Occurrence and fate of perfluoroalkyl and polyfluoroalkyl substances (PFAS) in an urban aquifer located at the Besòs River Delta (Spain).

Urban aquifers are at risk of contamination from persistent and mobile organic compounds (PMOCs), especially per- and polyfluoroalkyl substances (PFAS), which are artificial organic substances widely used across various industrial sectors. PFAS are considered toxic, mobile and persistent, and have therefore gained significant attention in environmental chemistry. Moreover, precursors could transform into more recalcitrant products under natural conditions. However, there is limited information about the processes which affect their behaviour in groundwater at the field-scale. In this context, the aim of this study is to assess the presence of PFAS in an urban aquifer in Barcelona, and identify processes that control their evolution along the groundwater flow. 21 groundwater and 6 river samples were collected revealing the presence of 16 PFAS products and 3 novel PFAS. Short and ultra-short chain PFAS were found to be ubiquitous, with the highest concentrations detected for perfluorobutanesulfonic acid (PFBS), trifluoroacetic acid (TFA) and trifluoromethanesulfonic acid (TFSA). Long chain PFAS and novel PFAS were found to be present in very low concentrations (<50 ng/L). It was observed that redox conditions influence the behaviour of a number of PFAS controlling their attenuation or recalcitrant behaviour. Most substances showed accumulation, possibly explained by sorption/desorption processes or transformation processes, highlighting the challenges associated with PFAS remediation. In addition, the removal processes of different intensities for three PFAS were revealed. Our results help to establish the principles of the evolution of PFAS along the groundwater flow, which are important for the development of conceptual models used to plan and adopt site specific groundwater management activities (e.g., Managed Aquifer Recharge).

First Report of the Joint Exposure to Glyphosate and Glufosinate of a Male Population in the Province of Córdoba (Argentina).

Despite potential health implications, data on the presence of Glyphosate (GLY) and other non-GLY herbicides in human matrices remain scarce. This study aimed to develop a simple and cost-effective methodology for detecting and quantifying GLY, its primary biodegradation product; aminomethylphosphonic acid (AMPA); and glufosinate (GLU) in plasma and urine of environmentally and occupationally exposed populations from the province of Córdoba (Argentina). Different alternatives of pre-treatment, derivatization with FMOC-Cl, solid phase extraction, and final sample conditioning steps were evaluated to improve the quantification of the herbicides by a high-performance liquid chromatography system coupled to a triple-quadrupole mass spectrometer. Recoveries ranged from 39 to 84% in both matrices, while limits of quantification were 3, 1, and 0.3 ng/mL and 3.6, 5.1, and 0.3 ng/mL for AMPA, GLY, and GLU in plasma and urine, respectively. In plasma samples, GLY was the most frequently detected analyte (32%), followed by GLU (10%). In urine samples, GLU was the most frequently detected herbicide (13%), followed by GLY (6%). No differences between group or matrix correlations were found. This study is the first report of GLU in human biological matrices and should be used to establish baseline values for future surveillance systems.

Exposure to micro(nano)plastics polymers in water stored in single-use plastic bottles.

Human exposure to micro (nano)plastics (MNPLs) has become a significant concern as a potential health threat. Exposure routes include ingestion, inhalation, and dermal contact, being food and drinking water the primary sources of oral exposure. Here we present the quantification of polymers of MNPLs particles from 700 nm to 20 µm in bottled water commercialised in Spain, including an estimation of the potential risk for daily consumers. We evaluated samples from 20 popular brands in 0.5 and 1.5 L plastic bottles. A double-suspect screening approach developed and validated in our research group for drinking water was adapted for bottled water samples. The identification and quantification of MNPLs-polymers in mass units and the tentative identification of plastic additives (PA) until the second level of confidence was carried out based on high-performance liquid chromatography coupled to high-resolution mass spectrometry (HPLC-HRMS). The results showed the presence of polypropylene (PP), polyethylene (PE) and polypropylene terephthalate (PET) in the samples. Among them, PE was the most frequently detected and quantified polymer (55% of samples) followed by PET which was detected in 33% of the samples and showing the highest concentration (4700 ng L-1). The median value of the sum of polymer concentrations was 359 ng L-1. In addition, 28 plastic additives were detected, where at least one of them was present in 100% of the samples. Stabilizers and plasticisers were the most frequently identified. A prioritisation study was performed using a multi-QSAR modelling software, where bis(2-ethylhexyl) adipate and bis(2-ethylhexyl) phthalate were estimated as the most potentially harmful compounds for human health. Overall, findings suggest that bottled water is a non-negligible route to exposure to MNPLs.

