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

[EN] 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.Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work has been financially supported by the Ministry of Science and Innovation of the Spanish Government through project PID2020-117686RB-C31, and by the Education Department of the Basque Government as a consolidated group of the Basque Research System (IT1213-19)

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.Authors gratefully acknowledge financial support from the State Research Agency of the Ministry of Science and Innovation (Government of Spain) through project PID2020–117686RB-C31 and the Basque Government as a consolidated group of the Basque Research System (IT-1446–22). M. Musatadi also acknowledges the Basque Government for his predoctoral grant

Multi-Target Analysis and Suspect Screening of Xenobiotics in Milk by UHPLC-HRMS/MS

The development of suspect or non-target screening methods to detect xenobiotics in biological fluids is essential to properly understand the exposome and assess its adverse health effects on humans. In order to fulfil that aim, the biomonitorization of human fluids is compulsory. However, these methods are not yet extensively developed, especially for polar organic xenobiotics in biofluids such as milk, as most works are only focused on certain analytes of interest. In this work, a multi-target analysis method to determine 245 diverse xenobiotics in milk by means of Ultra High Performance Liquid Chromatography (UHPLC)-qOrbitrap was developed. Under optimal conditions, liquid milk samples were extracted with acetonitrile in the presence of anhydrous Na2SO4 and NaCl, and the extracts were cleaned-up by protein precipitation at low temperature and Captiva Non-Drip (ND)—Lipids filters. The optimized method was validated at two concentration-levels (10 ng/g and 40 ng/g) obtaining satisfactory figures of merit for more than 200 compounds. The validated multi-target method was applied to several milk samples, including commercial and breast milk, provided by 4 healthy volunteers. Moreover, the method was extended to perform suspect analysis of more than 17,000 xenobiotics. All in all, several diverse xenobiotics were detected, highlighting food additives (benzothiazole) or phytoestrogens (genistein and genistin) in commercial milk samples, and stimulants (caffeine), plasticizers (phthalates), UV filters (benzophenone), or pharmaceuticals (orlistat) in breast milk samples.This research was funded by the Agencia Estatal de Investigación (AEI) of Spain, the European Regional Development Fund through projects CTM2017-84763-C3-1-R and CTM2017-90890-REDT (AEI/FEDER, EU) and the Basque Government through the financial support as consolidated group of the Basque Research System (IT1213-19). B.G. acknowledges a Juan de la Cierva-Formación fellowship by the Spanish Ministry for the Economy, Industry and Competitiveness (MINECO)

The influence of temperature in sea urchin embryo toxicity of crude and bunker oils alone and mixed with dispersant

This investigation deals with how temperature influences oil toxicity, alone or combined with dispersant (D). Larval lengthening, abnormalities, developmental disruption, and genotoxicity were determined in sea urchin embryos for assessing toxicity of low-energy water accommodated fractions (LEWAF) of three oils (NNA crude oil, marine gas oil –MGO-, and IFO 180 fuel oil) produced at 5–25 °C. PAH levels were similar amongst LEWAFs but PAH profiles varied with oil and production temperature. The sum of PAHs was higher in oil-dispersant LEWAFs than in oil LEWAFs, most remarkably at low production temperatures in the cases of NNA and MGO. Genotoxicity, enhanced after dispersant application, varied depending on the LEWAF production temperature in a different way for each oil. Impaired lengthening, abnormalities and developmental disruption were recorded, the severity of the effects varying with oil, dispersant application and LEWAF production temperature. Toxicity, only partially attributed to individual PAHs, was higher at lower LEWAF production temperatures.The authors would like to gratefully acknowledge financial support from the European Union's Horizon 2020 research and innovation program EU H2020-BG-2005-2 under grant agreement No 679266 (project GRACE), from the Spanish Ministry of Education, Culture and Sport through pre-doctoral fellowship FPU15/05517, and from the Basque Government through Consolidated Research Group GIC IT1302-19 & IT1743-22. The authors acknowledge Total Spain for kindly providing the dispersant

Characterization of the contamination fingerprint of wastewater treatment plant effluents in the Henares River Basin (central Spain) based on target and suspect screening analysis

