Occurrence of emerging contaminants in three river basins impacted by wastewater treatment plant effluents: Spatio-seasonal patterns and environmental risk assessment.

The concern on the fate and distribution of contaminants of emerging concern (CECs) is a burning topic due to their widespread occurrence and potential harmful effects. Particularly, antibiotics have received great attention due to their implications in antimicrobial resistance occurrence. The impact of wastewater treatment plants (WWTP) is remarkable, being one of the main pathways for the introduction of CECs into aquatic systems. The combination of novel analytical methodologies and risk assessment strategies is a promising tool to find out environmentally relevant compounds posing major concerns in freshwater ecosystems impacted by those wastewater effluents. Within this context, a multi-target approach was applied in three Spanish river basins affected by different WWTP treated effluents for spatio-temporal monitoring of their chemical status. Solid phase extraction followed by ultra-high-performance liquid chromatography were used for the quantification of a large panel of compounds (n = 270), including pharmaceuticals and other consumer products, pesticides and industrial chemicals. To this end, water samples were collected in four sampling campaigns at three locations in each basin: (i) upstream from the WWTPs; (ii) WWTP effluent discharge points (effluent outfall); and (iii) downstream from the WWTPs (500 m downriver from the effluent outfall). Likewise, 24-h composite effluent samples from each of the WWTPs were provided in all sampling periods. First the occurrence and distribution of these compounds were assessed. Diverse seasonal trends were observed depending on the group of emerging compounds, though COVID-19 outbreak affected variations of certain pharmaceuticals. Detection frequencies and concentrations in effluents generally exceeded those in river samples and concentrations measured upstream WWTPs were generally low or non-quantifiable. Finally, risks associated with maximum contamination levels were evaluated using two different approaches to account for antibiotic resistance selection as well. From all studied compounds, 89 evidenced environmental risk on at least one occasion in this study.

Sample preparation for suspect screening of persistent, mobile and toxic substances and their phase II metabolites in human urine by mixed-mode liquid chromatography.

Persistent, mobile and toxic substances have drawn attention nowadays due to their particular properties, but they are overlooked in human monitorization works, limiting the knowledge of the human exposome. In that sense, human urine is an interesting matrix since not only parent compounds are eliminated, but also their phase II metabolites that could act as biomarkers. In this work, 11 sample preparation procedures involving preconcentration were tested to ensure maximum analytical coverage in human urine using mixed-mode liquid chromatography coupled with high-resolution tandem mass spectrometry. The optimized procedure consisted of a combination of solid-phase extraction and salt-assisted liquid-liquid extraction and it was employed for suspect screening. Additionally, a non-discriminatory dilute-and-shoot approach was also evaluated. After evaluating the workflow in terms of limits of identification and type II errors (i.e., false negatives), a pooled urine sample was analysed. From a list of 1450 suspects and in-silico simulated 1568 phase II metabolites (i.e. sulphates, glucuronides, and glycines), 44 and 14 substances were annotated, respectively. Most of the screened suspects were diverse industrial chemicals, but biocides, natural products and pharmaceuticals were also detected. Lastly, the complementarity of the sample preparation procedures, columns, and analysis conditions was assessed. As a result, dilute-and-shoot and the Acclaim Trinity P1 column at pH = 3 (positive ionization) and pH = 7 (negative ionization) allowed the maximum coverage since almost 70 % of the total suspects could be screened using those conditions.

Development and evaluation of a comprehensive workflow for suspect screening of exposome-related xenobiotics and phase II metabolites in diverse human biofluids.

