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

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

Assessing wastewater-based epidemiology for the prediction of SARS-CoV-2 incidence in Catalonia.

While wastewater-based epidemiology has proven a useful tool for epidemiological surveillance during the COVID-19 pandemic, few quantitative models comparing virus concentrations in wastewater samples and cumulative incidence have been established. In this work, a simple mathematical model relating virus concentration and cumulative incidence for full contagion waves was developed. The model was then used for short-term forecasting and compared to a local linear model. Both scenarios were tested using a dataset composed of samples from 32 wastewater treatment plants and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) incidence data covering the corresponding geographical areas during a 7-month period, including two contagion waves. A population-averaged dataset was also developed to model and predict the incidence over the full geography. Overall, the mathematical model based on wastewater data showed a good correlation with cumulative cases and allowed us to anticipate SARS-CoV-2 incidence in one week, which is of special relevance in situations where the epidemiological monitoring system cannot be fully implemented.

Elimination of SARS-CoV-2 along wastewater and sludge treatment processes.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is shed in the feces of infected people. As a consequence, genomic RNA of the virus can be detected in wastewater. Although the presence of viral RNA does not inform on the infectivity of the virus, this presence of genetic material raised the question of the effectiveness of treatment processes in reducing the virus in wastewater and sludge. In this work, treatment lines of 16 wastewater treatment plants were monitored to evaluate the removal of SARS-CoV-2 RNA in raw, processed waters and sludge, from March to May 2020. Viral RNA copies were enumerated using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) in 5 different laboratories. These laboratories participated in proficiency testing scheme and their results demonstrated the reliability and comparability of the results obtained for each one. SARS-CoV-2 RNA was found in 50.5% of the 101 influent wastewater samples characterized. Positive results were detected more frequently in those regions with a COVID-19 incidence higher than 100 cases per 100,000 inhabitants. Wastewater treatment plants (WWTPs) significantly reduced the occurrence of virus RNA along the water treatment lines. Secondary treatment effluents showed an occurrence of SARS-CoV-2 RNA in 23.3% of the samples and no positive results were found after MBR and chlorination. Non-treated sludge (from primary and secondary treatments) presented a higher occurrence of SARS-CoV-2 RNA than the corresponding water samples, demonstrating the affinity of virus particles for solids. Furthermore, SARS-CoV-2 RNA was detected in treated sludge after thickening and anaerobic digestion, whereas viral RNA was completely eliminated from sludge only when thermal hydrolysis was applied. Finally, co-analysis of SARS-CoV-2 and F-specific RNA bacteriophages was done in the same water and sludge samples in order to investigate the potential use of these bacteriophages as indicators of SARS-CoV-2 fate and reduction along the wastewater treatment.

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.

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

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

Aquatic macroinvertebrates under stress: Bioaccumulation of emerging contaminants and metabolomics implications.

The current knowledge on bioaccumulation of emerging contaminants (ECs) in aquatic invertebrates exposed to the realistic environmental concentrations is limited. Even less is known about the effects of chemical pollution exposure on the metabolome of aquatic invertebrates. We conducted an in situ translocation experiment with passive filter-feeding caddisfly larvae (Hydropsyche sp.) in an effluent-influenced river in order to i) unravel the bioaccumulation (and recovery) dynamics of ECs in aquatic invertebrates, and ii) test whether exposure to environmentally realistic concentrations of ECs will translate into metabolic profile changes in the insects. The experiment was carried out at two sites, upstream and downstream of the discharge of an urban wastewater treatment plant effluent. The translocated animals were collected at 2-week intervals for 46 days. Both pharmaceuticals and endocrine disrupting compounds (EDCs) were detected in water (62 and 7 compounds, respectively), whereas in Hydropsyche tissues 5 EDCs accumulated. Overall, specimens from the upstream site translocated to the impacted site reached higher ECs concentrations in their tissues, as a reflection of the contaminants' water concentrations. However, bioaccumulation was a temporary process susceptible to change under lower contaminant concentrations. Non-targeted metabolite profiling detected fine metabolic changes in translocated Hydropsyche larvae. Both translocations equally induced stress, but it was higher in animals translocated to the impacted site.

Comprehensive study of sulfamethoxazole effects in marine mussels: Bioconcentration, enzymatic activities and metabolomics.

Antibiotics accumulation in aquatic organisms may be of great concern from an ecological point of view but also from a human perspective, especially when they are accumulated in edible animals like marine mussels. In this work, mussels (Mytilus galloprovincialis) were exposed to sulfamethoxazole antibiotic (SMX) at 10 µg/L during 96 h, followed by 24 h of depuration. The experiment was carried out at summer and winter conditions. SMX showed a bioconcentration factor in mussel of 1.5 L/kg (dry weight) and 69% of the compound was eliminated from the organism in 24 h. The metabolomics approach revealed alterations in amino acids levels (aspartate, phenylalanine, valine and tryptophan) pinpointing disturbances in osmotic regulation and energy metabolism. Besides, the levels of some nucleotides (guanosine and inosine) and a carboxylic acid were also affected. However, SMX exposed mussels did not show any significant alteration in the enzymatic activities related to the xenobiotic metabolism and oxidative stress. Moreover, some of the changes observed in mussel's metabolites suggested alterations in mussel's organoleptic characteristics that can affect its quality as seafood commodity. Overall, our results showed that SMX exposure to marine mussels may have ecological implications by provoking sub-lethal effects to exposed organisms. Nevertheless, no risk for consumers derived from mussel ingestion is expected due to the low bioconcentration capacity of SMX and fast depuration in this seafood type.

