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.

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.

Development, characterization and comparisons of targeted and non-targeted metabolomics methods.

The potential of a metabolomics method to detect statistically significant perturbations in the metabolome of an organism is enhanced by excellent analytical precision, unequivocal identification, and broad metabolomic coverage. While the former two metrics are usually associated with targeted metabolomics and the latter with non-targeted metabolomics, a systematic comparison of the performance of both approaches has not yet been carried out. The present work reports on the development and performance evaluation of separate targeted and non-targeted metabolomics methods. The targeted approach facilitated determination of 181 metabolites (quantitative analysis of 18 amino acids, 11 biogenic amines, 5 neurotransmitters, 5 nucleobases and semi-quantitative analysis of 50 carnitines, 83 phosphatidylcholines, and 9 sphingomyelins) using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and flow injection-tandem mass spectrometry (FI-MS/MS). Method accuracy and/or precision were assessed using replicate samples of NIST SRM1950 as well as fish liver and brain tissue from Gilthead Bream (Sparus aurata). The non-target approach involved UPLC-high resolution (Orbitrap) mass spectrometry (UPLC-HRMS). Testing of ionization mode and stationary phase revealed that a combination of positive electrospray ionization and HILIC chromatography produced the largest number of chromatographic features during non-target analysis. Furthermore, an evaluation of 4 different sequence drift correction algorithms, and combinations thereof, revealed that batchCorr produced the best precision in almost every test. However, even following correction of non-target data for signal drift, the precision of targeted data was better, confirming our existing assumptions about the strengths of targeted metabolomics. Finally, the accuracy of the online MS2-library mzCloud was evaluated using reference standards for 38 different metabolites. This is among the few studies that have systematically evaluated the performance of targeted and non-targeted metabolomics and provides new insight into the advantages and disadvantages of each approach.

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.

Ciprofloxacin by-products in seawater environment in the presence and absence of gilt-head bream.

The widespread use of pharmaceuticals has caused a growing concern on the presence of pharmaceuticals such as the antibiotic ciprofloxacin (CIPRO) in the aquatic environment, since they may exert adverse effects on non-target organisms, including fish. In order to study the uptake, distribution in different tissues (liver, muscle, brain and gill) and biofluids (plasma and bile), metabolism and elimination of CIPRO in gilt-head bream (Sparus aurata), controlled dosing experiments for 8 days at 200 µg/L concentration were carried out. CIPRO was only observed in bile at concentration up to 315 ±â€¯4 ng/mL, probably due to its low octanol-water partition coefficient (log P = -2.4 at pH 7.4) and the zwitterionic behavior (pKa1 = 5.76 and pKa2 = 8.68). CIPRO by-products (BPs) were also identified in seawater environment, both in presence and absence of fish. The analysis done by means of liquid chromatography-high resolution mass spectrometry (hybrid quadrupole-Orbitrap) permitted the annotation of up to 35 BPs of CIPRO in seawater and bile, from which 30 structures were reported for the first time. These results confirm that CIPRO is very susceptible to photolysis, and that it goes through various phase I and phase II metabolisms in the fish. All these results suggested that, for a complete characterization of CIPRO exposure, BPs should also be included in the biomonitoring campaigns since they might also be toxicologically relevant.

Simultaneous determination of 41 multiclass organic pollutants in environmental waters by means of polyethersulfone microextraction followed by liquid chromatography-tandem mass spectrometry.

A new procedure using polyethersulfone (PES) microextraction followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was developed in this work for the simultaneous determination of 41 multiclass priority and emerging organic pollutants including herbicides, hormones, personal care products, and pharmaceuticals, among others, in seawater, wastewater treatment plant (WWTP) effluents, and estuary samples. The optimization of the analysis included two different chromatographic columns and different variables (polarity, fragmentor voltage, collision energy, and collision cell accelerator) of the mass spectrometer. In the case of PES extraction, ion strength of the water, pH, addition of EDTA, and the amount of the polymeric material were thoroughly investigated. The developed procedure was compared with a previously validated one based on a standard solid-phase extraction (SPE). In contrast to the SPE protocol, the PES method allowed a cost-efficient extraction of complex aqueous samples with lower matrix effect from 120 mL of water sample. Satisfactory and comparable apparent recovery values (80-119 and 70-131%) and method quantification limits (MQLs, 0.4-26 and 0.2-23 ng/L) were obtained for PES and SPE procedures, respectively, regardless of the matrix. Repeatability values lower than 27% were obtained. Finally, the developed methods were applied to the analysis of real samples from the Basque Country and irbesartan, valsartan, acesulfame, and sucralose were the analytes most often detected at the highest concentrations (51-1096 ng/L). Graphical abstract Forty-one multiclass pollutant determination in environmental waters by means of PES/SPE-LC-MS/MS.