Suspect and non-target screening (SNTS) methods are being promoted in order to decode the human exposome since a wide chemical space can be analysed in a diversity of human biofluids. However, SNTS approaches in the exposomics field are infra-studied in comparison to environmental or food monitoring studies. In this work, a comprehensive suspect screening workflow was developed to annotate exposome-related xenobiotics and phase II metabolites in diverse human biofluids. Precisely, human urine, breast milk, saliva and ovarian follicular fluid were employed as samples and analysed by means of ultra-high performance liquid chromatography coupled with high resolution tandem mass spectrometry (UHPLC-HRMS/MS). To automate the workflow, the "peak rating" parameter implemented in Compound Discoverer 3.3.2 was optimized to avoid time-consuming manual revision of chromatographic peaks. In addition, the presence of endogenous molecules that might interfere with the annotation of xenobiotics was carefully studied as the employment of inclusion and exclusion suspect lists. To evaluate the workflow, limits of identification (LOIs) and type I and II errors (i.e., false positives and negatives, respectively) were calculated in both standard solutions and spiked biofluids using 161 xenobiotics and 22 metabolites. For 80.3 % of the suspects, LOIs below 15 ng/mL were achieved. In terms of type I errors, only two cases were identified in standards and spiked samples. Regarding type II errors, the 7.7 % errors accounted in standards increased to 17.4 % in real samples. Lastly, the use of an inclusion list for endogens was favoured since it avoided 18.7 % of potential type I errors, while the exclusion list caused 7.2 % of type II errors despite making the annotation workflow less time-consuming.

What is in the fish? Collaborative trial in suspect and non-target screening of organic micropollutants using LC- and GC-HRMS.

A collaborative trial involving 16 participants from nine European countries was conducted within the NORMAN network in efforts to harmonise suspect and non-target screening of environmental contaminants in whole fish samples of bream (Abramis brama). Participants were provided with freeze-dried, homogenised fish samples from a contaminated and a reference site, extracts (spiked and non-spiked) and reference sample preparation protocols for liquid chromatography (LC) and gas chromatography (GC) coupled to high resolution mass spectrometry (HRMS). Participants extracted fish samples using their in-house sample preparation method and/or the protocol provided. Participants correctly identified 9-69 % of spiked compounds using LC-HRMS and 20-60 % of spiked compounds using GC-HRMS. From the contaminated site, suspect screening with participants' own suspect lists led to putative identification of on average ∼145 and ∼20 unique features per participant using LC-HRMS and GC-HRMS, respectively, while non-target screening identified on average ∼42 and ∼56 unique features per participant using LC-HRMS and GC-HRMS, respectively. Within the same sub-group of sample preparation method, only a few features were identified by at least two participants in suspect screening (16 features using LC-HRMS, 0 features using GC-HRMS) and non-target screening (0 features using LC-HRMS, 2 features using GC-HRMS). The compounds identified had log octanol/water partition coefficient (KOW) values from -9.9 to 16 and mass-to-charge ratios (m/z) of 68 to 761 (LC-HRMS and GC-HRMS). A significant linear trend was found between log KOW and m/z for the GC-HRMS data. Overall, these findings indicate that differences in screening results are mainly due to the data analysis workflows used by different participants. Further work is needed to harmonise the results obtained when applying suspect and non-target screening approaches to environmental biota samples.

The role of sample preparation in suspect and non-target screening for exposome analysis using human urine.

