Preliminary hazard assessment of a new nature-inspired antifouling (NIAF) agent.

A recently synthesized aminated 3,4-dioxygenated xanthone (Xantifoul2) was found to have promising antifouling (AF) effects against the settlement of the macrofouler Mytilus galloprovincialis larvae. Preliminary assessment indicated that Xantifoul2 has reduced ecotoxicological impacts: e.g., being non-toxic to the marine crustacea Artemia salina (<10 % mortality at 50 µM) and showing low bioconcentration factor in marine organisms. In order to meet the EU Biocidal Product Regulation, a preliminary hazard assessment of this new nature-inspired antifouling (NIAF) agent was accomplished in this work. Xantifoul2 did not affect the swimming ability of the planktonic crustacean Daphnia magna, the growth of the diatom Phaeodactylum tricornutum, and the cellular respiration of luminescent Gram-negative bacteria Vibrio fischeri, supporting the low toxicity towards several non-target marine species. Regarding human cytotoxicity, Xantifoul2 did not affect the cell viability of retinal human cells (hTERT-RPE-1) and lipidomic studies revealed depletion of lipids involved in cell death, membrane modeling, lipid storage, and oxidative stress only at a high concentration (10 µM). Accelerated degradation studies in water were conducted under simulated sunlight to allow the understanding of putative transformation products (TPs) that could be generated in the aquatic ecosystems. Both Xantifoul2 and photolytic-treated Xantifoul2 in the aqueous matrix were therefore evaluated on several nuclear receptors (NRs). The results of this preliminary hazard assessment of Xantifoul2, combined with the high degradation rates in water, provide strong evidence of the safety of this AF agent under the evaluated conditions, and provide the support for future validation studies before this compound can be introduced in the market.

Microplastic distribution in different tissues of small pelagic fish of the Northeast Atlantic Ocean.

The accumulation of microplastics (MP) by marine species of ecological and commercial interest represents a major concern, particularly for those present in human diet. This study analysed the accumulation of MP in three species of coastal pelagic fish with high commercial value, European sardine (Sardina pilchardus), European anchovy (Engraulis encrasicolus) and horse mackerel (Trachurus trachurus), collected along the Western coast of the Iberian Peninsula. The gastrointestinal tract (GT), gills and muscle were analysed and a total of 504 particles were observed. MP were found in all target tissues of the studied species. Horse mackerel exhibited significantly higher concentrations of microplastics in GT compared to other tissues. On the other hand, anchovies and sardines had significantly lower microplastic concentrations in their muscle tissue. The accumulation of microplastics in the gills showed a significant difference between species, with anchovy having significantly higher concentrations compared to horse mackerel. Horse mackerel had the highest percentage of individuals with microplastics in their GT (92 %), followed by sardine (75 %) and anchovy (50 %). Horse mackerel was also the species that registered the highest percentage of individuals with particles in the muscle (63 %), followed by anchovy (40 %) and finally sardine (39 %). MP in the gills of European sardines and anchovies were similar to those found in water samples. The majority of MP found measured <0.5 mm and were blue fibers. Furthermore, the presence of MP in the GT showed a weak and moderated significant negative correlation with the Fulton Condition Index in horse mackerel and European sardine. Our study confirms the ubiquitous extent of MP contamination in the ocean and provides baseline evidence of MP tissue distribution in three small pelagic fish species with distinct feeding behaviour, while correlating this with the presence of MP in water. Importantly, the results of this study contribute to improve the understanding of biological partitioning of MP in open sea fish species with high commercial relevance, and the potential deleterious effects of our increasingly MP contaminated world.

Spatial distribution and temporal trends of butyltin compounds (TBT, DBT & MBT) in short sediment cores of the SW Portuguese Shelf (western Iberian Margin, NE Atlantic).

