Antibiotics in the Basque coast (N Spain): Occurrence in waste and receiving waters, and risk assessment (2017-2020).

The study of the presence of antibiotics in the aquatic environment is a preliminary step to analyse their possible harmful effects on aquatic ecosystems. In order to monitor their occurrence in the aquatic environment, the European Commission established in 2015, 2018, and 2020 three Watch Lists of substances for Union-wide monitoring (Decisions (EU) 2015/495, 2018/840, and 2020/1161), where some antibiotics within the classes of macrolides, fluoroquinolones and penicillins were included. In the Basque coast, northern Spain, three macrolide antibiotics (erythromycin, clarithromycin, azithromycin) and ciprofloxacin were monitored quarterly from 2017 to 2020 (covering a period before and after the COVID19 outbreak), in water samples collected from two Waste Water Treatment Plants (WWTPs), and three control points associated with receiving waters (transitional and coastal water bodies). This work was undertaken for the Basque Water Agency (URA). The three macrolide antibiotics in water showed a frequency of quantification >65 % in the Basque coast, with higher concentrations in the WWTP emission stations than in receiving waters. Their frequency of quantification decreased from 2017 to 2020, as did the consumption of antibiotics in Spanish primary care since 2015. Ciprofloxacin showed higher frequencies of quantification in receiving waters than in wastewaters, but the highest concentrations were observed in the WWTP emission stations. Although consumption of fluoroquinolones (among which is ciprofloxacin) in primary care in the Basque Country has decreased in recent years, this trend was not observed in the waters sampled in the present study. On the other hand, concentrations of clarithromycin, azithromycin, and ciprofloxacin in receiving waters exceeded their respective Predicted No-Effect Concentrations, so they could pose an environmental risk. These substances are widely used in human and animal medicine, so, although only ciprofloxacin is included in the third Watch List, it would be advisable to continue monitoring macrolides in the Basque coast as well.

Contaminants of emerging concern in the Basque coast (N Spain): Occurrence and risk assessment for a better monitoring and management decisions.

The study of the presence in the aquatic environment of certain substances considered as contaminants of emerging concern (CEC) is a preliminary step to the analysis of the possible harmful effects on aquatic ecosystems and the establishment of the corresponding environmental quality standards. In order to monitor the occurrence of CECs in the aquatic environment, the European Commission established in 2015 and 2018 two watch-list of substances for Union-wide monitoring in the field of water policy (Decision (EU) 2015/495 and Decision (EU) 2018/840). In the coast of the Basque Country, southeast of the Bay of Biscay, 19 of these watch list substances were monitored quarterly from May 2017 to March 2019. Water samples were collected at the effluent of three wastewater treatment plants and five control points associated with receiving waters (transitional and coastal water bodies). The most frequently quantified substances were azithromycin (91%), imidacloprid (82%), clarithromycin (80%), diclofenac (78%) and erythromycin (73%), with frequencies of quantification higher in wastewaters (83-100%) than in receiving waters (70-85%). In general, concentrations in wastewater were also higher than in receiving waters, indicating a dilution effect in the environment. In receiving waters, six out of the nineteen substances monitored exceeded their respective Predicted No-Effect Concentrations: azithromycin (34%), imidacloprid (9%), 17ß-estradiol (E2) (9%), clarithromycin (7%), ciprofloxacin (7%), and diclofenac (5%); and therefore, their levels could pose an environmental risk.

Using MALDI-TOF MS imaging and LC-HRMS for the investigation of the degradation of polycaprolactone diol exposed to different wastewater treatments.

