mass spectrometry (SPE-LC-MS/MS) method. Occurrence and frequency rates were investigated across all locations over a 10-week period, with the highest concentration obtained for the flame retardant tris(2-chloroethyl) phosphate (TCEP) at 4767 ng∙L-1 in the R. Thames in Central London. Geographical variations were observed between sites and were partially explained using principal component analysis (PCA) and hierarchical cluster analysis (HCA). In particular, discrimination between the R. Ter and the R. Thames was observed based on the presence and concentration of flame retardants, benzotriazole, and steroids. Environmental risk assessment (ERA) across sites showed that caffeine, a chemical marker, and bisphenol A (BPA), a plasticizer, were classified as high-risk for the R. Liffey and R. Thames, based on relative risk quotients (rRQs), and that caffeine was classified as high-risk for the R. Ter, based on RQs. The total risks at each location, namely ΣRQriver, and ΣrRQriver, were: 361, 455, and 723 for the rivers Liffey, Thames, and Ter, respectively. Caffeine, as expected, was ubiquitous in all 3 urban areas, though with the highest RQ observed in the R. Ter. High contributions of BPA were also observed across the three matrices. Therefore, these two compounds should be prioritized independently of location. This study represents a comprehensive EDC monitoring comparison between different European cities based on a single analytical method, which allowed for a geographically independent ERA prioritization to be performed.

A comprehensive study on diesel oil bioremediation under microcosm conditions using a combined microbiological, enzymatic, mass spectrometry, and metabarcoding approach.

This study aims at the application of a marine fungal consortium (Aspergillus sclerotiorum CRM 348 and Cryptococcus laurentii CRM 707) for the bioremediation of diesel oil-contaminated soil under microcosm conditions. The impact of biostimulation (BS) and/or bioaugmentation (BA) treatments on diesel-oil biodegradation, soil quality, and the structure of the microbial community were studied. The use of the fungal consortium together with nutrients (BA/BS) resulted in a TPH (Total Petroleum Hydrocarbon) degradation 42% higher than that obtained by natural attenuation (NA) within 120 days. For the same period, a 72 to 92% removal of short-chain alkanes (C12 to C19) was obtained by BA/BS, while only 3 to 65% removal was achieved by NA. BA/BS also showed high degradation efficiency of long-chain alkanes (C20 to C24) at 120 days, reaching 90 and 92% of degradation of icosane and heneicosane, respectively. In contrast, an increase in the levels of cyclosiloxanes (characterized as bacterial bioemulsifiers and biosurfactants) was observed in the soil treated by the consortium. Conversely, the NA presented a maximum of 37% of degradation of these alkane fractions. The 5-ringed PAH benzo(a)pyrene, was removed significantly better with the BA/BS treatment than with the NA (48 vs. 38 % of biodegradation, respectively). Metabarcoding analysis revealed that BA/BS caused a decrease in the soil microbial diversity with a concomitant increase in the abundance of specific microbial groups, including hydrocarbon-degrading (bacteria and fungi) and also an enhancement in soil microbial activity. Our results highlight the great potential of this consortium for soil treatment after diesel spills, as well as the relevance of the massive sequencing, enzymatic, microbiological and GC-HRMS analyses for a better understanding of diesel bioremediation.

Analysis of microplastics in the environment: Identification and quantification of trace levels of common types of plastic polymers using pyrolysis-GC/MS.

This work describes the development of analytical workflows based on pyrolysis coupled with gas chromatography-mass spectrometry (Pyr-GC/MS) for the qualitative and quantitative analysis of 12 of the most common plastic polymers in environmental samples. The most suitable characteristic pyrolyzate compounds and respective indicator ions were selected for each polymer in order to obtain the most appropriate response for analytical purposes. Additionally, commercial pyrolyzates and polymers libraries were used to confirm the identity of the detected microplastics. The method was validated, showing a good linearity for all the plastic polymers (R2 > 0.97) and limits of detection between 0.1 (polyurethane) to 9.1 µg (polyethylene). The developed methodology was successfully applied for the analysis of plastic polymers in environmental microplastic samples collected in three Mediterranean beaches (NE Spain).•Fast and reproducible Pyr-GC/MS method for the analysis of the 12 most common plastic polymers in a single GC/MS run•Straightforward analytical workflows using pyrolyzates and polymers libraries enable a fast identification and quantification of microplastics in environmental samples.