Micro(nano)plastics in the atmosphere of the Atlantic Ocean.

The occurrence, long-range atmospheric transport and deposition of micro and nano plastics (MNPLs) remains un-quantified for the oceanic atmosphereopen ocean. Here we show the characterisation of MNPLs and the aerosol composition (PM10) in a north-south Atlantic transect from Vigo (Spain) to Punta Arenas (Chile). The analytical procedure to assess the composition of MNPLs consisted of a double suspect screening approach of the polymers and additives, the two constituents of plastics. Polymers were analysed by size exclusion chromatography coupled with high-resolution mass spectrometry using an atmospheric pressure photoionization source operated in positive and negative conditions (HPLC(SEC)-APPI(+/-)-HRMS). Plastic additives were screened with high-performance liquid chromatography coupled to high-resolution mass spectrometry using an electrospray ionisation source (HPLC-ESI(+/-)-HRMS). The most common polymers were polyethylene (PE), polypropylene (PP), polyisoprene (PI), and polystyrene (PS), with the highest polymer concentration being 51.7 ng·m-3 of PI. The air mass back trajectories showed the variable influence of oceanic and terrestrial air masses. These differences were reflected in the aerosol composition with different contributions of Saharan dust, sea spray aerosol, organic/elemental carbon, and MNPLs. Results showed that samples largely influenced by sea-spray and air masses originating from coastal South America and the north Atlantic subtropical gyre were more contaminated by MNPLs. Moreover, this information was complemented by the characterisation of the largest particles using scanning electron microscopy (SEM) and µ-Fourier Transform Infrared Spectroscopy (µ-FTIR). This work provides the first field evidence of the long-range transport of MNPLs in most of the Atlantic Ocean, as the result of dynamic coupling between the lower atmosphere and the surface ocean. Sea-spray formation arises as a key driver for the aerosolisation of MNPLs, and atmospheric transport followed by dry deposition may modulate the occurrence of MNPLs in large oceanic regions, issues that will require future research efforts.

Per- and Poly-Fluoroalkyl Substances in Portuguese Rivers: Spatial-Temporal Monitoring.

Eighteen per-and polyfluoroalkyl substances (PFASs) were investigated in surface waters of four river basins in Portugal (Ave, Leça, Antuã, and Cértima) during the dry and wet seasons. All sampling sites showed contamination in at least one of the seasons. In the dry season, perfluorooctanoate acid (PFOA) and perfluoro-octane sulfonate (PFOS), were the most frequent PFASs, while during the wet season these were PFOA and perfluobutane-sulfonic acid (PFBS). Compounds detected at higher concentrations were PFOS (22.6 ng L-1) and perfluoro-butanoic acid (PFBA) (22.6 ng L-1) in the dry and wet seasons, respectively. Moreover, the prospective environmental risks of PFASs, detected at higher concentrations, were evaluated based on the Risk Quotient (RQ) classification, which comprises acute and chronic toxicity. The results show that the RQ values of eight out of the nine PFASs were below 0.01, indicating low risk to organisms at different trophic levels in the four rivers in both seasons, wet and dry. Nevertheless, in the specific case of perfluoro-tetradecanoic acid (PFTeA), the RQ values calculated exceeded 1 for fish (96 h) and daphnids (48 h), indicating a high risk for these organisms. Furthermore, the RQ values were higher than 0.1, indicating a medium risk for fish, daphnids and green algae (96 h).

Cytotoxicity assessment and suspected screening of PLASTIC ADDITIVES in bioplastics of single-use household items.