The use of suspect and non-target screening (SNTS) for the characterization of the chemical exposome employing human biofluids is gaining attention. Among the biofluids, urine is one of the preferred matrices since organic xenobiotics are excreted through it after metabolization. However, achieving a consensus between selectivity (i.e. preserving as many compounds as possible) and sensitivity (i.e. minimizing matrix effects by removing interferences) at the sample preparation step is challenging. Within this context, several sample preparation approaches, including solid-phase extraction (SPE), liquid-liquid extraction (LLE), salt-assisted LLE (SALLE) and dilute-and-shoot (DS) were tested to screen not only exogenous compounds in human urine but also their phase II metabolites using liquid-chromatography coupled to high-resolution tandem mass spectrometry (LC-HRMS/MS). Additionally, enzymatic hydrolysis of phase II metabolites was evaluated. Under optimal conditions, SPE resulted in the best sample preparation approach in terms of the number of detected xenobiotics and metabolites since 97.1% of the total annotated suspects were present in samples extracted by SPE. In LLE and SALLE, pure ethyl acetate turned out to be the best extractant but fewer suspects than with SPE (80.7%) were screened. Lastly, only 52.5% of the suspects were annotated in the DS approach, showing that it could only be used to detect compounds at high concentration levels. Using pure standards, the presence of diverse xenobiotics such as parabens, industrial chemicals (benzophenone-3, caprolactam and mono-2-ethyl-5-hydroxyhexyl phthalate) and chemicals related to daily habits (caffeine, cotinine or triclosan) was confirmed. Regarding enzymatic hydrolysis, only 10 parent compounds of the 44 glucuronides were successfully annotated in the hydrolysed samples. Therefore, the screening of metabolites in non-hydrolysed samples through SNTS is the most suitable approach for exposome characterization.

Development of an analytical method for the simultaneous determination of 50 semi-volatile organic contaminants in wastewaters.

This work describes the development of a robust analytical methodology for the simultaneous determination of 50 semi-volatile organic compounds (SVOCs) in wastewater effluent samples by solid-phase extraction (SPE) followed by gas chromatography coupled to mass spectrometry (GC-MS) analysis. In this work, we studied extensively whether the validated SPE method used for the analysis of polar compounds in wastewaters could be extended to the analysis of non-polar compounds in the same analytical run. To that aim, the effect of different organic solvents in the SPE process (i.e., sample conditioning prior to SPE, elution solvent and evaporation steps) was evaluated. In this sense, the addition of methanol to wastewater samples before the extraction, the use of hexane:toluene (4:1, v/v) mixture for the quantitative elution of target compounds, and the addition of isooctane during the evaporation were required to minimize analyte losses during SPE and enhance extraction yields. Overall, the developed methodology showed a good performance for the determination of 50 SVOCs, and was further applied to the analysis of real wastewater effluent samples.•A validated SPE method for polar compounds was extended to the analysis of non-polar compounds.•Elution with hex:tol (4:1, v/v) and the addition of isooctane during the evaporation yield good recoveries.•The developed methodology was suitable for the determination of 50 SVOCs in aqueous samples.

Dilute-and-shoot coupled to mixed mode liquid chromatography-tandem mass spectrometry for the analysis of persistent and mobile organic compounds in human urine.

In this work, a comprehensive method for the simultaneous determination of 33 diverse persistent and mobile organic compounds (PMOCs) in human urine was developed by dilute-and-shoot (DS) followed by mixed-mode liquid chromatography coupled with tandem mass spectrometry (MMLC-MS/MS). In the sample preparation step, DS was chosen since it allowed the quantification of all targets in comparison to lyophilization. For the chromatographic separation, Acclaim Trinity P1 and P2 trimodal columns provided greater capacity for retaining PMOCs than reverse phase and hydrophilic interaction liquid chromatography. Therefore, DS was validated at 5 and 50 ng/mL in urine with both mixed mode columns at pH = 3 and 7. Regarding figures of merit, linear calibration curves (r2 > 0.999) built between instrumental quantification limits (mostly below 5 ng/mL) and 500 ng/mL were achieved. Despite only 60% of the targets were recovered at 5 ng/mL because of the dilution, all PMOCs were quantified at 50 ng/mL. Using surrogate correction, apparent recoveries in the 70-130% range were obtained for 91% of the targets. To analyse human urine samples, the Acclaim Trinity P1 column at pH = 3 and 7 was selected as a consensus between analytical coverage (i.e. 94% of the targets) and chromatographic runs. In a pooled urine sample, industrial chemicals (acrylamide and bisphenol S), biocides and their metabolites (2-methyl-4-isothiazolin-3-one, dimethyl phosphate, 6-chloropyridine-3-carboxylic acid, and ammonium glufosinate) and an artificial sweetener (aspartame) were determined at ng/mL levels. The outcomes of this work showed that humans are also exposed to PMOCs due to their persistence and mobility, and therefore, further human risk assessment is needed.