Linking chemical exposure and fish metabolome: Discovering new biomarkers of environmental exposure of Argyrosomus regius to the antidepressant venlafaxine.

In this study, a non-target metabolomic approach was used to investigate changes in the metabolome of juvenile meagre (Argyrosomus regius) exposed to venlafaxine (20 µg/L). A total of 24, 22 and 8 endogenous metabolites tentatively identified in liver, brain and plasma, respectively, were significantly changed in venlafaxine exposed meagre, showing tissue-dependent variations in the metabolic profile. The amino acids tryptophan, tyrosine and phenylalanine, which are related to the synthesis, availability, and expression of neurotransmitters (e.g., serotonin, dopamine, epinephrine), showed to be dysregulated by venlafaxine exposure. A high impact was observed in fish brain metabolome that showed a trend of up-regulation for most of the tentatively identified metabolites. In conclusion, the identification of possible biomarkers of exposure in fish metabolome to environmental stressors such as venlafaxine is crucial to assess early signal changes at molecular level, enabling the prevention of deleterious effects at the organism and population levels.

Effects of sulfamethoxazole exposure on mussels (Mytilus galloprovincialis) metabolome using retrospective non-target high-resolution mass spectrometry and chemometric tools.

In this work, the Regions of Interest-Multivariate Curve Resolution (ROIMCR) method is proposed for the analysis of non-target metabolomics data. Samples from marine mussels (Mytilus galloprovincialis) exposed to a sublethal concentration (10 µg/L) of sulfamethoxazole (SMX) during 4 days in different seasonal conditions (summer and winter) were analyzed by High-Performance Liquid Chromatography - High-Resolution Mass Spectrometry (HPLC-HRMS) to study the effect of their exposure to SMX and the different seasonal conditions. The Regions of Interest (ROI) procedure has been applied for data filtering, compression, preprocessing and storage steps. Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) is then applied to the previously MS ROI preprocessed data sets to resolve the elution profiles and spectral fingerprints of the chemical constituents of the analyzed samples. The peak areas of the elution profiles of the chemical constituents resolved by the combined ROIMCR procedure were analyzed by Principal Component Analysis (PCA) and samples were clustered according to their experimental seasonal and SMX exposure. The effects of the two investigated factors and of their interaction on the concentrations of the metabolites were statistically assessed by ANOVA simultaneous component analysis (ASCA). Both types of analyses, PCA clustering and ASCA, confirmed that the seasonal conditions (summer versus winter) produced larger effects than those produced by the exposure to SMX and by the interaction of these two factors. The concentration changes of 16 identified endogenous metabolites were validated individually using a Wilcoxon statistical test, which confirmed the presence of significant disturbances in the levels of some of these metabolites (free fatty acids, amino acids and nucleic acids), and indicated the possible alteration of six different biological pathways, affected by the investigated seasonal and SMX exposure factors.

Antibiotics, antibiotic resistance and associated risk in natural springs from an agroecosystem environment.

This study investigates the occurrence, transport, and risks associated to antibiotic residues, antibiotic resistance genes (ARGs) and antibiotic resistant Escherichia coli (AR-E. coli) in eleven natural springs in an agroecosystem environment with intense livestock production, where groundwater nitrate concentration usually sets above 50 mg L-1. Out of 23 multiple-class antibiotics monitored, tetracycline and sulfonamide residues were the most ubiquitous, and they were detected at concentrations ranging from ng L-1 to µg L-1. Five ARGs were monitored, conferring resistance to the antibiotic classes of major use in livestock production. Thus, genes conferring resistance to sulfonamides (sul1 and sul2) and tetracyclines (tetW) as well as a gene proxy for anthropogenic pollution (intI1) were present in most springs. sul1 was the most abundant, with absolute concentrations ranging from 4 × 102 to 5.6 × 106 gene copies L-1 water. AR-E. coli showing resistance to sulfonamides and tetracyclines was also detected, with a prevalence up to approximately 40 % in some sites but with poor correlations with the concentration of antibiotic residues and ARGs. The occurrence of antibiotics, ARGs and AR-E. coli was characterized by large seasonal variations which were mostly associated to both hydrological factors and reactive transport processes. Finally, a risk assessment approach pointed out towards low risk for both the groundwater environment and human health, when spring water is used for direct human consumption, associated with the occurrence of antibiotics, ARGs and AR-E. coli. However, long-term effects cannot be neglected, and proper actions must be taken to preserve groundwater quality.