Bioplastics made of renewable sources provide an excellent alternative to fossil-based materials. However, similar or greater quantities of plastic additives than fossil-based plastics are used in the formulations of bioplastics to improve their performance and barrier properties. Nowadays, there is an increasing concern about sources of chemical exposure. However, there is an important knowledge gap regarding complex additive mixtures, particularly in bio-based materials. In this study, we have characterised the presence of plastic additives in single-use materials (collected from retail shops in Spain), which are made of the most common bio-based biodegradable materials, poly(lactic acid) (PLA) and poly(hydroxybutyrate) (PHB), in contrast with a fossil-based plastic material that is extensively made from high-density polyethylene (HDPE). The approach consisted of the pulverization of material in the nano-micro range (100 nm-10 µm), with the materials being extracted using different solvents and ultrasonic-assisted solvent extraction (UASE). 100% of the additives in the material cannot be extracted, but since they were performed in the same condition for all materials can inform about the fingerprint of primary organics and the relative abundances between the different materials. The extracts were analysed by high-performance liquid chromatography coupled with high-resolution mass spectrometry equipped with a heated electrospray ionisation source operated in positive and negative ionisation conditions (HPLC-HESI(+/-)-HRMS), separately, using a suspect screening approach. A total number of 203 additives were tentatively identified (confidence level 2) in the bioplastics items of this study. An average of 123 plastic additives were found in PLA items and 121 in PHB items. Plasticisers were the most abundant additives; the phthalates group was the most commonly found, while 63 plastic additives were confirmed by standards and quantified. In parallel, the cytotoxicity of plastic particles in terms of cell viability and oxidative stress was studied using A549 alveolar basal epithelial cells, and the toxicity of the different extracts was also established using HepG2 adenocarcinoma cells. The main results of this study demonstrate that the plastic particles did not show a significant reduction in cell viability, but oxidative stress was significant, with PLA being the material that showed the highest effect. On the other hand, extracts of plastic particles did not show inhibition of cell viability except for HDPE extract, but the different extracts produced oxidative stress, with PLA showing the highest effect. Although the item showing the highest concentrations of additives was the extract of PLA material while also showing the most elevated oxidative stress, the low migration of toxicants from plastic materials ensures their safe use. However, this also supports the idea that bioplastics can contain many toxic substances in their formulations, some of which are unknown and should be studied in more depth.

Polymers of micro(nano) plastic in household tap water of the Barcelona Metropolitan Area.

Microplastics (MPLs) are emerging persistent pollutants affecting drinking water systems, and different studies have reported their presence in tap water. However, most of the work has a focus on particles in the 100-5 µm range. Here, a workflow to identify and quantify polymers of micro and nanoplastics (MNPLs), with sizes from 0.7 to 20 µm in tap water, is presented. The analytical method consisted of water fractionated filtration followed by toluene ultrasonic-assisted extraction and size-exclusion chromatography, using an advanced polymer chromatography column coupled to high-resolution mass spectrometry with atmospheric pressure photoionization source with negative and positive ionization conditions (HPLC(APC)-APPI(±)-HRMS) and normal phase chromatography HILIC LUNA® column and electrospray ionisation source in positive and negative mode (HPLC(HILIC)-ESI(±)-HRMS). The acquisition was performed in full scan mode, and the subsequent tentative identification of MNPLs polymers has been based on increasing the confirmation level, including the characterisation of monomers by using Kendrick Mass Defect (KMD) analysis, and confirmation and quantification using standards. This approach was applied to assess MNPLs in tap water samples of the Barcelona Metropolitan Area (BMA), that were collected from August to October 2020 from home taps of volunteers distributed in the 42 postal codes of the BMA. Polyethylene (PE), polypropylene (PP), polyisoprene (PI), polybutadiene (PBD), polystyrene (PS), polyamide (PA), and polydimethylsiloxanes (PDMS) were identified. PE, PP, and PA were the most highly detected polymers, and PI and PBD were found at the highest concentrations (9,143 and 1,897 ng/L, respectively). A principal component analysis (PCA) was conducted to assess differences in MNPLs occurrence in drinking water, that was provided from the two drinking water treatment plants (DWTPs) suppliers. Results showed that no significant differences (at 95% confidence level) were established between the drinking water supplies to the different areas of the BMA.

Assessment of Micro- and Nanoplastic Composition (Polymers and Additives) in the Gastrointestinal Tracts of Ebro River Fishes.