The interest in contaminants of emerging concern (CECs) has increased lately due to their continued emission and potential ecotoxicological hazards. Wastewater treatment plants (WWTPs) are generally not capable of eliminating them and are considered the main pathway for CECs to the aquatic environment. The number of CECs in WWTPs effluents is often so large that complementary approaches to the conventional target analysis need to be implemented. Within this context, multitarget quantitative analysis (162 compounds) and a suspect screening (>40,000 suspects) approaches were applied to characterize the CEC fingerprint in effluents of five WWTPs in the Henares River basin (central Spain) during two sampling campaigns (summer and autumn). The results indicated that 76% of the compounds quantified corresponded to pharmaceuticals, 21% to pesticides and 3% to industrial chemicals. Apart from the 82 compounds quantified, suspect screening increased the list to 297 annotated compounds. Significant differences in the CEC fingerprint were observed between summer and autumn campaigns and between the WWTPs, being those serving the city of Alcala de Henares the ones with the largest number of compounds and concentrations. Finally, a risk prioritization approach was applied based on risk quotients (RQs) for algae, invertebrates, and fish. Azithromycin, diuron, chlortoluron, clarithromycin, sertraline and sulfamethoxazole were identified as having the largest risks to algae. As for invertebrates, the compounds having the largest RQs were carbendazim, fenoxycarb and eprosartan, and for fish acetaminophen, DEET, carbendazim, caffeine, fluconazole, and azithromycin. The two WWTPs showing higher calculated Risk Indexes had tertiary treatments, which points towards the need of increasing the removal efficiency in urban WWTPs. Furthermore, considering the complex mixtures emitted into the environment and the low dilution capacity of Mediterranean rivers, we recommend the development of detailed monitoring plans and stricter regulations to control the chemical burden created to freshwater ecosystems.Authors acknowledge financial support from the Agencia Estatal de Investigación (AEI) of Spain and the European Regional Development Fund through project CTM2017-84763-C3-1-R project and the Basque Government through the financial support as consolidated group of the Basque Research System (IT1213-19). NLH is grateful to the Spanish Ministry of Economy, Industry and Competitivity for her predoctoral scholarship FPI 2018. BGG acknowledge an EHU/UPV postdoctoral fellowship. AR is supported by the Talented Researcher Support Programme - Plan GenT (CIDEGENT/2020/043) of the Generalitat Valenciana. Finally, the authors acknowledge support from the AEI and the Ministry of Science, Innovation and Universities (MICIU) to support the Thematic Network of Excellence (NET4SEA) on emerging contaminants in marine settings (CTM2017-90890-REDT, MICIU/AEI/ FEDER, EU)

Comparison of conventional and dispersive solid phase extraction clean-up approaches for the simultaneous analysis of tetracyclines and sulfonamides in a variety of fresh vegetables

he extensive use of antibiotics in agriculture has led to the occurrence of residual drugs in different vegetables frequently consumed by humans. This could pose a potential threat to human health, not only because of the possible effects after ingestion but also because the transmission of antibiotic-resistant genes could occur. In this work, two accurate sample preparation procedures were developed and validated for the simultaneous analysis of sulfonamides (SAs) and tetracyclines (TCs) in four of the most widely consumed vegetables (lettuce, onion, tomato, and carrot) in Europe. The evaluated protocols were based on QuECHERS for extraction and subsequent clean-up by SPE (solid phase extraction) or dispersive SPE. Parameters affecting both extraction and clean-up were carefully evaluated and selected for accuracy of results and minimal matrix effect. Overall, apparent recoveries were above 70% for most of the target analytes with both analytical procedures, and adequate precision (RSD<30%) was obtained for all the matrices. The procedural limits of quantification (LOQPRO) values for SPE clean-up remained below 4.4 μg kg−1 for TCs in all vegetables except for chlortetracycline (CTC) in lettuce (11.3 μg kg−1) and 3.0 μg kg−1 for SAs, with the exception of sulfadiazine (SDZ) in onion (3.9 μg kg−1) and sulfathiazole (STZ) in carrot (5.0 μg kg−1). Lower LOQPRO values (0.1–3.7 μg kg−1) were obtained, in general, when dSPE clean-up was employed. Both methods were applied to twenty-five market vegetable samples from ecological and conventional agriculture and only sulfamethazine (SMZ) and sulfapyridine (SPD) were detected in lettuce at 1.2 μg kg−1 and 0.5 μg kg−1, respectively.Authors acknowledge financial support from the Elkartek project entitled “Emergencia y diseminación de resistencia a los antibióticos: vínculos entre salud humana, ganadería, alimentación y medioambiente (elkartek 20/88)”, the projects “Evaluación del riesgo de aparición y diseminación de resistencias a antibióticos en productos vegetales frescos y suelos de cultivo de la comunidad autónoma del País Vasco (PA21/05 and PA22/05)” inside the “Research projects targeted to agriculture 2020 program” of the Basque Government (Basque Country, Spain); and the Basque Government through the financial support as consolidated group of the Basque Research System (IT1446-22). I. Vergara-Luis and B. Gonzalez-Gaya are grateful to the University of the Basque Country (UPV/EHU) for their pre-doctoral and post-doctoral fellowships. I. Baciero thanks to the Basque Government for her pre-doctoral fellowship

Comprehensive micropollutant characterization of wastewater during Covid-19 crisis in 2020: Suspect screening and environmental risk prioritization strategy