The Complex Interplay Between Antibiotic Resistance and Pharmaceutical and Personal Care Products in the Environment.

Antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) are important environmental contaminants. Nonetheless, what drives the evolution, spread, and transmission of antibiotic resistance dissemination is still poorly understood. The abundance of ARB and ARGs is often elevated in human-impacted areas, especially in environments receiving fecal wastes, or in the presence of complex mixtures of chemical contaminants, such as pharmaceuticals and personal care products. Self-replication, mutation, horizontal gene transfer, and adaptation to different environmental conditions contribute to the persistence and proliferation of ARB in habitats under strong anthropogenic influence. Our review discusses the interplay between chemical contaminants and ARB and their respective genes, specifically in reference to co-occurrence, potential biostimulation, and selective pressure effects, and gives an overview of mitigation by existing man-made and natural barriers. Evidence and strategies to improve the assessment of human health risks due to environmental antibiotic resistance are also discussed. Environ Toxicol Chem 2023;00:1-16. © 2022 SETAC.

Applicability of an on-line solid-phase extraction liquid chromatography - tandem mass spectrometry for the wastewater-based assessment of human exposure to chemicals from personal care and household products.

Wastewater-based epidemiology (WBE) can be a useful complementary approach to assess human exposure to potentially harmful chemicals, including those from personal care and household products. In this work, a fully automated multiresidue method, based on on-line solid-phase extraction liquid chromatography - tandem mass spectrometry, was developed for the determination of 27 biomarkers of human exposure to selected chemicals from personal care and household products, including parabens, UV filters, phthalates and alternative plasticizers, phosphorous flame retardants/plasticizers (PFRs), and bisphenols. These biomarkers include both the parent compounds and their human metabolites. In addition, two oxidative stress biomarkers, 8-epi-prostaglandin F2α and 4-hydroxy nonenal mercapturic acid, were also considered in the study. The method was carefully optimized to tackle the challenges of analyzing compounds with different physico-chemical properties in a highly complex raw wastewater matrix, while model experiments were performed to investigate filtration losses and analyte stability. The applicability of the developed method was tested by analyzing raw wastewater from four European cities: Antwerp, Brussels (Belgium), Girona (Spain), and Zagreb (Croatia). Twenty-one biomarkers (10 parent compounds and 11 metabolites) were detected in all analyzed wastewater samples. The parent compounds with the highest mass loads were PFRs, parabens, and bisphenol S, while phthalate monoesters were the most prominent metabolites. The mass loads of most compounds were quite similar across cities, but geographic differences were observed for some biomarkers, such as metabolites of phthalates and alternative plasticizers. Exposure was then assessed for seven substances for which quantitative urinary excretion data are known. Our results indicate that safe reference values were exceeded for several contaminants, including butylated phthalates, bisphenol A, and tris(2-butoxyethyl) phosphate, particularly for toddlers. With this relatively simple method, which requires less sample manipulation, it is possible to promptly identify and monitor exposure to harmful chemicals at the population level using the WBE approach.

Prospects on coupling UV/H2O2 with activated sludge or a fungal treatment for the removal of pharmaceutically active compounds in real hospital wastewater.

Conventional active sludge (AS) process at municipal centralized wastewater treatment facilities may exhibit little pharmaceuticals (PhACs) removal efficiencies when treating hospital wastewater (HWW). Therefore, a dedicated efficient wastewater treatment at the source point is recommended. In this sense, advanced oxidation processes (AOPs) and fungal treatment (FG) have evidenced promising results in degrading PhACs. The coupling of the AOP based on UV/H2O2 treatment with biological treatment (AS or FG) treating a real non-sterile HWW, was evaluated in this work. In addition, a coagulation-flocculation pretreatment was applied to improve the efficiency of all approaches. Twenty-two PhACs were detected in raw HWW, which were effectively removed (93-95%) with the combination of any of the biological treatment followed by UV/H2O2 treatment. Similar removal results (94%) were obtained when placing UV/H2O2 treatment before FG, while a lower removal (83%) was obtained in the combination of UV/H2O2 followed by AS. However, the latest was the only treatment combination that achieved a decrease in the toxicity of water. Moreover, deconjugation of conjugated PhACs has been suggested for ofloxacin and lorazepam after AS treatment, and for ketoprofen after fungal treatment. Monitoring of carbamazepine and its transformation products along the treatment allowed to identify the same carbamazepine degradation pathway in UV/H2O2 and AS treatments, unlike fungal treatment, which followed another degradation route.