One of the main routes of fish exposure to micro- and nanoplastics (MNPLs) is their ingestion. MNPLs can act as reservoirs of organic contaminants that are adsorbed onto their surfaces, or that can leach from their complex formulations, with potential impacts on biota and along the aquatic food chain. While MNPLs have been reported in fishes worldwide, complete information on MNPL compositions, polymers and additives continues to be scarce. In this work, the presence of MNPLs in the gastrointestinal tracts (GIT) of fish from the Ebro River (Spain) was investigated using a double suspected screening approach to assess and quantify polymers and additives. The sample-preparation procedure consisted of sequential alkaline and acidic digestions with KOH and HNO3, followed by ultrasonic-assisted extraction (USAE) with toluene. The analysis of polymers was carried out with size-exclusion chromatography followed by high-resolution mass spectrometry using an atmospheric pressure photoionization source, operating in negative and positive ionisation modes (SEC-(±)-APPI-HRMS) using full-scan acquisition (FS). Plastic additives were assessed using high-performance liquid chromatography with a C18 analytical column coupled to HRMS equipped with an electrospray ionisation source operating under positive and negative conditions (LC-(±ESI)-HRMS). The acquisition was performed in parallel with full-scan (FS) and data-dependent scan (ddMS2) modes, working under positive and negative ionisation modes. The polymers most frequently detected and quantified in fish GITs were polysiloxanes, polyethylene (PE), polypropylene (PP) and polystyrene (PS). PE was detected in 84% of the samples, with a concentration range from 0.55 to 3545 µg/g. On the other hand, plasticisers such as phthalates and stabilisers such as benzotriazoles were the most frequently identified plastic additives.

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.

Current Insights into Potential Effects of Micro-Nanoplastics on Human Health by in-vitro Tests.

Humans are exposed to micro and nanoplastics (MNPLs) through inhalation, ingestion and, to a lesser extent, dermal contact. In recent years, new insights indicate the potential of MNPLs to cause damages to human health. Particle toxicity can include oxidative stress, inflammatory lesions, and then increased internalization or translocation through tissues. On the other hand, plastic additives are used in plastic particles, once internalized, can release toxic substances. It is noteworthy that the potential effects of MNPLs encompass a wide range of polymers and chemical additives, showing various physicochemical and toxicological properties, and the size, shape and surface properties are other variables influencing their effects. In spite of the research carried out recently, MNPLs research is in its early stages, and further investigation is required. In this review article, the knowledge of human exposure routes and the recent results on the toxicological effects of MNPLs in human health are presented and discussed. Finally, the current limitations and the main gaps in the body of knowledge are summarised.

Priority and emerging organic microcontaminants in three Mediterranean river basins: Occurrence, spatial distribution, and identification of river basin specific pollutants.

There is a worldwide growing use of chemicals by our developed, industrialized, and technological society. More than 100,000 chemical substances are thus commonly used both by industry and households. Depending on the amount produced, physical-chemical properties, and mode of use, many of them may reach the environment and, notably, the aquatic receiving systems. This may result in undesirable and harmful side-effects on both the human and the ecosystem's health. Mediterranean rivers are largely different from Northern and Central European rivers in terms of hydrological regime, climate conditions (e.g. air temperature, solar irradiation, precipitation), and socio-economics (e.g. land use, tourism, crop types, etc.), with all these factors leading to differences in the relative importance of the environmental stressors, in the classes and levels of the pollutants found and their environmental fate. Furthermore, water scarcity might be critical in affecting water pollution because of the lowered dilution capacity of chemicals. This work provides raw chemical data from different families of microcontaminants identified in three selected Mediterranean rivers (the Sava, Evrotas, and Adige) collected during two sampling campaigns conducted in 2014 and 2015 in three different matrices, namely, water, sediments, and biota (fish). More than 200 organic micropollutants were analyzed, including relevant groups like pharmaceuticals, personal care products, perfluorinated compounds, pesticides, pyrethroid insecticides, flame retardants, and persistent organic pollutants. Data obtained were summarized with some basic statistics for all compound families and matrices analyzed. Observed occurrence and spatial patterns were interpreted both in terms of compound physical-chemical properties and local environmental pressures. Finally, their spatial distribution was examined and their ecotoxicological risk in the water phase was assessed. This allowed locating, at each basin, the most polluted sites ("hot spots") and identifying the respective river basin specific pollutants (RBSPs), prioritizing them in terms of the potential ecotoxicological risk posed to the aquatic ecosystems.