Determination of fluoroquinolones in fish tissues, biological fluids, and environmental waters by liquid chromatography tandem mass spectrometry.

This work describes the optimization, validation, and application in real samples of accurate and precise analytical methods to determine ten fluoroquinolones (FQs) (norfloxacin, enoxacin, pefloxacin, ofloxacin, levofloxacin, ciprofloxacin, danofloxacin, lomefloxacin, enrofloxacin, and sparfloxacin) in different environmental matrices, such as water (estuarine, seawater, and wastewater treatment plant effluent), fish tissues (muscle and liver), and fish biofluids (plasma and bile). The analysis step performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was fully optimized to improve the separation and detection steps. The extraction of analytes from fish tissues was accomplished using focused ultrasound solid-liquid extraction using methanol/acetic acid (95:5 v/v) as extractant. The preconcentration and clean-up steps were optimized in terms of extraction efficiency and cleanliness and the best strategy for each matrix was selected: (i) Oasis HLB for seawater and muscle, (ii) liquid-liquid extraction combined with Oasis HLB for the lipid-rich liver, (iii) the combination of Evolute-WAX and Oasis HLB for estuarine water and wastewater treatment plant effluent, and (iv) molecular imprinted polymers for biofluids. The methods afforded satisfactory apparent recoveries (80-126%) and repeatability (RSD < 15%), except for sparfloxacin, which showed a lack of correction with the available isotopically labeled surrogates ([2H8]-ciprofloxacin and [2H5]-enrofloxacin). Ciprofloxacin, norfloxacin, and ofloxacin were detected in both water and fish liver samples from the Biscay Coast at concentrations up to 278 ng/L and 4 ng/g, respectively. To the best of our knowledge, this work is one of the few analyzing up to ten FQs and in so many fish tissues and biofluids. Graphical abstract Determination of fluoroquinolones in different environmental matrices, such as water (estuarine, seawater, and wastewater treatment plant effluent), fish tissues (muscle and liver), and fish biofluids (plasma and bile).

Bioconcentration and Biotransformation of Amitriptyline in Gilt-Head Bream.

Extensive global use of the serotonin-norepinephrine reuptake inhibitor Amitriptyline (AMI) for treatment of mental health problems has led to its common occurrence in the aquatic environment. To assess AMI bioconcentration factors, tissue distribution, and metabolite formation in fish, we exposed gilt-head bream (Sparus aurata) to AMI in seawater for 7 days at two concentrations (0.2 µg/L and 10 µg/L). Day 7 proportional bioconcentration factors (BCFs) ranged from 6 (10 µg/L dose, muscle) to 127 (0.2 µg/L dose, brain) and were consistently larger at the low dose level. The relative tissue distribution of AMI was consistent at both doses, with concentrations decreasing in the order brain ≈ gill > liver > plasma > bile ≫ muscle. Using a suspect screening workflow based on liquid chromatography-high resolution (Orbitrap) mass spectrometry we identified 33 AMI metabolites (both Phase I and Phase II), occurring mostly in bile, liver and plasma. Ten structures are reported for the first time. Remarkably, all 33 metabolites retained the tricyclic ring structure common to tricyclic antidepressants, which may be toxicologically relevant. Collectively these data indicate that, in addition to AMI, a broad suite of metabolites should be included in biomonitoring campaigns in order to fully characterize exposure in aquatic wildlife.

Determination of tricyclic antidepressants in biota tissue and environmental waters by liquid chromatography-tandem mass spectrometry.

This work describes the optimization, validation, and application in real samples of accurate and precise analytical methods to determine tricyclic antidepressants (TCAs), including amitriptyline, nortriptyline, imipramine, and clomipramine in different environmental matrices, such as water (estuary, seawater, and wastewater treatment plant effluent) and biota (fish muscle, fish liver, and mussels), which would lead to supplement the scarce information on the presence of TCAs in aquatic organisms. The analysis step performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was fully optimized to improve the sensitivity of the separation and detection steps. The extraction of solid samples was accomplished using focused ultrasonic solid-liquid extraction (FUSLE), which required a low amount of sample (0.5 g), solvent (7 mL acetonitrile/H2O, 95:5 v/v) and short extraction time (30 s). In the optimisation of the clean-up step, mixed mode solid-phase extraction (SPE) using a strong cation exchanger rendered clean extracts and the best results in terms of extraction efficiency and matrix effect. The same SPE mode was also used for the extraction and pre-concentration of TCAs from environmental water matrices. The methods afforded satisfactory apparent recovery values (86-122%) and repeatability (RSD < 5%), regardless of the matrix. Finally, the developed methods were applied to the analysis of real samples from the Biscay Coast, where TCAs were detected in both water and biota samples up to 25.9 ng/L and 1.8 ng/g, respectively. Up to our knowledge, this is the first work using FUSLE for the determination of TCAs and one of the few analyzing TCAs in biota samples.