Micropollutants monitoring in wastewater can serve as a picture of what is consuming society and how it can impact the aquatic environment. In this work, a suspect screening approach was used to detect the known and unknown contaminants in wastewater samples collected from two wastewater treatment plants (WWTPs) located in the Basque Country (Crispijana in Alava, and Galindo in Vizcaya) during two weekly sampling campaigns, which included the months from April to July 2020, part of the confinement period caused by COVID-19. To that aim, high-resolution mass spectrometry was used to collect full-scan data-dependent tandem mass spectra from the water samples using a suspect database containing >40,000 chemical substances. The presence of > 80 contaminants was confirmed (level 1) and quantified in both WWTP samples, while at least 47 compounds were tentatively identified (2a). Among the contaminants of concern, an increase in the occurrence of some compounds used for COVID-19 disease treatment, such as lopinavir and hydroxychloroquine, was observed during the lockdown. A prioritization strategy for environmental risk assessment was carried out considering only the compounds quantified in the effluents of Crispijana and Galindo WWTPs. The compounds were scored based on the removal efficiency, estimated persistency, bioconcentration factor, mobility, toxicity potential and frequency of detection in the samples. With this approach, 33 compounds (e.g. amantadine, clozapine or lopinavir) were found to be considered key contaminants in the analyzed samples based on their concentration, occurrence and potential toxicity. Additionally, antimicrobial (RQ-AR) and antiviral (EDRP) risk of certain compounds was evaluated, where ciprofloxacin and fluconazole represented medium risk for antibiotic resistance (1 > RQ-AR > 0.1) in the aquatic ecosystems. Regarding mixture toxicity, the computed sum of toxic unit values of the different effluents (> 1) suggest that interactions between the compounds need to be considered for future environmental risk assessments.This study was funded by the Basque Government through financial support as a consolidated group of the Basque Research System (IT1446-22), the Agencia Estatal de Investigación (AEI) of Spain, the 2020 call for the generation of knowledge and scientific and technological strengthening of the R&D&i system and the R&D&i focused on society's challenges, through project PID2020-117686RB-C31 and the Council of Vitoria-Gasteiz and Fundación Vital. The authors are grateful to the Consorcio de Aguas de Bilbao and especially to Iñigo González. Naroa Lopez-Herguedas is grateful to the Spanish Ministry of Economy, Industry and Competitivity for her predoctoral scholarship FPI 2018. Iker Alvarez-Mora is grateful to the University of the Basque Country and the Université de Pau et des Pays de L' Adour for his cotutelle predoctoral scholarship. Finally, the authors acknowledge support from the AEI and the Ministry of Science, Innovation and Universities (MICIU) to support the Thematic Network of Excellence (NET4SEA) on emerging contaminants in marine settings (CTM2017-90890-REDT, MICIU/AEI/FEDER, EU)

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.Authors gratefully acknowledge financial support from the State Research Agency of the Ministry of Science and Innovation (Government of Spain) through project PID 2020-117686RB-C31 and the Basque Government as a consolidated group of the Basque Research System (IT-1446-22). M. Musatadi also acknowledges the Basque Government for his predoctoral grant

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.Authors acknowledge financial support from the Agencia Estatal de Investigación (AEI) of Spain and the European Regional Development Fund through CTM2017-84763-C3-1-R and CTM2020-117686RB-C31 projects and the Basque Government through the financial support as a consolidated group of the Basque Research System (IT1213-19). The authors are grateful to the Consorcio de Aguas de Bilbao and especially to Iñigo González. Naroa Lopez is grateful to the Spanish Ministry of Economy, Industry and Competitivity for her predoctoral scholarship FPI 2018. Belen González-Gaya and Leire Mijangos acknowledge the University of the Basque Country for their postdoctoral scholarships (FPI 2018). Iker Alvarez-Mora is grateful to the University of the Basque Country and the Université de Pau et des Pays de L' Adour for his cotutelle predoctoral scholarship. Finally, the authors acknowledge support from the AEI and the Ministry of Science, Innovation and Universities (MICIU) to support the Thematic Network of Excellence (NET4SEA) on emerging contaminants in marine settings (CTM2017-90890-REDT, MICIU/AEI/FEDER, EU)

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.Authors acknowledge financial support from the Agencia Estatal de Investigación (AEI) of Spain and the European Regional Development Fund through CTM2017-84763-C3-1-R and CTM2020-11686RB-C31 projects and the Basque Government through the financial support as a consolidated group of the Basque Research System (IT1446-22). The authors are grateful to the Consorcio de Aguas de Bilbao and especially to Iñigo González. Naroa Lopez-Herguedas is grateful to the Spanish Ministry of Economy, Industry and Competitivity for her predoctoral scholarship FPI 2018 (PRE2018-086493)