Screening of suspected micro(nano)plastics in the Ebro Delta (Mediterranean Sea).

This is the first work reporting the use of a double suspect-screening to assess most common polymers and additives in micro(nano)plastics (NPLs/MPLs) found in environmental waters. The method consisted of water filtration followed by ultrasonic-assisted extraction with toluene and analysis employing size exclusion chromatography using an advanced polymer chromatography column coupled to high-resolution mass spectrometry with an atmospheric pressure photoionisation source by negative ionisation conditions (LC(APC)-APPI(-)-HRMS). The identification of NPL/MPLs polymers has been based on increasing confirmation level, including the monomers characterisation by the Kendrick Mass Defect and confirmation and quantification when standards were available. In parallel, the identification of main additives in NPL/MPLs composition, as well organic contaminants adsorbed onto the plastic particles were carried out by analysis of the extracts by LC(C18)-APPI (+/-)-HRMS. To assess the impact of plastic pollution it is necessary to assess the composition in terms of polymers but also the additives. This screening approach has been employed to study composition of NPL/MPLs in the Ebro Delta. Two sampling campaigns including freshwater and seawater samples have been investigated to assess plastic composition in the top 5 cm. Polystyrene (PS), polyethylene (PE), polyisoprene (PI), polybutadiene (PBD), polypropylene (PP) and polysiloxanes were the most detected polymers and PP and PE, sizing between < 1000 and 2000 Da, were found at concentrations reaching up to 7000 ng/L in some areas. The pentadecanoic acid, 1,2,3-benzotriazoles, 2-ethylhexanoic acid (2-EHA), and phthalates such as dimethyl phthalate, mono(2-ethylhexyl) phthalate (MEHP) and the phthalimide were more frequently detected plastic additives. Finally, series of organic contaminants were as well detected in the particulate fraction. These organic contaminants cannot be associated to plastic compositions but can be associated to their adsorption to the particulate matter, in particular to NPL/MPLs, due to their non-polar character. Among these organic contaminants, the more frequently detected were pharmaceutical compounds, food additives and pesticides.

Adsorption and Desorption Behaviour of Polychlorinated Biphenyls onto Microplastics' Surfaces in Water/Sediment Systems.

The potential of microplastics (MPLs) in marine ecosystems to adsorb and transport other micropollutants to biota, contributing to their entry in the food chain, is a primary cause of concern. However, these interactions remain poorly understood. Here, we have evaluated the adsorption/desorption behaviour of marker polychlorinated biphenyls (PCBs), onto MPL surfaces of three widely used polymers-polystyrene (PS), polyethylene (PE), and polyethylene terephthalate (PET). The range of MPL sizes ranged from 1 to 600 µm. The adsorption/desorption was evaluated in sediment/water systems in marine microcosms emulating realistic environmental conditions for 21 days. The adsorption percentages ranged from 20 to 60%. PCBs with a lower degree of chlorination showed higher adsorption percentages because of conformational impediments of PCBs with high-degree chlorination, and also by their affinity to be adsorbed in sediments. Glassy plastic polymers as PET and PS showed a superior affinity for PCBs than rubbery polymers, such as PE. The polymers that can bond PCBs by π-π interactions, rather than van der Waals forces showed better adsorption percentages, as expected. Finally, the adsorption/desorption behaviour of selected PCBs onto MPLs was fitted to a Freundlich isotherm model, with correlations higher than 0.8 in most of the cases.

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.

Trace analysis of polystyrene microplastics in natural waters.