Spatial patterns and temporal trends of the butyltin compounds tributyltin (TBT), dibutyltin (DBT), and monobutyltin (MBT) were investigated in a set of sediment samples collected along the SW Portuguese continental shelf. This region did not reach the Good Environmental Status (GES) in accordance with the Marine Strategy Framework Directive (MSFD) during a first evaluation carried out in 2012. Overall, MBT and DBT were the predominant organotin species detected, but high concentrations of TBT were found in and around disposal sites for dredge sludge derived from the dredging in navigation channels, harbours, and shipyard facilities of the Tagus and Sado estuaries. Although Portuguese regulations for monitoring sediment quality in relation to dredging activities consider only PAH, PCB and HCB, they also dictate that other organic contaminants such as butyltin compounds (BTs) should be monitored if suspicion of high values exists, but no action limits are defined for these (MAOTDR, 2007). Without action limits, the monitoring recommendation given in the regulations is not put into practice. Considering their toxicity, BT derivates should be integrated in the legislation, because they represent an environmental threat in the relocation of dredged material, especially when derived from harbour and shipyards areas. Based on this study, we recommend giving more attention to the amounts and impacts of BTs in sediments at dredged material disposal sites (DMDS) and their surroundings. Or even better, in order to be more efficient, monitoring should be done at the source of the dredged materials and not at the sink. In case it is not done, the monitoring of concentrations of TBT (and other BTs) in sediments and organisms, including imposex studies, at all Portuguese sites for disposal of dredged material receiving slightly to strongly contaminated dredged material must be developed.

In Vitro Antimicrobial Studies of Mesoporous Silica Nanoparticles Comprising Anionic Ciprofloxacin Ionic Liquids and Organic Salts.

The combination of active pharmaceutical ingredients in the form of ionic liquids or organic salts (API-OSILs) with mesoporous silica nanoparticles (MSNs) as drug carriers can provide a useful tool in enhancing the capabilities of current antibiotics, especially against resistant strains of bacteria. In this publication, the preparation of a set of three nanomaterials based on the modification of a MSN surface with cholinium ([MSN-Chol][Cip]), 1-methylimidazolium ([MSN-1-MiM][Cip]) and 3-picolinium ([MSN-3-Pic][Cip]) ionic liquids coupled with anionic ciprofloxacin have been reported. All ionic liquids and functionalized nanomaterials were prepared through sustainable protocols, using microwave-assisted heating as an alternative to conventional methods. All materials were characterized through FTIR, solution 1H NMR, elemental analysis, XRD and N2 adsorption at 77 K. The prepared materials showed no in vitro cytotoxicity in fibroblasts viability assays. The minimum inhibitory concentration (MIC) for all materials was tested against Gram-negative K. pneumoniae and Gram-positive Enterococcus spp., both with resistant and sensitive strains. All sets of nanomaterials containing the anionic antibiotic outperformed free ciprofloxacin against resistant and sensitive forms of K. pneumoniae, with the prominent case of [MSN-Chol][Cip] suggesting a tenfold decrease in the MIC against sensitive strains. Against resistant K. pneumoniae, a five-fold decrease in the MIC was observed for all sets of nanomaterials compared with neutral ciprofloxacin. Against Enterococcus spp., only [MSN-1-MiM][Cip] was able to demonstrate a slight improvement over the free antibiotic.

Ozone membrane contactor for tertiary treatment of urban wastewater: Chemical, microbial and toxicological assessment.

A membrane ozone contactor, operated under continuous mode, was applied to promote the tertiary treatment of urban wastewater (UWW), targeting the removal of contaminants of emerging concern (CECs), bacterial disinfection, and toxicity reduction. This system relies on the homogeneous radial distribution of ozone (O3) in the reaction zone by "titration" through a microfiltration borosilicate tubular membrane, while the UWW swirls around the membrane and drags the O3 microbubbles generated in the membrane shell-side. The membrane is coated with titanium dioxide (TiO2-P25) and radiation can be externally supplied via four UV lamps. The ozonation tests were carried out with secondary-treated UWW collected in different seasons (winter and summer) and spiked with a mix of 19 CECs (10 µg L-1 each). For an O3 dose of 18 g m-3, the best performance was obtained by increasing the O3 concentration (maximum [O3]G,inlet of 200 g Nm-3) and decreasing the gas flow rate (minimum QG of 0.15 Ndm3 min-1), providing the highest ozone transfer yield (88 %) and, thus higher specific ozone dose (g O3 per g dissolved organic carbon). Under these conditions, removals >80 % or concentrations below the limit of quantification were obtained for up to 13 of the 19 CECs and reductions up to 5 log units for total heterotrophs and below the limit of detection for enterobacteria and enterococci. Tests including a UVC dose of 0.10 kJ L-1 enhanced disinfection ability but had no impact on CECs oxidation. After ozonation, the abundance of antibiotic resistant bacteria was reduced but not eliminated, and microbial regrowth after 3-day storage was observed. No toxic effect was detected on zebrafish embryos using a dilution factor of 4 for the ozonized UWW and when granular activated carbon adsorption was subsequently applied the dilution factor decreased to 2.