Suspect Screening of Chemicals in Hospital Wastewaters Using Effect-Directed Analysis Approach as Prioritization Strategy.

The increasing number of contaminants in the environment has pushed water monitoring programs to find out the most hazardous known and unknown chemicals in the environment. Sample treatment-simplification methods and non-target screening approaches can help researchers to not overlook potential chemicals present in complex aqueous samples. In this work, an effect-directed analysis (EDA) protocol using the sea urchin embryo test (SET) as a toxicological in vivo bioassay was used as simplified strategy to identify potential unknown chemicals present in a very complex aqueous matrix such as hospital effluent. The SET bioassay was used for the first time here to evaluate potential toxic fractions in hospital effluent, which were obtained after a two-step fractionation using C18 and aminopropyl chromatographic semi-preparative columns. The unknown compounds present in the toxic fractions were identified by means of liquid chromatography coupled to a Q Exactive Orbitrap high-resolution mass spectrometer (LC-HRMS) and using a suspect analysis approach. The results were complemented by gas chromatography-mass spectrometry analysis (GC-MS) in order to identify the widest range of chemical compounds present in the sample and the toxic fractions. Using EDA as sample treatment simplification method, the number of unknown chemicals (>446 features) detected in the raw sample was narrowed down to 94 potential toxic candidates identified in the significantly toxic fractions. Among them, the presence of 25 compounds was confirmed with available chemical standards including 14 pharmaceuticals, a personal care product, six pesticides and four industrial products. The observations found in this work emphasize the difficulties in identifying potential toxicity drivers in complex water samples, as in the case of hospital wastewater.

Effect-directed analysis of a hospital effluent sample using A-YES for the identification of endocrine disrupting compounds.

An effect-directed analysis (EDA) approach was used to identify the compounds responsible for endocrine disruption in a hospital effluent (Basque Country). In order to facilitate the identification of the potentially toxic substances, a sample was collected using an automated onsite large volume solid phase extraction (LV-SPE) system. Then, it was fractionated with a two-step orthogonal chromatographic separation and tested for estrogenic effects with a recombinant yeast (A-YES) in-vitro bioassay. The fractionation method was optimized and validated for 184 compounds, and its application to the hospital effluent sample allowed reducing the number of unknowns from 292 in the raw sample to 35 after suspect analysis of the bioactive fractions. Among those, 7 of them were confirmed with chemical standards. In addition, target analysis of the raw sample confirmed the presence of mestranol, estrone and dodemorph in the fractions showing estrogenic activity. Predictive estrogenic activity modelling using quantitative structure-activity relationships indicated that the hormones mestranol (5840 ng/L) and estrone (128 ng/L), the plasticiser bisphenol A (9219 ng/L) and the preservative butylparaben (1224 ng/L) were the main contributors of the potential toxicity. Derived bioanalytical equivalents (BEQs) pointed mestranol and estrone as the main contributors (56 % and 43 %, respectively) of the 50 % of the sample's explained total estrogenic activity.

From target analysis to suspect and non-target screening of endocrine-disrupting compounds in human urine.