Polymers are used in high amounts in a wide range of applications from biomedicine to industry. Because of the growing awareness of the increasing amounts of plastic wastes in the aquatic environment during recent years, the evaluation of their biodegradability deserves special attention. In the past, most efforts were dedicated to studying the biodegradation of polyesters in soil and compost, while very little research has been conducted on their fate in wastewater. Here, we assessed the ability of bacterial communities residing in the aerobic and denitrification tank from a wastewater treatment plant (WWTP) to degrade the polymeric ester polycaprolactone diol (PCLD; average molecular weight of 1250 Da). Following the incubation of the solid polymer in WWTP tanks, matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) was used to provide evidence for hydrolytic reactions and to study differences in the spatial degradation on the PCLD surface. It was demonstrated that regardless of the wastewater type, the chemical structure on the PCLD surface underwent modifications after 7 days of exposure. Apart from the parent PCLD peak series in MALDI-MSI mass spectra, the presence of a second oligomer series with mass peaks spaced by m/z 114 (as in PCLD) was observed. It was proposed to correspond to polycaprolactone (PCL) originating from the hydrolytic cleavage of the diethylene glycol from PCLD. Their ion masses were detected at m/z 104 below the PCLD peaks and their structures were proposed as PCL cyclized oligomers. Differences in the spatial distribution of low MW ions (<800) between the aerobic and denitrifying exposed samples in MALDI MSI were also noticeable. While the ions at m/z 221.1, 247.1 and 449.2 predominated in the aerobic exposed sample, those at m/z 475.5 and 677.4 were characteristic of the denitrifying one. The MALDI-MSI measurements in the low mass range were complemented with LC-HRMS analysis to determine plausible structures of the major degradation products. Ten transformation products (TPs) were detected in the denitrifying wastewater experiment, five of them were the result of ester hydrolysis forming caprolactone oligomers (TPs 220, 334, 448, 562, and 676) while the other series corresponded to formation of PCL chain with a terminal diethylene glycol, likewise formed by ester hydrolysis (TPs 246, 360, 474, 588, and 702). Graphical abstract Investigation of the polymer degradation in WWTPs by MALDI-MSI and LC-HRMS.

Biomarkers of Morbid Obesity and Prediabetes by Metabolomic Profiling of Human Discordant Phenotypes.

Metabolomic studies aimed to dissect the connection between the development of type 2 diabetes and obesity are still scarce. In the present study, fasting serum from sixty-four adult individuals classified into four sex-matched groups by their BMI [non-obese versus morbid obese] and the increased risk of developing diabetes [prediabetic insulin resistant state versus non-prediabetic non-insulin resistant] was analyzed by LC- and FIA-ESI-MS/MS-driven metabolomic approaches. Altered levels of [lyso]glycerophospholipids was the most specific metabolic trait associated to morbid obesity, particularly lysophosphatidylcholines acylated with margaric, oleic and linoleic acids [lysoPC C17:0: R=-0.56, p=0.0003; lysoPC C18:1: R=-0.61, p=0.0001; lysoPC C18:2 R=-0.64, p<0.0001]. Several amino acids were biomarkers of risk of diabetes onset associated to obesity. For instance, glutamate significantly associated with fasting insulin [R=0.5, p=0.0019] and HOMA-IR [R=0.46, p=0.0072], while glycine showed negative associations [fasting insulin: R=-0.51, p=0.0017; HOMA-IR: R=-0.49, p=0.0033], and the branched chain amino acid valine associated to prediabetes and insulin resistance in a BMI-independent manner [fasting insulin: R=0.37, p=0.0479; HOMA-IR: R=0.37, p=0.0468]. Minority sphingolipids including specific [dihydro]ceramides and sphingomyelins also associated with the prediabetic insulin resistant state, hence deserving attention as potential targets for early diagnosis or therapeutic intervention.

Chemical and toxicological characterisation of anticancer drugs in hospital and municipal wastewaters from Slovenia and Spain.

Anticancer drugs are continuously released into hospital and urban wastewaters, where they, most commonly, undergo conventional treatment in wastewater treatment plants (WWTPs). Wastewaters contain complex mixtures of substances including parent compounds, their metabolites and transformation products (TPs). In this study, samples of hospital effluents and WWTP influents and effluents from Slovenia and Spain were analyzed for twenty-two selected anticancer drugs, their metabolites and transformation products. Acute and chronic toxicity tests were performed on the crustacean Ceriodaphnia dubia, genotoxicity was determined with Tradescantia and Allium cepa micronucleus (MN) assays and in vitro comet assay in zebrafish (Danio rerio) liver cell line (ZFL cells). Sixty of the two hundred-twenty determinations revealed detectable levels of anticancer drug residues. Among the targeted compounds, platinum based were most frequently detected (90%). Furthermore, erlotinib was detected in 80%, cyclophosphamide and tamoxifen in 70% and methotrexate in 60% of the samples. Seven of ten samples were toxic to C. dubia after acute exposure, whereas after chronic exposure all samples reduced reproduction of C. dubia at high sample dilutions. Allium cepa proved insensitive to the potential genotoxicity of the tested samples, while in Tradescantia increased MN frequencies were induced by a hospital effluent and WWTP influents. In ZFL comet assay all but one sample induced a significant increase of DNA strand breaks. Correlations of chemotherapeutics or their TPs were detected for all bioassays except for Allium cepa genotoxicity test, however for each test the highest correlations were found for different substances indicating differential sensitivities of the test organisms.