First evidence of microplastic ingestion in the ocean giant sunfish (Mola mola).

Mola mola is the largest teleost inhabiting our ocean and the presence of microplastics (MP) in this flagship species was, before this study, never described. Thus, this investigation focused on analysing MP ingestion in 53 ocean giant sunfish in the Northeast Atlantic Ocean. A total of 116 MP were found in 79% of the specimens, with a median of 1 MP.ind-1, ranging from 0 to 11 MP.ind-1. Seasonal differences were observed, with more fibers registered in specimens caught in autumn. Among the different size classes observed, the smallest category (<300 µm) was the most frequent (43%). Blue (43%) was the most prevalent color, followed by green (29%) and black (10%). The majority of fragments were styrene acrylic copolymer (53%), while most fibers were rayon (78%). These findings emphasize that the ocean sunfish population crossing the southern waters of Portugal is exposed to microplastic pollution and highlight the need for effective management policies to address plastic pollution in marine ecosystems.

Occurrence of persistent and mobile chemicals and other contaminants of emerging concern in Spanish and Portuguese wastewater treatment plants, transnational river basins and coastal water.

This work investigated, during one year, the occurrence and fate of 52 contaminants of emerging concern (CECs) in transnational river basins and coastal areas of the North of Portugal and Galicia (NW Spain) and the wastewater treatment plants (WWTPs) discharging on these environments. The different CECs investigated included pharmaceuticals, personal care products, industrial chemicals, among others, of which ca. 90 % would fulfill the persistence, mobility and toxicity criteria proposed by the German Environmental Agency. The results showed the ubiquitous presence of these CECs and an incomplete removal of over 60 % of them with current conventional WWTPs. These findings highlight the requirement of a prominent and coordinated upgrade of WWTP treatments in order to meet the future European Union regulations on urban wastewater treatment and surface water quality. In fact, even some compounds exhibiting high removals, such as caffeine or xylene sulfonate, were frequently detected in river and estuarine waters at the high ng L-1 level. Thus, our preliminary risk assessment study concluded that 18 of the CECs presented a potential risk for the environment, being caffeine, sulpiride, perfluorooctanoic acid (PFOA), diclofenac, fipronil and perfluorobutanoic acid (PFBA) the most concerning ones. Yet, additional toxicity data as well as a more robust information on persistence and mobility of CECs are necessary for better estimating the magnitude of the problem and improve risk assessment. As an example, in the case of the antidiabetic metformin, recent research has revealed toxicity for model fish species at concentration levels below those found in 40 % of the river water samples analyzed in this work.

A modelling framework to assess multiple metals impacts on marine food webs: Relevance for assessing the ecological implications of deep-sea mining based on a systematic review.

Industrial deep-sea mining will release plumes containing metals that may disperse over long distances; however, there is no general understanding of metal effects on marine ecosystems. Thus, we conducted a systematic review in search of models of metal effects on aquatic biota with the future perspective to support Environmental Risk Assessment (ERA) of deep-sea mining. According to results, the use of models to study metal effects is strongly biased towards freshwater species (83% freshwater versus 14% marine); Cu, Hg, Al, Ni, Pb, Cd and Zn are the best-studied metals, and most studies target few species rather than entire food webs. We argue that these limitations restrain ERA on marine ecosystems. To overcome this gap of knowledge, we suggest future research directions and propose a modelling framework to predict the effects of metals on marine food webs, which in our view is relevant for ERA of deep-sea mining.