Pharmaceuticals and endocrine disruptors in raw and cooked seafood from European market: Concentrations and human exposure levels.

Pharmaceuticals (PhACs) and endocrine disrupting compounds (EDCs) are chemicals of emerging concern that can accumulate in seafood sold in markets. These compounds may represent a risk to consumers through effects on the human reproductive system, metabolic disorders, pathogenesis of breast cancer or development of microbial resistance. Measuring their levels in highly consumed seafood is important to assess the potential risks to human health. Besides, the effect of cooking on contaminant levels is relevant to investigate. Therefore, the objectives of this research were to study the presence and levels of PhACs and EDCs in commercially available seafood in the European Union market, to investigate the effect of cooking on contaminant levels, and to evaluate the dietary exposure of humans to these compounds through seafood consumption. A sampling survey of seafood from 11 European countries was undertaken. Twelve highly consumed seafood types were analysed raw and cooked with 3 analytical methods (65 samples, 195 analysis). PhACs were mostly not detectable or below quantification limits in seafood whereas EDCs were a recurrent group of contaminants quantified in the majority of the samples. Besides, cooking by steaming significantly increased their levels in seafood from 2 to 46-fold increase. Based on occurrence and levels, bisphenol A, methylparaben and triclosan were selected for performing a human exposure assessment and health risk characterisation through seafood consumption. The results indicate that the Spanish population has the highest exposure to the selected EDCs through seafood consumption, although the exposure via seafood remained below the current toxicological reference values.

Antidepressants in a changing ocean: Venlafaxine uptake and elimination in juvenile fish (Argyrosomus regius) exposed to warming and acidification conditions.

The presence of antidepressants, such as venlafaxine (VFX), in marine ecosystems is increasing, thus, potentially posing ecological and human health risks. The inherent mechanisms of VFX uptake and elimination still require further understanding, particularly accounting for the impact of climate change-related stressors, such as warming and acidification. Hence, the present work aimed to investigate, for the first time, the effects of increased seawater temperature (ΔT°C = +5 °C) and pCO2 levels (ΔpCO2 ∼1000 µatm, equivalent to ΔpH = -0.4 units) on the uptake and elimination of VFX in biological tissues (muscle, liver, brain) and plasma of juvenile meagre (Argyrosomus regius) exposed to VFX through two different exposure pathways (via water, i.e. [VFX ] ∼20 µg L-1, and via feed, i.e. [VFX] ∼160 µg kg-1 dry weight, dw). Overall, results showed that VFX can be uptaken by fish through both water and diet. Fish liver exhibited the highest VFX concentration (126.7 ±â€¯86.5 µg kg-1 and 6786.4 ±â€¯1176.7 µg kg-1 via feed and water exposures, respectively), as well as the highest tissue:plasma concentration ratio, followed in this order by brain and muscle, regardless of exposure route. Both warming and acidification decreased VFX uptake in liver, although VFX uptake in brain was favoured under warming conditions. Conversely, VFX elimination in liver was impaired by both stressors, particularly when acting simultaneously. The distinct patterns of VFX uptake and elimination observed in the different scenarios calls for a better understanding of the effects of exposure route and abiotic conditions on emerging contaminants' toxicokinetics.

Effects of water warming and acidification on bioconcentration, metabolization and depuration of pharmaceuticals and endocrine disrupting compounds in marine mussels (Mytilus galloprovincialis).

Warming and acidification are expected impacts of climate change to the marine environment. Besides, organisms that live in coastal areas, such as bivalves, can also be exposed to anthropogenic pollutants like pharmaceuticals (PhACs) and endocrine disrupting compounds (EDCs). In this study, the effects of warming and acidification on the bioconcentration, metabolization and depuration of five PhACs (sotalol, sulfamethoxazole, venlafaxine, carbamazepine and citalopram) and two EDCs (methylparaben and triclosan) were investigated in the mussel species (Mytilus galloprovincialis), under controlled conditions. Mussels were exposed to warming and acidification, as well as to the mixture of contaminants up to 15.7 µg L-1 during 20 days; followed by 20 days of depuration. All contaminants bioconcentrated in mussels with levels ranging from 1.8 µg kg-1 dry weight (dw) for methylparaben to 12889.4 µg kg-1 dw for citalopram. Warming increased the bioconcentration factor (BCF) of sulfamethoxazole and sotalol, whereas acidification increased the BCF of sulfamethoxazole, sotalol and methylparaben. In contrast, acidification decreased triclosan levels, while both stressors decreased venlafaxine and citalopram BCFs. Warming and acidification facilitated the elimination of some of the tested compounds (i.e. sotalol from 50% in control to 60% and 68% of elimination in acidification and warming respectively). However, acidification decreased mussels' capacity to metabolize contaminants (i.e. venlafaxine). This work provides a first insight in the understanding of aquatic organisms' response to emerging contaminants pollution under warming and acidification scenarios.