The development of quantitative and qualitative analytical methods to assess micro-plastics (MPLs) and nano-plastics (NPLs) content in the environment is a central issue for realistic risk assessment studies. However, the quantitative analysis continues being a critical issue, in particular for MPLs from 100 µm down to the nano-sized range in complex environmental samples. This paper evaluates the potential of mass spectrometry for the analysis of MPLs and NPLs. The performance of different techniques including matrix-assisted laser desorption ionisation (MALDI) coupled to time-of-flight mass spectrometry (TOF-MS), liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS), and the ambient ionisation approaches as desorption electrospray ionisation (DESI) and direct analysis real-time (DART), were assessed for the study of polystyrene (PS) MPLs and NPLs in natural waters. A method based on LC-HRMS, equipped with an atmospheric pressure photoionisation source (APPI), operated in negative conditions for the quantitative analysis of PS MPLs and NPLs in natural waters, was developed. The chromatographic separation was achieved using an advanced polymer chromatographic (APC) column using toluene isocratic as the mobile phase. The optimal analytical method showed an instrumental limit of detection (ILOD) of 20 pg and methods limits of detection and quantification around 30 pg L-1 and 100 pg L-1, respectively. And, recoveries of 60 and 70% in samples from rivers and the marine coast, respectively. The performance of the new method was proved by the analysis of fortified samples and natural seawater samples.

Assessing molecular subtypes of gastric cancer: microsatellite unstable and Epstein-Barr virus subtypes. Methods for detection and clinical and pathological implications.

BACKGROUND: The molecular classification of gastric cancer recognises two subtypes prone to immune checkpoint blockade: the microsatellite unstable and the Epstein-Barr virus (EBV)-related tumours. We aim to assess the concordance between immunohistochemistry and PCR for microsatellite status evaluation, and explore the value of microsatellite instability (MSI) and EBV as predictive survival factors. MATERIAL AND METHODS: We collected 246 consecutively diagnosed gastric cancer cases in all stages and evaluated the microsatellite status using immunohistochemistry for mismatched repair (MMR) proteins and PCR. EBV expression was studied through in situ hybridisation. RESULTS: Forty-five (18%) cases presented MSI and 13 (6%) were positive for EBV. MSI was associated with female sex, older age, distal location and distal non-diffuse type of the modified Lauren classification. EBV expression was most frequent in proximal location and proximal non-diffuse type. The sensitivity, specificity, positive predictive value and negative predictive value of immunohistochemistry for the microsatellite study were 91%, 98%, 91% and 98%, respectively. In the multivariate analysis, MSI was an independent predictor of favourable tumour-specific survival (TSS) in stages I-III (MSI: HR: 0.37, 95% CI 0.12 to 0.95, p=0.04). CONCLUSIONS: The MSI status and the EBV expression should be incorporated in routine pathological report for two reasons. First, MSI defines a different pathological entity with a better outcome. Second, MSI and EBV may be useful biomarkers to identify patients who will respond to immune checkpoint blockade inhibitors. For this purpose, immunohistochemical study for MMR proteins and in situ hybridisation study for EBV evaluation are feasible and cost-effective methods.

Fungal treatment of metoprolol and its recalcitrant metabolite metoprolol acid in hospital wastewater: Biotransformation, sorption and ecotoxicological impact.

Hospital wastewater (HWW) effluents represent an important source of contaminants such as pharmaceutical compounds and their human metabolites. To better evaluate dedicated treatment of hospital effluents for pollutant mitigation, not only the parent compounds should be considered but also the intermediates generated during treatment. The metabolite metoprolol acid (MTPA) has been found in urban wastewaters at higher concentration than its parent compound metoprolol (MTP), being more recalcitrant to biodegradation. The aim of this study was to investigate degradation, transformation and sorption of the ß-blocker MTP, and its recalcitrant metabolite MTPA, during water treatment based on the fungi Ganoderma lucidum, Trametes versicolor and Pleurotus ostreatus. Fourteen intermediates were identified in MTP biotransformation while five of them also attributed to MTPA biodegradation and two to MTPA only. Their identification allowed their correlation in separate biotransformation pathways suggested. The highest degradation rate of metoprolol (up to 51%) and metoprolol acid (almost 77%) was found after 15-days treatment with Ganoderma lucidum, with an increase in toxicity up to 29% and 4%, respectively. This fungus was further selected for treating real HWW in a batch fluidized bed bioreactor (FBB). Treated wastewater and fungal biomass samples were used to evaluate the distribution of the target compounds and the intermediates identified between solid and liquid phases. While similar elimination capabilities were observed for the removal of metoprolol, and even higher for its persistent metabolite metoprolol acid, the extent on compound transformation diminished considerably compared with the study treating purified water: a high level of the persistent α-HMTP and TP240 were still present in effluent samples (15% and 6%, respectively), being both TPs present at high proportion (up to 28%) in fungal biomass. This is the first time that pharmaceutical TPs have been investigated in the fungal biomass.