New insights on the combined removal of antibiotics and ARGs in urban wastewater through the use of two configurations of vertical subsurface flow constructed wetlands.

The occurrence and removal of 49 antibiotics and 11 selected antibiotic resistance genes (ARGs) were investigated in 2 vertical subsurface flow (VF) constructed wetlands (1.5 m2 each): an unsaturated (UVF) unit and a partially saturated (SVF) unit (0.35 m saturated out of 0.8 m) operating in parallel and treating urban wastewater. Thirteen antibiotics were detected in influent wastewater, 6 of which were present in all samples. The SVF showed statistical significance on the removal of 4 compounds (namely ciprofloxacin, ofloxacin, pipemidic acid and azithromycin), suggesting that the wider range of pH and/or redox conditions of this configuration might promote the microbial degradation of some antibiotics. In contrast, the concentration of the latter (except pipemidic acid) and also clindamycin was higher in the effluent than in the influent of the UVF. Five ARGs were detected in influent wastewater, sul1 and sul2, blaTEM, ermB and qnrS. All of them were detected also in the biofilm of both wetlands, except qnrS. Average removal rates of ARGs showed no statistical differences between both wetland units, and ranged between 46 and 97% for sul1, 33 and 97% for sul2, 9 and 99% for ermB, 18 and 97% for qnrS and 11 and 98% for blaTEM.

Combining an effect-based methodology with chemical analysis for antibiotics determination in wastewater and receiving freshwater and marine environment.

Two different methodologies were combined to evaluate the risks that antibiotics can pose in the environment; i) an effect-based methodology based on microbial growth inhibition and ii) an analytical method based on liquid-chromatography coupled to mass spectrometry (LC-MS). The first approach was adapted and validated for the screening of four antibiotic families, specifically macrolides/ß-lactams, quinolones, sulfonamides and tetracyclines. The LC-MS method was applied for the identification and quantification of target antibiotics; then, the obtained results were combined with ecotoxicological data from literature to determine the environmental risk. The two methodologies were used for the analysis of antibiotics in water samples (wastewater, river water and seawater) and biofluids (fish plasma and mollusk hemolymph) in two monitoring campaigns undertaken in the Ebro Delta and Mar Menor Lagoon (both in the Mediterranean coast of Spain). Both approaches highlighted macrolides (azithromycin) and quinolones (ciprofloxacin and ofloxacin) as the main antibiotics in wastewater treatment plant (WWTP) effluents with potential risk for the environment. However, no risk for the aquatic life was identified in the river, lagoon and seawater as antibiotic levels were much lower than those in WWTP effluents. Fish from Ebro River were the organisms presenting the highest antibiotic concentration when compared with bivalves (mussels) from the Mediterranean Sea and gastropods (marine snails) from the Mar Menor Lagoon. The effect-based methodology successfully determined antibiotic risk in wastewater, but its applicability was less clear in environmental waters such as seawater, due to its high detection limits. Improving sample preconcentration could increase the method sensibility. Overall, combination of both methodologies provides comprehensive insights in antibiotic occurrence and risk associated in areas under study.

Distribution of antibiotics in water, sediments and biofilm in an urban river (Córdoba, Argentina, LA).