Stressors of emerging concern in deep-sea environments: microplastics, pharmaceuticals, personal care products and deep-sea mining.

Although most deep-sea areas are remote in comparison to coastal zones, a growing body of literature indicates that many sensitive ecosystems could be under increased stress from anthropogenic sources. Among the multiple potential stressors, microplastics (MPs), pharmaceuticals and personal care products (PPCPs/PCPs) and the imminent start of commercial deep-sea mining have received increased attention. Here we review recent literature on these emerging stressors in deep-sea environments and discuss cumulative effects with climate change associated variables. Importantly, MPs and PPCPs have been detected in deep-sea waters, organisms and sediments, in some locations in comparable levels to coastal areas. The Atlantic Ocean and the Mediterranean Sea are the most studied areas and where higher levels of MPs and PPCPs have been detected. The paucity of data for most other deep-sea ecosystems indicates that many more locations are likely to be contaminated by these emerging stressors, but the absence of studies hampers a better assessment of the potential risk. The main knowledge gaps in the field are identified and discussed, and future research priorities are highlighted to improve hazard and risk assessment.

Dephosphorylation of the pre-initiation complex is critical for origin firing.

In eukaryotes, cyclin-dependent kinase (CDK) ensures that the genome is duplicated exactly once by inhibiting helicase loading factors before activating origin firing. CDK activates origin firing by phosphorylating two substrates, Sld2 and Sld3, forming a transient and limiting intermediate-the pre-initiation complex (pre-IC). Here, we show in the budding yeast Saccharomyces cerevisiae that the CDK phosphorylations of Sld3 and Sld2 are rapidly turned over during S phase by the PP2A and PP4 phosphatases. PP2ARts1 targets Sld3 specifically through an Rts1-interaction motif, and this targeted dephosphorylation is important for origin firing genome-wide, for formation of the pre-IC at origins and for ensuring that Sld3 is dephosphorylated in G1 phase. PP2ARts1 promotes replication in vitro, and we show that targeted Sld3 dephosphorylation is critical for viability. Together, these studies demonstrate that phosphatases enforce the correct ordering of replication factor phosphorylation and in addition to kinases are also key drivers of replication initiation.

Are Fish Populations at Risk? Metformin Disrupts Zebrafish Development and Reproductive Processes at Chronic Environmentally Relevant Concentrations.

The antidiabetic drug Metformin (MET), one of the most prevalent pharmaceuticals in the environment, is currently detected in surface waters in the range of ng/L to low µg/L. As current knowledge regarding the long-term effects of environmentally relevant concentrations of MET in nontarget organisms is limited, the present study aimed at investigating the generational effects of MET, in concentrations ranging from 390 to 14 423 ng/L in the model organism Danio rerio (up to 9 mpf), including the effects on its nonexposed offspring (until 60 dpf). We integrate several apical end points, i.e., embryonic development, survival, growth, and reproduction, with qRT-PCR and RNA-seq analyses to provide additional insights into the mode of action of MET. Reproductive-related parameters in the first generation were particularly sensitive to MET. MET parental exposure impacted critical molecular processes involved in the metabolism of zebrafish males, which in turn affected steroid hormone biosynthesis and upregulated male vtg1 expression by 99.78- to 155.47-fold at 390 and 14 432 MET treatment, respectively, pointing to an estrogenic effect. These findings can potentially explain the significant decrease in the fertilization rate and the increase of unactivated eggs. Nonexposed offspring was also affected by parental MET exposure, impacting its survival and growth. Altogether, these results suggest that MET, at environmentally relevant concentrations, severely affects several biological processes in zebrafish, supporting the urgent need to revise the proposed Predicted No-Effect Concentration (PNEC) and the Environmental Quality Standard (EQS) for MET.

Development of multi-residue gas chromatography coupled with mass spectrometry methodologies for the measurement of 15 chemically different disinfection by-products (DBPs) of emerging concern in drinking water from two different Portuguese water treatment plants.