In the present work, a target analysis method for simultaneously determining 24 diverse endocrine-disrupting compounds (EDCs) in urine (benzophenones, bisphenols, parabens, phthalates and antibacterials) was developed. The target analysis approach (including enzymatic hydrolysis, clean-up by solid-phase extraction and analysis by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)) was optimized, validated and applied to volunteers' samples, in which 67% of the target EDCs were quantified. For instance, benzophenone-3 (0.2-13 ng g-1), bisphenol A (7.7-13.7 ng g-1), methyl 3,5-dihydroxybenzoate (8-254 ng g-1), mono butyl phthalate (2-17 ng g-1) and triclosan (0.3-9 ng g-1) were found at the highest concentrations, but the presence of other analogues was detected as well. The developed target method was further extended to suspect and non-target screening (SNTS) by means of LC coupled to high-resolution MS/MS. First, well-defined workflows for SNTS were validated by applying the previously developed method to an extended list of compounds (83), and then, to the same real urine samples. From a list of approximately 4000 suspects, 33 were annotated at levels from 1 to 3, with food additives/ingredients and personal care products being the most abundant ones. In the non-target approach, the search was limited to molecules containing S, Cl and/or Br atoms, annotating 4 pharmaceuticals. The results from this study showed that the combination of the lower limits of detection of MS/MS and the identification power of high-resolution MS/MS is still compulsory for a more accurate definition of human exposome in urine samples.

Assessing alcohol consumption through wastewater-based epidemiology: Spain as a case study.

BACKGROUND: In this study, an alternative and complementary method to those approaches currently used to estimate alcohol consumption by the population is described. This method, known as wastewater-based epidemiology (WBE), allows back-calculating the alcohol consumption rate in a given population from the concentrations of a selected biomarker measured in wastewater. METHODS: Composite (24-h) wastewater samples were collected at the inlet of 17 wastewater treatment plants located in 13 Spanish cities for seven consecutive days in 2018. The sampled area covered 12.8% of the Spanish population. Wastewater samples were analyzed to determine the concentration of ethyl sulfate, the biomarker used to back-calculate alcohol consumption. RESULTS: Alcohol consumption ranged from 4.5 to 46 mL/day/inhabitant. Differences in consumption were statistically significant among the investigated cities and between weekdays and weekends. WBE-derived estimates of alcohol consumption were comparable to those reported by its corresponding region in the Spanish National Health Survey in most cases. At the national level, comparable results were obtained between the WBE-derived annual consumption rate (5.7 ± 1.2 L ethanol per capita (aged 15+)) and that reported by the National Health Survey (4.7 L ethanol per capita (aged 15+)). CONCLUSIONS: This is the largest WBE study carried out to date in Spain to estimate alcohol consumption rates. It confirms that this approach is useful for establishing spatial and temporal patterns of alcohol consumption, which could contribute to the development of health care management plans and policies. Contrary to established methods, it allows obtaining information in a fast and relatively economical way.

Application of the Sea Urchin Embryo Test in Toxicity Evaluation and Effect-Directed Analysis of Wastewater Treatment Plant Effluents.

Sea urchin embryo assay was used to assess general toxicity at four wastewater treatment plant effluents of Biscay (Gorliz, Mungia, Gernika, and Galindo), and within the tested range, all the extracts showed embryo growth inhibition and skeleton malformation activities with EC50 values, in relative enrichment factor units, between 1.1-16.8 and 1.1-8.8, respectively. To identify the causative compounds, effect-directed analysis was successfully applied for the first time using a sea urchin embryo test to the secondary treatment of the Galindo effluent. To this end, two subsequent fractionation steps were performed using C18 (21 fractions) and aminopropyl columns (15 fractions). By this fractionation, the number of features detected by LC-HRMS in the raw sample was drastically reduced from 1500 to 9, and among them, two pesticides (mexacarbate, 17 ng/L, and fenpropidin, 23 ng/L), two antidepressants (amitriptyline, 304 ng/L, and paroxetine, 26 ng/L), and two anthelmintic agents (mebendazole, 65 ng/L, and albendazole, 48 ng/L) could be identified in the two toxic fractions. The artificial mixture of the identified six compounds could explain 79% of the observed effect, with albendazole and paroxetine as the predominant contributors (49% and 49%, respectively) affecting the sea urchin embryogenesis activity.

Occurrence of per- and polyfluorinated compounds in paper and board packaging materials and migration to food simulants and foodstuffs.