In this study, we evaluated the distribution of up to forty-three antibiotics and 4 metabolites residues in different environmental compartments of an urban river receiving both diffuse and point sources of pollution. This is the first study to assess the fate of different antibiotic families in water, biofilms and sediments simultaneously under a real urban river scenario. Solid phase extraction, bead-beating disruption and pressurized liquid extraction were applied for sample preparation of water, biofilm and sediment respectively, followed by the quantification of target antibiotics by UPLC-ESI-MS/MS. Twelve antibiotics belonging to eight chemical families were detected in Suquía River samples (67% positive samples). Sites downstream the WWTP discharge were the most polluted ones. Concentrations of positive samples ranged 0.003-0.29 µg L-1 in water (max. cephalexin), 2-652 µg kg-1d.w. in biofilm (max. ciprofloxacin) and 2-34 µg kg-1d.w. in sediment (max. ofloxacin). Fluoroquinolones, macrolides and trimethoprim were the most frequently detected antibiotics in the three compartments. However cephalexin was the prevalent antibiotic in water. Antibiotics exhibited preference for their accumulation from water into biofilms rather than in sediments (bioaccumulation factors > 1,000 L kg-1d.w. in biofilms, while pseudo-partition coefficients in sediments < 1,000 L kg-1d.w.). Downstream the WWTP there was an association of antibiotics levels in biofilms with ash-free dry weight, opposite to chlorophyll-a (indicative of heterotrophic communities). Cephalexin and clarithromycin in river water were found to pose high risk for the aquatic ecosystem, while ciprofloxacin presented high risk for development of antimicrobial resistance. This study contributes to the understanding of the fate and distribution of antibiotic pollution in urban rivers, reveals biofilm accumulation as an important environmental fate, and calls for attention to government authorities to manage identified highly risk antibiotics.

Extended suspect screening to identify contaminants of emerging concern in riverine and coastal ecosystems and assessment of environmental risks.

A suspect screening methodology was developed for the fast and reliable identification of 360 contaminants of emerging concern (CECs) of anthropogenic origin in the vulnerable area of the Ebro Delta (Catalonia, Spain) and to track for potential contamination sources. The suspect screening methodology was combined with a risk assessment approach to prioritize the most ecologically relevant CECs. Out of the 360 suspects, 37 compounds were tentatively identified, 22 of which were fully confirmed using isotopically labelled standards. The detected suspect compounds included pesticides, pharmaceuticals, personal care products, stimulants and their metabolites. Pesticides were more ubiquitous in irrigation and drainage channels, while pharmaceuticals, stimulants, and personal care products were the most common in effluent wastewaters, in the receiving freshwater systems as well as in the marine environment. Ten compounds were found to be of high ecological concern, including the pharmaceuticals telmisartan, venlafaxine, and carbamazepine, the herbicides terbuthylazine, desethylterbuthylazine, and terbutryn, the fungicides azoxystrobin, tebuconazole and prochloraz and the insecticide tebufenozide. These compounds could be used as markers of anthropogenic contamination in riverine and coastal ecosystems.

Sustainable microalgae-based technology for biotransformation of benzalkonium chloride in oil and gas produced water: A laboratory-scale study.

Many countries have implemented stringent regulatory standards for discharging produced water (PW) from the oil and gas extraction process. Among the different chemical pollutants occurring in PW, surfactants are widely applied in the oil and gas industry to provide a barrier from metal corrosion. However, the release of these substances from the shale formation can pose serious hazardous impacts on the aquatic environment. In this study, a low-cost and eco-friendly microalgae laboratory-scale technology has been tested for biotransformation of benzalkonium chloride (BACC12 and BACC14) in seawater and PW during 14-days of treatment (spiked at 5 mg/L). From the eight microalgae strains selected, Tetraselmis suecica showed the highest removal rates of about 100% and 54% in seawater and PW, respectively. Suspect screening analysis using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) allowed the identification of 12 isomeric intermediates generated coming from biotransformation mechanisms. Among them, the intermediate [OH-BACC12] was found as the most intense compound generated from BACC12, while the intermediate [2OH-BACC14] was found as the most intense compound generated from BACC14. The suggested chemical structures demonstrated a high reduction on their amphiphilic properties, and thus, their tendency to be adsorbed into sediments after water discharge. In this study, Tetraselmis suecica was classified as the most successful specie to reduce the surfactant activity of benzalkonium chloride in treated effluents.

Effect-Based Identification of Hazardous Antibiotic Transformation Products after Water Chlorination.