In water treatment plants (WTPs), chemical agents, such as chlorine and ozone, might react with organic matter and anthropogenic contaminants, forming a high diversity of disinfection by-products (DBPs). Due to the potential toxicological effects, the identification of unregulated DBPs (UR-DBPs) is critical to help water managers in the selection of effective water treatment processes, contributing to improving water safety plans. Given the limited validated analytical methods to detect UR-DBPs, here we developed new multi-residue gas chromatography coupled with mass spectrometry methodologies for the detection and quantification of 15 UR-DBPs, including aldehydes, haloketones (HKs), nitrosamines and alcohols, in drinking water matrices. Solid-phase extraction (SPE), for the nitrosamine group, and solid-phase micro extraction (SPME), for the remaining DBPs, were used as sample preparation methods. The developed methodologies allowed the quantification of target UR-DBPs at trace concentration levels (ng L-1), with method quantification limits (MQLs) ranging from 14.4 ng L-1 to 26.0 ng L-1 (SPE-GC-MS) and 2.3 ng L-1 and 1596 ng L-1 (SPME-GC-MS). The methods were applied to different drinking water matrices, considering distinct delivery points of EPAL - Empresa Portuguesa das Águas Livres WTPs. Overall, the aldehyde group, represented by decanal, nonanal and 2-ethylheaxanal, showed the highest occurrence, followed by HKs and nitrosamines. The results of this study suggested that the formation of these UR-DBPs should be further monitored in WTPs.

Global early replication disrupts gene expression and chromatin conformation in a single cell cycle.

BACKGROUND: The early embryonic divisions of many organisms, including fish, flies, and frogs, are characterized by a very rapid S-phase caused by high rates of replication initiation. In somatic cells, S-phase is much longer due to both a reduction in the total number of initiation events and the imposition of a temporal order of origin activation. The physiological importance of changes in the rate and timing of replication initiation in S-phase remains unclear. RESULTS: Here we assess the importance of the temporal control of replication initiation using a conditional system in budding yeast to drive the early replication of the majority of origins in a single cell cycle. We show that global early replication disrupts the expression of over a quarter of all genes. By deleting individual origins, we show that delaying replication is sufficient to restore normal gene expression, directly implicating origin firing control in this regulation. Global early replication disrupts nucleosome positioning and transcription factor binding during S-phase, suggesting that the rate of S-phase is important to regulate the chromatin landscape. CONCLUSIONS: Together, these data provide new insight into the role of the temporal control of origin firing during S-phase for coordinating replication, gene expression, and chromatin establishment as occurs in the early embryo.

Novel Organic Salts Based on Mefloquine: Synthesis, Solubility, Permeability, and In Vitro Activity against Mycobacterium tuberculosis.

The development of novel pharmaceutical tools to efficiently tackle tuberculosis is the order of the day due to the rapid development of resistant strains of Mycobacterium tuberculosis. Herein, we report novel potential formulations of a repurposed drug, the antimalarial mefloquine (MFL), which was combined with organic anions as chemical adjuvants. Eight mefloquine organic salts were obtained by ion metathesis reaction between mefloquine hydrochloride ([MFLH][Cl]) and several organic acid sodium salts in high yields. One of the salts, mefloquine mesylate ([MFLH][MsO]), presented increased water solubility in comparison with [MFLH][Cl]. Moreover, all salts with the exception of mefloquine docusate ([MFLH][AOT]) showed improved permeability and diffusion through synthetic membranes. Finally, in vitro activity studies against Mycobacterium tuberculosis revealed that these ionic formulations exhibited up to 1.5-times lower MIC values when compared with [MFLH][Cl], particularly mefloquine camphorsulfonates ([MFLH][(1R)-CSA], [MFLH][(1S)-CSA]) and mefloquine HEPES ([MFLH][HEPES]).

Antifouling Marine Coatings with a Potentially Safer and Sustainable Synthetic Polyphenolic Derivative.