This work describes the screening of twenty three per- and polyfluoroalkyl substances (PFASs) in twenty five paper and board (P/B) packaging materials and their migration to several food simulants (50% ethanol, 95% ethanol and Tenax®) at different conditions of time and temperature. A different migration pattern depending on the carbon chain length was observed; while short carbon chain PFASs tend to migrate more to 50% ethanol than to 95% ethanol, long chain PFASs showed the opposite trend. On the other hand, very low migration percentages of all PFASs to Tenax® were found. Finally, migration of 9 PFASs into real foods (cereals, rice and infant milk powder) for 6 months was quantified and compared with the results obtained with the simulants. As a result, significant underestimations of the PFASs migration to foodstuffs were obtained using Tenax®, especially for short carbon chain PFASs and milk powder.

Short-term stability assessment for the analysis of emerging contaminants in seawater.

This paper describes the stability study performed in seawater and seawater extracts (spiked at ~ 200 ng/L) for 23 emerging contaminants. Four different alternatives were tested at six different times (0, 3, 10, 17, 24 and 31 days): (i) seawater at 4 °C, (ii) mixed-mode solid-phase extraction cartridge (Bond Elute Plexa and Strata X-AW) stored at - 20 °C, (iii) polyethersulfone hollow fibre stored at - 20 °C and (iv) methanol extracts once the samples were extracted from PES hollow fibre and stored at - 20 °C. Moreover, the integrity of the supporting polymeric phases was studied by Raman, optical microscopy, differential scanning calorimetric and thermogravimetric analysis. As may be expected, seawater samples showed the lowest stability (losses between 21 and 99%) while methanol extract provides stable results (losses < 30%) over the tested period. In the case of solid-phase cartridges, the stability profile showed an average loss of 7% while, in polyethersulfone hollow fibres, losses up to 58% were observed. Finally, we were able to relate the lower efficiency of polyethersulfone fibres with the wettability of this material based on the thermogravimetric analysis.

Amitriptyline at an Environmentally Relevant Concentration Alters the Profile of Metabolites Beyond Monoamines in Gilt-Head Bream.

The antidepressant amitriptyline is a widely used selective serotonin reuptake inhibitor that is found in the aquatic environment. The present study investigates alterations in the brain and the liver metabolome of gilt-head bream (Sparus aurata) after exposure at an environmentally relevant concentration (0.2 µg/L) of amitriptyline for 7 d. Analysis of variance-simultaneous component analysis is used to identify metabolites that distinguish exposed from control animals. Overall, alterations in lipid metabolism suggest the occurrence of oxidative stress in both the brain and the liver-a common adverse effect of xenobiotics. However, alterations in the amino acid arginine are also observed. These are likely related to the nitric oxide system that is known to be associated with the mechanism of action of antidepressants. In addition, changes in asparagine and methionine levels in the brain and pantothenate, uric acid, and formylisoglutamine/N-formimino-L-glutamate levels in the liver could indicate variation of amino acid metabolism in both tissues; and the perturbation of glutamate in the liver implies that the energy metabolism is also affected. These results reveal that environmentally relevant concentrations of amitriptyline perturb a fraction of the metabolome that is not typically associated with antidepressant exposure in fish. Environ Toxicol Chem 2019;00:1-13. © 2019 SETAC.

Multiresidue analytical method for the determination of 41 multiclass organic pollutants in mussel and fish tissues and biofluids by liquid chromatography coupled to tandem mass spectrometry.