Levels of regulated POPs in fish samples from the Sava River Basin. Comparison to legislated quality standard values.

Fish samples of different species (i.e. rainbow trout (Onchorhynchus mykiss), barbel (Barbus barbus) and European chub (Squalius cephalus)) were collected from the Sava River Basin for a preliminary investigation of the levels of PCDD/Fs, PCBs, PBDEs and PFAS as a whole. Concentrations of PCDD/Fs, in terms of pg WHO-TEQ/g ww, were below the maximum limit established at the Commission Regulation (EU) No 1259/2011. On the contrary, when DL-PCBs were also included, levels increase up to 11.7 pg WHO-TEQPCDD/Fs+DL-PCBs/g ww in a particular case, with two samples out of a total of ten exceeding the maximum set at this EU Regulation and the EQS established at the European Directive regarding priority substances in the field of water policy (0.0065 ng WHO-TEQPCDD/Fs+DL-PCBs/g ww). A similar trend was also observed for NDL-PCBs, whit the same two samples, from the lower stretch of the river basin, exceeding the maximum limit allowed at the EU Regulation (125 ng/g ww). For PBDEs, levels found in all the samples exceeded the EQS (0.0085 ng/g ww) up to more than a thousand times and 40% of the samples presented PFOS values above the EQS. Data from this study were compared to values reported at the literature for fish from other geographical areas.

Fungal biodegradation of the N-nitrosodimethylamine precursors venlafaxine and O-desmethylvenlafaxine in water.

Antidepressant drugs such as Venlafaxine (VFX) and O-desmethylvenlafaxine (ODMVFX) are emerging contaminants that are commonly detected in aquatic environments, since conventional wastewater treatment plants are unable to completely remove them. They can be precursors of hazardous by-products, such as the carcinogenic N-nitrosodimethylamine (NDMA), generated upon water chlorination, as they contain the dimethylamino moiety, necessary for the formation of NDMA. In this study, the capability of three white rot fungi (Trametes versicolor, Ganoderma lucidum and Pleurotus ostreatus) to remove both antidepressants from water and to decrease NDMA formation potential was investigated. Furthermore, transformation by-products (TPs) generated along the treatment process were elucidated and also correlated with their NDMA formation potential. Very promising results were obtained for T. versicolor and G. lucidum, both being able to remove up to 100% of ODMVFX. In the case of VFX, which is very recalcitrant to conventional wastewater treatment, a 70% of removal was achieved by T. versicolor, along with a reduction in NDMA formation potential, thus decreasing the associated problems for human health and the environment. However, the NDMA formation potential remained practically constant during treatment with G. lucidum despite of the equally high VFX removal (70%). This difference was attributed to the generation of different TPs during both fungal treatments. For example, G. lucidum generated more ODMVFX, which actually has a higher NDMA formation potential than the parent compound itself.

Occurrence and persistence of carbapenemases genes in hospital and wastewater treatment plants and propagation in the receiving river.

This study aims to investigate the prevalence of clinically relevant carbapenemases genes (blaKPC, blaNDM and blaOXA-48) in water samples collected over one-year period from hospital (H), raw and treated wastewater of two wastewater treatment plants (WWTPs) as well as along the Zenne River (Belgium). The genes were quantified in both particle-attached (PAB) and free-living (FLB) bacteria. Our results showed that absolute abundances were the highest in H waters. Although absolute abundances were significantly reduced in WWTP effluents, the relative abundance (normalized per 16S rRNA) was never lowered through wastewater treatment. Particularly, for the PAB the relative abundances were significantly higher in the effluents respect to the influents of both WWTPs for all the genes. The absolute abundances along the Zenne River increased from upstream to downstream, peaking after the release of WWTPs effluents, in both fractions. Our results demonstrated that blaKPC, blaNDM and blaOXA-48 are widely distributed in the Zenne as a consequence of chronic discharge from WWTPs. To conclude, the levels of carbapenemases genes are significantly lower than other genes conferring resistance to more widely used antibiotics (analyzed in previous studies carried out at the same sites), but could raise up to the levels of high prevalent resistance genes.