Abiotic amidine and guanidine hydrolysis of lamotrigine-N2-glucuronide and related compounds in wastewater: The role of pH and N2-substitution on reaction kinetics.

The stability of lamotrigine (LMG) and its principal human metabolite, lamotrigine N2-glucuronide (LMG-N2-G), was studied as a function of pH (4-9). While LMG was stable across the entire pH range, under neutral-basic conditions, LMG-N2-G was converted to three transformation products (TPs) which were identified using high resolution mass spectrometry (HRMS). The MS fragmentation studies indicated that two TPs were the result of the hydrolysis of the amidine and guanidine moieties. The third TP detected was an intermediate in the guanidine hydrolysis reaction. In order to evaluate the transformation kinetics of the LMG-N2-G degradation, another set of pH-dependent experiments was carried out in hospital effluent, wastewater influent and effluent spiked at 20 and 200 nM after pH adjustment (pH 6.5, 7, 8, 8.5 and 9), demonstrating that, at higher pH, LMG-N2-G is degraded at higher rate. Later, the pH-dependent stability of related compounds with different nitrogen N2-substituents (N2-R) on the 1,2,4-triazine ring was studied. This revealed that because of different imino tautomer equilibrium LMG (N2-H) and LMG-N2-oxide ((+)N2-O(-)) were stable at all pHs but N2-methyl-LMG (N2-CH3) as well as LMG-N2-G were susceptible to amidine and guanidine hydrolysis at basic pH. Finally, hospital effluent samples collected over the course of one week were monitored for their presence. LMG, LMG-N2-G and two of its TPs were detected with concentrations ranging between 0.01 and 1 µgL(-1).

Toxicity of the mixture of selected antineoplastic drugs against aquatic primary producers.

The residues of antineoplastic drugs are considered as new and emerging pollutants in aquatic environments. Recent experiments showed relatively high toxicity of 5-fluorouracil (5-FU), imatinib mesylate (IM), etoposide (ET) and cisplatin (CP) that are currently among most widely used antineoplastic drugs, against phytoplankton species. In this study, we investigated the toxic potential of the mixture of 5-FU + IM + ET against green alga Pseudokirchneriella subcapitata and cyanobacterium Synechococcus leopoliensis, and the stability and sorption of these drugs to algal cells. Toxic potential of the mixture was predicted by the concepts of 'concentration addition' and 'independent action' and compared to the experimentally determined toxicity. In both test species, the measured toxicity of the mixture was at effects concentrations EC10-EC50 higher than the predicted, whereas at higher effect concentration (EC90), it was lower. In general, P. subcapitata was more sensitive than S. leopoliensis. The stability studies of the tested drugs during the experiment showed that 5-FU, IM and CP are relatively stable, whereas in the cultures exposed to ET, two transformation products with the same mass as ET but different retention time were detected. The measurements of the cell-linked concentrations of the tested compounds after 72 h exposure indicated that except for CP (1.9 % of the initial concentration), these drugs are not adsorbed or absorbed by algal cells. The results of this study showed that in alga and cyanobacteria exposure to the mixture of 5-FU + ET + IM, in particular at low effect concentration range, caused additive or synergistic effect on growth inhibition, and they suggest that single compound toxicity data are not sufficient for the proper toxicity prediction for aquatic primary producers.

Human Metabolite Lamotrigine-N(2)-glucuronide Is the Principal Source of Lamotrigine-Derived Compounds in Wastewater Treatment Plants and Surface Water.