In this work, the full optimisation and validation procedure to analyse a wide set of emerging organic contaminants in biotissues (mussel and fish muscle, liver, gills and brain) and biofluids (fish plasma and bile) is described. The target families include artificial sweeteners, industrial products, hormones, pharmaceutical and personal care products, pesticides and phytoestrogens. Different clean-up strategies (hydrophilic-lipophilic-balanced (HLB) solid-phase extraction, Florisil solid-phase extraction and liquid-liquid extraction followed by HLB solid-phase extraction and microextraction based on polyethersulfone polymer) were evaluated for the clean-up of focused ultrasonic solid-liquid extraction (FUSLE) extracts before the analysis by liquid chromatography-triple quadrupole tandem mass spectrometry (LC-QqQ-MS/MS). The methods afforded satisfactory apparent recovery values (71-126%) using isotopically labelled analytes and matrix-matched calibration approach, regardless of the matrix. Method detection limits in the range of 4-48 ng/g and 0.3-111 ng/L were obtained for biotissues and biofluids, respectively. The developed method was applied to determine the uptake and tissue distribution in juvenile gilt-head bream (Sparus aurata) during 7 days in seawater, and unexpectedly, perfluoro-1-butanesulfonate tended to accumulate in liver and, to a lesser extent, in muscle and gills. Furthermore, real mussel samples collected in the Basque coast were also analysed and the presence of the highly consumed valsartan (7 ng/g) and telmisartan (6.8 ng/g) compounds in bivalves is reported for the first time here. Graphical abstract ᅟ.

Occurrence of emerging pollutants in estuaries of the Basque Country: Analysis of sources and distribution, and assessment of the environmental risk.

In this study, the spatial and temporal-distribution of 41-multiclass organic compounds were assessed in three estuaries of the Basque Country, from winter 2016 to winter 2017 by grab (active) sampling methods and an extra campaign combining both, grab and passive sampling methods. Wastewater treatment plant (WWTP) effluents were also evaluated to assess their impact on the estuaries. Moreover, the physicochemical features (phosphate and nitrate concentrations, pH, etc.) of each site were measured and included in the statistical analysis. Anti-inflammatory drugs (diclofenac and acetaminophen), hypertensive drugs (irbesartan and valsartan), a stimulant (caffeine), an artificial sweetener (acesulfame) and a corrosion inhibitor (2-hydroxybenzothiazole) were the most ubiquitous compounds. Due to the stratification of the waters in the estuary of Bilbao two independent sources were identified: WWTP and harbour activities. In the case of Gernika and Plentzia, both are estuaries with a high tidal dilution, and the main sources were localized in the effluents of the WWTPs. In addition to this, the use of POCIS provides an efficient way to monitor emerging pollutants over a relatively long sampling period. Finally, risk quotient (RQ) values of each contaminant were estimated from the maximum values determined at each estuary and WWTP effluent for acute and chronic effects. In the case of acute toxicity the highest RQ values (¼1) were obtained for the angiotensin II receptor blockers (telmisartan, eprosartan, etc.), diuron and diclofenac. In the case of the chronic toxicity the highest RQ values (¼1) were estimated for caffeine, diclofenac, bezafibrate and sulfadiazine.

Non-targeted metabolomics reveals alterations in liver and plasma of gilt-head bream exposed to oxybenzone.

The extensive use of the organic UV filter oxybenzone has led to its ubiquitous occurrence in the aquatic environment, causing an ecotoxicological risk to biota. Although some studies reported adverse effects, such as reproductive toxicity, further research needs to be done in order to assess its molecular effects and mechanism of action. Therefore, in the present work, we investigated metabolic perturbations in juvenile gilt-head bream (Sparus aurata) exposed over 14 days via the water to oxybenzone (50 mg/L). The non-targeted analysis of brain, liver and plasma extracts was performed by means of UHPLC-qOrbitrap MS in positive and negative modes with both C18 and HILIC separation. Although there was no mortality or alterations in general physiological parameters during the experiment, and the metabolic profile of brain was not affected, the results of this study showed that oxybenzone could perturb both liver and plasma metabolome. The pathway enrichment suggested that different pathways in lipid metabolism (fatty acid elongation, α-linolenic acid metabolism, biosynthesis of unsaturated fatty acids and fatty acid metabolism) were significantly altered, as well as metabolites involved in phenylalanine and tyrosine metabolism. Overall, these changes are signs of possible oxidative stress and energy metabolism modification. Therefore, this research indicates that oxybenzone has adverse effects beyond the commonly studied hormonal activity, and demonstrates the sensitivity of metabolomics to assess molecular-level effects of emerging contaminants.