The development of harmless substances to replace biocide-based coatings used to prevent or manage marine biofouling and its unwanted consequences is urgent. The formation of biofilms on submerged marine surfaces is one of the first steps in the marine biofouling process, which facilitates the further settlement of macrofoulers. Anti-biofilm properties of a synthetic polyphenolic compound, with previously described anti-settlement activity against macrofoulers, were explored in this work. In solution this new compound was able to prevent biofilm formation and reduce a pre-formed biofilm produced by the marine bacterium, Pseudoalteromonas tunicata. Then, this compound was applied to a marine coating and the formation of P. tunicata biofilms was assessed under hydrodynamic conditions to mimic the marine environment. For this purpose, polyurethane (PU)-based coating formulations containing 1 and 2 wt.% of the compound were prepared based on a prior developed methodology. The most effective formulation in reducing the biofilm cell number, biovolume, and thickness was the PU-based coating containing an aziridine-based crosslinker and 2 wt.% of the compound. To assess the marine ecotoxicity impact of this compound, its potential to disrupt endocrine processes was evaluated through the modulation of two nuclear receptors (NRs), peroxisome proliferator-activated receptor γ (PPARγ), and pregnane X receptor (PXR). Transcriptional activation of the selected NRs upon exposure to the polyphenolic compound (10 µM) was not observed, thus highlighting the eco-friendliness towards the addressed NRs of this new dual-acting anti-macro- and anti-microfouling agent towards the addressed NRs.

Metformin disrupts Danio rerio metabolism at environmentally relevant concentrations: A full life-cycle study.

Metformin (MET), an anti-diabetic pharmaceutical of large-scale consumption, is increasingly detected in surface waters. However, current knowledge on the long-term effects of MET on non-target organisms is limited. The present study aimed to investigate the effects of MET in the model freshwater teleost Danio rerio, following a full life-cycle exposure to environmentally relevant concentrations (390 to 14 423 ng/L). Considering that the mode of action (MoA) of MET on non-target organisms remains underexplored and that MET may act through similar human pathways, i.e., lipid and energy metabolisms, biochemical markers were used to determine cholesterol and triglycerides levels, as well as mitochondrial complex I activity in zebrafish liver. Also, the hepatosomatic index as an indication of metabolic disruption, and the expression levels of genes involved in MET's putative MoA, i.e. acaca, acadm, cox5aa, idh3a, hmgcra, prkaa1, were determined, the last by qRT-PCR. A screening of mRNA transcripts, associated with lipid and energy metabolisms, and other signaling pathways potentially involved in MET-induced toxicity were also assessed using an exploratory RNA-seq analysis. The findings here reported indicate that MET significantly disrupted critical biochemical and molecular processes involved in zebrafish metabolism, such as cholesterol and fatty acid biosynthesis, mitochondrial electron transport chain and tricarboxylic acid cycle, concomitantly to changes on the hepatosomatic index. Likewise, MET impacted other relevant pathways mainly associated with cell cycle, DNA repair and steroid hormone biosynthesis, here reported for the first time in a non-target aquatic organism. Non-monotonic dose response curves were frequently detected in biochemical and qRT-PCR data, with higher effects observed at 390 and 2 929 ng/L MET treatments. Collectively, the results suggest that environmentally relevant concentrations of MET severely disrupt D. rerio metabolism and other important biological processes, supporting the need to revise the proposed environmental quality standard (EQS) and predicted no-effect concentration (PNEC) for MET.

Disruptions of circadian rhythms, sleep, and stress responses in zebrafish: New infrared-based activity monitoring assays for toxicity assessment.