Fluvial biofilms exposed to desiccation and pharmaceutical pollution: New insights using metabolomics.

In many arid and semi-arid systems, biological communities in river ecosystems are submitted to flow interruption and desiccation, as well as to the impact of urban wastewaters. In this work, we studied (using a LC-LTQ-Orbitrap) the metabolomic response of biofilm communities exposed to both hydrological and chemical stressors. Fluvial biofilms were exposed to a mixture of 9 pharmaceuticals at a total concentration of 5000ng/L (mimicking concentrations and compounds found in polluted aquatic environments) and/or to seven days of desiccation, under laboratory conditions. The biosynthesis of fatty acids was the main metabolic pathway disrupted in biofilms. Endogenous biofilm's metabolites (metabolome) altered due to these stressors were identified. The metabolites that significantly changed only due to one of the stressors could be proposed as potential specific biomarkers. A biomarker of pharmaceutical exposure was the lysophosphatidic acid, which decreased a 160%, while for desiccation stearidonic acid (increased 160%), 16-Oxohexadecanoic acid (increased 340%) and palmitoleic acid (decreased 290%) were the biomarkers proposed. Besides, other metabolites showed different responses depending on the treatment, such as palmitic acid, linolenic acid, behenic acid, lignoceric acid and azelaic acid. The Carbon:Phosphorus (C:P) molar ratio increased due to all stress factors, whereas the algal community composition changed mainly due to desiccation. A possible relationship between those changes observed in structural parameters and the metabolome of biofilms was explored. Overall, our findings support the use of metabolomics to unravel at molecular level the effects from chemical and physical stressors on complex microbial communities, such as biofilms, and pinpoint biomarkers of exposure.

Multi-residue method for the determination of antibiotics and some of their metabolites in seafood.

The presence of antibiotics in seafood for human consumption may pose a risk for consumers. A methodology for the analysis of antibiotics in seafood based on QuEChERS (quick, easy, cheap, effective, rugged, and safe) extraction, followed by detection and quantification using liquid chromatography coupled to mass spectrometry was developed. The analytical method was evaluated for the determination of 23 antibiotics (including parent compounds and some metabolites) in fish, mussels and clams. Recoveries ranged between 30% and 70% for most of the compounds and method detection and quantification limits (MDLs and MQLs) were between 0.01 and 0.31 ng/g dry weigh (dw) and 0.02-1.03 ng/g (dw) respectively. Real seafood samples were analysed using this method. Nine antibiotics were found at levels above MDLs; however none of them exceed the maximum residue limits (MRL) established by the authorities. Tetracycline was the most ubiquitous compound, presenting also the highest concentration: 5.63 ng/g (dw) in fish from Netherlands. In addition, an alternative technique based on microbial growth inhibition was explored as semiquantitative detection method of antibiotics in seafood. This methodology could be applied as a fast screening technique for the detection of macrolides and ß-lactams in seafood but further research is needed for other antibiotics families.

Effects of flow intermittency and pharmaceutical exposure on the structure and metabolism of stream biofilms.

Increasing concentrations of pharmaceutical compounds occur in many rivers, but their environmental risk remains poorly studied in stream biofilms. Flow intermittency shapes the structure and functions of ecosystems, and may enhance their sensitivity to toxicants. This study evaluates the effects of a long-term exposure of biofilm communities to a mixture of pharmaceutical compounds at environmental concentrations on biofilm bioaccumulation capacity, the structure and metabolic processes of algae and bacteria communities, and how their potential effects were enhanced or not by the occurrence of flow intermittency. To assess the interaction between those two stressors, an experiment with artificial streams was performed. Stream biofilms were exposed to a mixture of pharmaceuticals, as well as to a short period of flow intermittency. Results indicate that biofilms were negatively affected by pharmaceuticals. The algal biomass and taxa richness decreased and unicellular green algae relatively increased. The structure of the bacterial (based on denaturing gradient gel electrophoresis of amplified 16S rRNA genes) changed and showed a reduction of the operational taxonomic units (OTUs) richness. Exposed biofilms showed higher rates of metabolic processes, such as primary production and community respiration, attributed to pharmaceuticals stimulated an increase of green algae and heterotrophs, respectively. Flow intermittency modulated the effects of chemicals on natural communities. The algal community became more sensitive to short-term exposure of pharmaceuticals (lower EC50 value) when exposed to water intermittency, indicating cumulative effects between the two assessed stressors. In contrast to algae, the bacterial community became less sensitive to short-term exposure of pharmaceuticals (higher EC50) when exposed to water intermittency, indicating co-tolerance phenomena. According to the observed effects, the environmental risk of pharmaceuticals in nature is high, but different depending on the flow regime, as well as the target organisms (autotrophs vs heterotrophs).