Study of bioconcentration of oxybenzone in gilt-head bream and characterization of its by-products.

The widespread occurrence of UV filters such as oxybenzone (OXY) in the aquatic ecosystems has raised social and scientific concern due to their high bioaccumulation potential and possible adverse effects in organisms. Within this context, the aim of the present work was to study the uptake, distribution, metabolization and elimination of OXY in different tissues (liver, gill and muscle) and biofluids (bile and plasma) of gilt-head bream (Sparus aurata) in a controlled seawater ecosystem (50 ng/mL OXY) within a 14-day exposure. The highest OXY concentrations in all the tissue/biofluids were found at the end of the experiment. The highest OXY levels were found in bile (1.8-17 µg/mL). In the case of liver, the concentrations found (9-160 ng/g) were lower than those expected for a lipidic matrix, which could be explained by a high OXY metabolization. Up to 20 Phase I and Phase II by-products of OXY were annotated by means of liquid chromatography-high resolution mass spectrometry, of which 12 were reported for the first time. In addition to OXY, its by-products might also cause adverse effects and their biomonitoring is advisable in order to fully characterize OXY exposure.

Evaluation of polar organic chemical integrative and hollow fibre samplers for the determination of a wide variety of organic polar compounds in seawater.

The calibration of two passive samplers for the determination of 20 emerging organic compounds in seawater is described in this work: i) a new version of polar organic chemical integrative sampler (POCIS) containing 100 mg of mixed-mode anion exchanger (Strata X-AW) and 100 mg of polymeric HLB (Plexa) sorbent materials and using a highly porous Nylon membrane (30-µm pore size) and ii) polyethersulfone (PES) hollow fibre. Among the studied contaminants, herbicides, hormones, life style products (stimulants and artificial sweeteners), industrial chemicals (corrosion inhibitor and fluorinated compounds), personal care products and several pharmaceuticals were included. In the case of POCIS, both the sorbents and the Nylon membranes were extracted and analysed independently. The calibration set up consisted on a continuous-flow tank that was fed with a continuous flow of seawater (2 L/h) and a stock mixture of contaminants (20 mL/h), assuring a nominal concentration of ~ 600 ng/L (each analyte) in the tank. The uptake was linear in POCIS sorbent and Nylon membranes but exponential for PES hollow fibres. Furthermore, the highest sampling rates (Rs) values were obtained in POCIS sorbent (between 2.7 for acetaminophen and 491 mL/day for perfluoro-n-octanoic acid, PFOA) followed by Nylon membranes (between 3.6 for OBT and 50 mL/day for telmisartan) and the lowest were those from PES fibres (between 1.7 for bezafibrate and 157 mL/day for butylparaben). Additionally, five deuterated compounds ([2H5]-atrazine, [2H3]-amitriptyline, [2H7]-irbesartan, [2H3]-ketoprofen and [2H9]-progesterone) were studied as candidates for performance reference compounds (PRCs) in both POCIS and PES, and though [2H5]-atrazine, [2H9]-progesterone and [2H3]-amitriptyline showed acceptable results in the case of POCIS, only [2H5]-atrazine provided a good validation. In the case of PES fibres, the PRC corrections did not provide acceptable results due to a low dissipation of the PRCs. Finally, POCIS were deployed in two sites of the low part of the estuary of Bilbao (northern Spain) from where water samples were also taken and analysed. As a result, in addition to the overall good agreement between the passive and active samplings, passive samplers allowed the determination of several compounds that were below the detection limits in the active sampling.