Behavioural disruptions are sensitive indicators of alterations to normal animal physiology and can be used for toxicity assessment. The small vertebrate zebrafish is a leading model organism for toxicological studies. The ability to continuously monitor the toxicity of drugs, pollutants, or environmental changes over several days in zebrafish can have high practical application. Although video-recordings can be used to monitor short-term zebrafish behaviour, it is challenging to videorecord prolonged experiments (e.g. circadian behaviour over several days) because of the darkness periods (nights) and the heavy data storage and image processing requirements. Alternatively, infrared-based activity monitors, widely used in invertebrate models such as drosophila, generate simple and low-storage data and could optimize large-scale prolonged behavioural experiments in zebrafish, thus favouring the implementation of high-throughput testing strategies. Here, we validate the use of a Locomotor Activity Monitor (LAM) to study the behaviour of zebrafish larvae, and we characterize the behavioural phenotypes induced by abnormal light conditions and by the Parkinsonian toxin MPP+. When zebrafish were deprived from daily light-cycle synchronization, the LAM detected various circadian disruptions, such as increased activity period, phase shifts, and decreased inter-daily stability. Zebrafish exposed to MPP+ (10, 100, 500 µM) showed a concentration-dependent decrease in activity, sleep disruptions, impaired habituation to repetitive startles (visual-motor responses), and a slower recovery to normal activity after the startle-associated stress. These phenotypes evidence the feasibility of using infrared-based LAM to assess multi-parameter behavioural disruptions in zebrafish. The procedures in this study have wide applicability and may yield standard methods for toxicity testing.

Use of illicit drugs, alcohol and tobacco in Spain and Portugal during the COVID-19 crisis in 2020 as measured by wastewater-based epidemiology.

The COVID-19 pandemic spread rapidly worldwide in the year 2020, which was initially restrained by drastic mobility restrictions. In this work, we investigated the use of illicit drugs (amphetamine, methamphetamine, ecstasy, cocaine and cannabis), and licit substances of abuse (alcohol and tobacco) during the earlier months (March-July 2020) of the pandemic restrictions in four Spanish (Bilbao and its metropolitan area, Vitoria-Gasteiz, Castellón and Santiago de Compostela) and two Portuguese (Porto and Vila do Conde) locations by wastewater-based epidemiology (WBE). The results show that no methamphetamine was detected in any of the locations monitored, while amphetamine use was only detectable in the two locations from the Basque Country (Bilbao and its metropolitan area and Vitoria-Gasteiz), with high estimated average usage rates (700-930 mg day-1 1000 inhabitant-1). The remaining substances were detected in all the investigated catchment areas. In general, no remarkable changes were found in population normalized loads compared to former years, except for cocaine (i.e. its main metabolite, benzoylecgonine). For this drug, a notable decrease in use was discernible in Castellón, while its usage in Porto and Santiago de Compostela seemed to continue in a rising trend, already initiated in former years. Furthermore, two events of ecstasy (3,4-methylenedioxymethamphetamine, MDMA) dumping in the sewage network were confirmed by enantiomeric analysis, one in Santiago de Compostela just prior the lockdown and the second one in the Bilbao and its metropolitan area in July after relieving the more stringent measures. The latter could also be associated with a police intervention. The comparison of WBE with (web) survey data, which do not provide information at a local level, points towards contradictory conclusions for some of the substances, thereby highlighting the need for stable WBE networks capable of near real-time monitoring drug use.

Enhanced In Vitro Antiviral Activity of Hydroxychloroquine Ionic Liquids against SARS-CoV-2.

The development of effective antiviral drugs against SARS-CoV-2 is urgently needed and a global health priority. In light of the initial data regarding the repurposing of hydroxychloroquine (HCQ) to tackle this coronavirus, herein we present a quantitative synthesis and spectroscopic and thermal characterization of seven HCQ room temperature ionic liquids (HCQ-ILs) obtained by direct protonation of the base with two equivalents of organic sulfonic, sulfuric and carboxylic acids of different polarities. Two non-toxic and hydrophilic HCQ-ILs, in particular, [HCQH2][C1SO3]2 and [HCQH2][GlcCOO]2, decreased the virus-induced cytopathic effect by two-fold in comparison with the original drug, [HCQH2][SO4]. Despite there being no significant differences in viral RNA production between the three compounds, progeny virus production was significantly affected (p < 0.05) by [HCQH2][GlcCOO]2. Overall, the data suggest that the in vitro antiviral activities of the HCQ-ILs are most likely the result of specific intra- and intermolecular interactions and not so much related with their hydrophilic or lipophilic character. This work paves the way for the development of future novel ionic formulations of hydroxychloroquine with enhanced physicochemical properties.