Antibiotic transformation products (TPs) generated during water treatment can be considered as an environmental concern, since they can retain part of the bioactivity of the parent compound. Effect-directed analysis (EDA) was applied for the identification of bioactive intermediates of azithromycin (AZI) and ciprofloxacin (CFC) after water chlorination. Fractionation of samples allowed the identification of bioactive intermediates by measuring the antibiotic activity and acute toxicity, combined with an automated suspect screening approach for chemical analysis. While the removal of AZI was in line with the decrease of bioactivity in chlorinated samples, an increase of bioactivity after complete removal of CFC was observed (at >0.5 mgCl2/L). Principal component analysis (PCA) revealed that some of the CFC intermediates could contribute to the overall toxicity of the chlorinated samples. Fractionation of bioactive samples identified that the chlorinated TP296 (generated from the destruction of the CFC piperazine ring) maintained 41%, 44%, and 30% of the antibiotic activity of the parent compound in chlorinated samples at 2.0, 3.0, and 4.0 mgCl2/L, respectively. These results indicate the spectrum of antibacterial activity can be altered by controlling the chemical substituents and configuration of the CFC structure with chlorine. On the other hand, the potential presence of volatile DBPs and fractionation losses do not allow for tentative confirmation of the main intermediates contributing to the acute toxic effects measured in chlorinated samples. Our results encourage further development of new and advanced methodologies to study the bioactivity of isolated unknown TPs to understand their hazardous effects in treated effluents.

Insights on the metabolization of the antidepressant venlafaxine by meagre (Argyrosomus regius) using a combined target and suspect screening approach.

Bioaccumulation of pharmaceuticals in fish exposed to contaminated water can be shaped by their capability to metabolize these xenobiotics, affecting their toxicity and animal welfare. In this study the in vivo metabolization of the antidepressant venlafaxine by the juvenile marine fish meagre (Argyrosomus regius) was evaluated using a combined target and suspect screening analytical approach. Thirteen venlafaxine metabolites were identified, namely N-desmethylvenlafaxine and N,N-didesmethylvenlafaxine, which were unequivocally identified using analytical standards, and 11 more tentatively identified by suspect screening analysis, including two Phase II metabolites formed by amino acid conjugation. All of them were detected in the liver, while in plasma and brain only 9 and 6 metabolites, respectively, were detected. Based on these findings, for the first time, a tentative metabolization pathway of venlafaxine by A. regius is proposed. Contrarily to what happen in humans, N-demethylation was identified as the main route of metabolization of venlafaxine by fish. Our findings highlight species-specificity in the metabolization of venlafaxine and allow a better understanding of venlafaxine's toxicokinetic in fish. These results emphasize the need to investigate the biotransformation of xenobiotics by non-target organisms to have an integrated overview of their environmental exposure and to improve future evaluations of environmental risk assessment.

Antibiotic residues in final effluents of European wastewater treatment plants and their impact on the aquatic environment.

A comprehensive monitoring of a broad set of antibiotics in the final effluent of wastewater treatment plants (WWTPs) of 7 European countries (Portugal, Spain, Ireland, Cyprus, Germany, Finland, and Norway) was carried out in two consecutive years (2015 and 2016). This is the first study of this kind performed at an international level. Within the 53 antibiotics monitored 17 were detected at least once in the final effluent of the WWTPs, i.e.: ciprofloxacin, ofloxacin, enrofloxacin, orbifloxacin, azithromycin, clarithromycin, sulfapyridine, sulfamethoxazole, trimethoprim, nalidixic acid, pipemidic acid, oxolinic acid, cefalexin, clindamycin, metronidazole, ampicillin, and tetracycline. The countries exhibiting the highest effluent average concentrations of antibiotics were Ireland and the southern countries Portugal and Spain, whereas the northern countries (Norway, Finland and Germany) and Cyprus exhibited lower total concentration. The antibiotic occurrence data in the final effluents were used for the assessment of their impact on the aquatic environment. Both, environmental predicted no effect concentration (PNEC-ENVs) and the PNECs based on minimal inhibitory concentrations (PNEC-MICs) were considered for the evaluation of the impact on microbial communities in aquatic systems and on the evolution of antibiotic resistance, respectively. Based on this analysis, three compounds, ciprofloxacin, azithromycin and cefalexin are proposed as markers of antibiotic pollution, as they could occasionally pose a risk to the environment. Integrated studies like this are crucial to map the impact of antibiotic pollution and to provide the basis for designing water quality and environmental risk in regular water monitoring programs.