Wastewater and surface water samples, extracted with four solid-phase extraction cartridges of different chemistries, were suspect-screened for the anticonvulsant lamotrigine (LMG), its metabolites, and related compounds. LMG, three human metabolites, and a LMG synthetic impurity (OXO-LMG) were detected. Preliminary results showed significantly higher concentrations of OXO-LMG in wastewater effluent, suggesting its formation in the wastewater treatment plants (WWTPs). However, biodegradation experiments with activated sludge demonstrated that LMG is resistant to degradation and that its human metabolite lamotrigine-N(2)-glucuronide (LMG-N2-G) is the actual source of OXO-LMG in WWTPs. In batch reactors, LMG-N2-G was transformed, following pseudo-first-order kinetics to OXO-LMG and LMG, but kinetic experiments suggested an incomplete mass balance. A fragment ion search applied to batch-reactor and environmental samples revealed another transformation product (TP), formed by LMG-N2-G oxidation, which was identified by high-resolution mass spectrometry. Accounting for all TPs detected, a total mass balance at two concentration levels in batch reactors was closed at 86% and 102%, respectively. In three WWTPs, the total mass balance of LMG-N2-G ranged from 71 to 102%. Finally, LMG-N2-G and its TPs were detected in surface water samples with median concentration ranges of 23-139 ng L(-1). The results of this study suggest that glucuronides of pharmaceuticals might also be sources of yet undiscovered, but environmentally relevant, transformation products.

Degradation kinetics and pathways of three calcium channel blockers under UV irradiation.

Calcium channel blockers (CCBs) are a group of pharmaceuticals widely prescribed to lower blood pressure and treat heart diseases. They have been frequently detected in wastewater treatment plant (WWTP) effluents and downstream river waters, thus inducing a potential risk to aquatic ecosystems. However, little is known about the behavior and fate of CCBs under UV irradiation, which has been adopted as a primary disinfection method for WWTP effluents. This study investigated the degradation kinetics and pathways of three commonly-used CCBs, including amlodipine (AML), diltiazem (DIL), and verapamil (VER), under UV (254 nm) irradiation. The chemical structures of transformation byproducts (TBPs) were first identified to assess the potential ecological hazards. On that basis, a generic solid-phase extraction method, which simultaneously used four different cartridges, was adopted to extract and enrich the TBPs. Thereafter, the photo-degradation of target CCBs was performed under UV fluences typical for WWTP effluent disinfection. The degradation of all three CCBs conformed to the pseudo-first-order kinetics, with rate constants of 0.031, 0.044 and 0.011 min(-1) for AML, DIL and VER, respectively. By comparing the MS(2) fragments and the evolution (i.e., formation or decay) trends of identified TBPs, the degradation pathways were proposed. In the WWTP effluent, although the target CCBs could be degraded, several TBPs still contained the functional pharmacophores and reached peak concentrations under UV fluences of 40-100 mJ cm(-2).

LC-HRMS suspect screening for detection-based prioritization of iodinated contrast media photodegradates in surface waters.

The objective of the study was to demonstrate the applicability of suspect screening for the detection of six iodinated contrast media (ICM) and their phototransformation products (TPs) in surface waters. First, a photodegradation study of ICM in surface water using a sunlight lab-scale simulator was performed. By means of a guided differential sample analysis, the exact masses of the molecular ions and the retention times of TPs were identified. Positive findings were filtered manually generating a suspect list of 108 photoproducts. Following a generic solid-phase extraction of surface water samples, LC-HRMS was used to screen for the presence of the compounds previously detected in the photodegradation samples. On the basis of detection frequencies (>50% of the samples), 11 TPs were prioritized and their structures elucidated by HRMS and NMR. In the real surface water samples, median concentration of parent compounds was 110 ng/L reaching up to 6 µg/L for iomeprol, while TPs were found at median concentration of 8 ng/L, reaching up to 0.4 µg/L for iomeprol TP651-B. In summary, the proposed screening approach facilitates the evaluation of the degradation of polar compounds at a real scale with a fast detection of TPs without prior availability of the standards.

A modified recombineering protocol for the genetic manipulation of gene clusters in Aspergillus fumigatus.

