Determination of endocrine disrupting compounds in fish liver, brain, and muscle using focused ultrasound solid-liquid extraction and dispersive solid phase extraction as clean-up strategy.

This study describes a new method for the simultaneous extraction of several endocrine disrupting compounds, including alkylphenols (APs), estrogen, bisphenol-A (BPA) and one phthalate metabolite (mono-2-ethylhexyl ester, MEHP) in fish liver, brain, and muscle. Parameters affecting the extraction (extraction solvent and temperature) and the clean-up (dispersive phase nature and amount) steps were evaluated. The extraction was performed by means of focused ultrasound solid-liquid extraction (FUSLE) using 10 mL of n-hexane:acetone (50:50, v/v) for 5 min at ~0 °C, and the clean-up was done by means of dispersive solid phase extraction (dSPE) using 100 mg of ENVI-CARB and 100 mg of MgSO4 for the cleaning of brain and muscle extracts together with 100 mg of PSA in the case of liver extracts. Good apparent recoveries were obtained in the case of liver (62-132 %), brain (66-120 %), and muscle (74-129 %), relative standard deviation (RSD%) was always below 26 %, and the method detection limits (MDLs) were at low ng/g level. The developed method was applied to fish captured in Urdaibai estuary (Bay of Biscay) in December 2015, and the concentrations obtained were in the range MDL-1115 ng/g in brain, MDL-962 ng/g in muscle, and MDL-672 ng/g in liver. In general, the highest concentrations were measured in liver, followed by brain and muscle. In addition, diethylstilbestrol was only detected in fish brain. Graphical Abstract MS method scheme for the/MS method scheme for the determination of EDCs in fish liver, brain and muscle.

Simultaneous enzymatic hydrolysis and extraction of endocrine-disrupting chemicals in fish bile using polyethersulfone polymer.

This study describes a new method for the simultaneous extraction and enzymatic hydrolysis of alkylphenols, estrogens, bisphenol-A and phthalate metabolite (mono-2-ethylhexyl ester, MEHP) in fish bile using polyethersulfone (PES) polymer as sorptive material. Parameters affecting the hydrolysis (enzyme amount) and extraction (nature of polymeric material, PES desorption solvent nature and time, PES amount and time profile) were optimised. The optimum conditions were fixed as: 5 PES tubes (1.5 cm length × 0.7 mm o.d.) were added to a vessel with 100 µL of sample, 800 µL of ultrapure water, 1.5 mL phosphate buffer (0.1 mol L(-1), pH 6) and 200 µL of ß-glucuronidase (1000 U mL(-1)) enzyme and the mixture was stirred at 37 °C and 550 rpm for 3 h. Quantitative results were obtained after desorption of PES material using 500 µL of ethyl acetate. The extracts were reconstituted in 250 µL of methanol and analysed by liquid chromatography-tandem mass spectrometry, obtaining apparent recoveries in the range of 73-134 % using deuterated compounds surrogates corrections. Relative standard deviations below 27 % were obtained for all target analytes and the method detection limits (MDLs) were in low nanograms per mililliter level for all the studied compounds, except in the case of MEHP which was detected at higher concentration levels (ng µL(-1)) in bile samples that do not allow its MDL determination. Bisphenol A (MDL-10.8 ng mL(-1)), diethylstilbestrol (MDL-1.4 ng mL(-1)) and MEHP (975-2604 ng mL(-1)) were detected in grey mullets captured nearby the wastewater treatment plant of Gernika (Biosphere Reserve of Urdaibai).

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.

Determination of endocrine disrupting compounds and their metabolites in fish bile.

This work describes a new methodology for the simultaneous determination of a large variety of emerging and persistent organic compounds and some of their metabolites in fish bile samples. The target compounds were musk fragrances, alkyl phenols, hormones, pesticides, phthalate esters and bisphenol-A, all of them with a known endocrine disrupting effect. To achieve the determination these three steps were optimized: i) an enzymatic hydrolysis of the metabolites to render the unconjugated compounds; ii) the solid phase extraction of the target analytes (Plexa cartridges 200-mg); and, iii) a clean-up of the extracts (Florisil cartridges 1-g). The samples were analyzed by gas-chromatography-mass spectrometry (GC-MS), though the polar fraction required a previous derivatization with O-bis (trimethylsilyl) trifluoroacetamide. Good apparent recoveries (63-122%), repeatability (<20%) and limits of detection (LODs) ranging between 0.04 and 459 ng/mL were obtained. This method was applied to the analysis of the target analytes in bile samples of thicklip grey mullets (Chelon labrosus) from five different populations of the Basque Coast (South East Bay of Biscay) during the period of May-June 2012. The target analytes were found at concentrations ranging from

Assessment of commercially available polymeric materials for sorptive microextraction of priority and emerging nonpolar organic pollutants in environmental water samples.

Among the different organic pollutants, persistent organic pollutants and emerging organic contaminants (EOCs) are of particular concern due to their potentially dangerous effects on the ecosystems and on human health. In the framework of the analysis of some of these organic pollutants in water samples, sorptive extraction devices have proven to be adequate for their monitoring. The efficiency of four commercially available and low-cost polymeric materials [polypropylene, poly(ethylene terephthalate), Raffia, and polyethersulfone (PES)] for the simultaneous extraction of 16 organic compounds from five different families from environmental water samples was evaluated in this work. Firstly, the homogeneity of the sorbent materials was confirmed by means of Raman spectroscopy. After the optimization of the parameters affecting the extraction and the liquid desorption steps, it was found that PES showed the largest efficiencies for slightly polar analytes and, to a lesser extent, for nonpolar analytes. Additionally, Raffia rendered good extraction efficiencies for nonpolar compounds. Thus sorptive extraction methods followed by large volume injection-programmable temperature vaporizer-gas chromatography-mass spectrometry were validated using PES and Raffia as sorbent materials. The validation of the method provided good linearity (0.978 < r (2) < 0.999 for PES and 0.977 < r (2) < 0.999 for Raffia), adequate repeatability (below 19 % and 14 % for PES and Raffia, respectively), and low method detection limits (low ng · l(-1) level). Finally, these materials were applied to the analysis of contaminants in environmental water samples.