Genomic analyses of fungal genome structure have revealed the presence of physically-linked groups of genes, termed gene clusters, where collective functionality of encoded gene products serves a common biosynthetic purpose. In multiple fungal pathogens of humans and plants gene clusters have been shown to encode pathways for biosynthesis of secondary metabolites including metabolites required for pathogenicity. In the major mould pathogen of humans Aspergillus fumigatus, multiple clusters of co-ordinately upregulated genes were identified as having heightened transcript abundances, relative to laboratory cultured equivalents, during the early stages of murine infection. The aim of this study was to develop and optimise a methodology for manipulation of gene cluster architecture, thereby providing the means to assess their relevance to fungal pathogenicity. To this end we adapted a recombineering methodology which exploits lambda phage-mediated recombination of DNA in bacteria, for the generation of gene cluster deletion cassettes. By exploiting a pre-existing bacterial artificial chromosome (BAC) library of A. fumigatus genomic clones we were able to implement single or multiple intra-cluster gene replacement events at both subtelomeric and telomere distal chromosomal locations, in both wild type and highly recombinogenic A. fumigatus isolates. We then applied the methodology to address the boundaries of a gene cluster producing a nematocidal secondary metabolite, pseurotin A, and to address the role of this secondary metabolite in insect and mammalian responses to A. fumigatus challenge.

Simultaneous determination of diclofenac, its human metabolites and microbial nitration/nitrosation transformation products in wastewaters by liquid chromatography/quadrupole-linear ion trap mass spectrometry.

An analytical method was developed and validated for the first determination of five major human metabolites of the non-steroidal anti-inflammatory drug diclofenac as well as two microbial transformation products in wastewater. The method was based on the extraction of diclofenac and the chemically synthetized compounds by solid-phase extraction (SPE), using a hydrophilic-lipophilic balanced polymer followed by liquid chromatography (LC) coupled to hybrid quadrupole-linear ion trap mass spectrometry (QqLIT-MS). Quantitation was performed by the internal standard approach, to correct for matrix effects. The accuracy of the method was generally higher than 40% for raw and treated wastewater with a precision below 12%. In wastewater influent and effluent samples the detection limits for the majority of target compounds were 0.3-2.5ngL(-1) and 0.1-3.1ngL(-1), respectively. The method was applied to the analysis of influent and effluent wastewater samples from urban wastewater treatment plants. Moreover, to obtain an extra tool for confirmation and identification of the studied diclofenac-derived compounds, Information-Dependent Acquisition (IDA) experiments were performed, with selected reaction monitoring (SRM) as the survey scan and an enhanced product ion (EPI) scan as the dependent scan. Diclofenac and its major human metabolite, 4'-hydroxydiclofenac were detected in all samples at concentrations of 331-1150ngL(-1) and 585-6000ngL(-1), respectively. Neither microbial transformation product of diclofenac was detected in any of the influent samples analyzed, but in effluents, their concentrations ranged from 4 to 105ngL(-1).

Evaluation of the phototransformation of the antiviral zanamivir in surface waters through identification of transformation products.

The antiviral zanamivir has been recently reported to occur in surface waters where its presence may lead to the selection of resistant strains of virus in aquatic fauna. In order to evaluate the fate of zanamivir in surface waters, its susceptibility to phototransformation was evaluated using simulated and natural sunlight. Upon exposure of aqueous solutions (20µgL(-1)) to simulated sunlight, zanamivir in surface water degraded at t1/23.6h. Under natural sunlight in surface water about 30% of the initial concentration of the antiviral disappeared within 18 days. The experiments with surface water showed similar effect as humic acid addition with expected decreasing effect on degradation while nitrate addition showed increasing effect. In the experiments with artificial sunlight at high concentrations of zanamivir, four photoproducts were tentatively identified by hydrophilic interaction chromatography-LTQ-Orbitrap-MS, showing [M+H](+) ions at m/z 112 (TP111), m/z 275 (TP274), m/z 323 (TP322), and m/z 333 (TP332). However at 20µgL(-1) only the formation of the recalcitrant TP111 was observed. The proposed structures were rationalized by photolysis mechanisms. Photoproduct TP111 was confirmed with a commercially available standard (isocytosine). In summary, the findings suggest that the photodegradation of zanamivir in surface waters proceeds with slow kinetics.