Integrated assessment of the presence of emerging compounds and their toxicological effects in estuaries of Biscay

306 p.The concern about the widespread presence of ECs in the marine environment and their possible toxic effects is gaining attention. In this PhD we get a close insight about the impact of ECs in estuarine waters and the contribution of wastewater treatment plant by focusing on the development of multiresidue methods for environmental aqueous samples and marine organism as well as passive sampling. After a year of monitoring, we were able to identify the main hot spots and the distribution of emerging compounds in the estuaries of Bilbao, Plentzia and Urdaibai using the procedures developed. Besides, by implementing in the effect directed analysis a bioassay designed for coastal waters as the way to drive the non-targeted analysis, we identify the anthelmintic mebendazole and amitriptyline as the main toxic emerging contaminants in Galindo WWTP effluent.IBEA: Ikerkuntza eta Berrikuntza Analitiko

Integrated assessment of the presence of emerging compounds and their toxicological effects in estuaries of Biscay

306 p.The concern about the widespread presence of ECs in the marine environment and their possible toxic effects is gaining attention. In this PhD we get a close insight about the impact of ECs in estuarine waters and the contribution of wastewater treatment plant by focusing on the development of multiresidue methods for environmental aqueous samples and marine organism as well as passive sampling. After a year of monitoring, we were able to identify the main hot spots and the distribution of emerging compounds in the estuaries of Bilbao, Plentzia and Urdaibai using the procedures developed. Besides, by implementing in the effect directed analysis a bioassay designed for coastal waters as the way to drive the non-targeted analysis, we identify the anthelmintic mebendazole and amitriptyline as the main toxic emerging contaminants in Galindo WWTP effluent.IBEA: Ikerkuntza eta Berrikuntza Analitiko

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

Integrated biological response to environmentally-relevant concentration of amitriptyline in Sparus aurata

[EN]Amitriptyline (AMI) is a commonly tricyclic antidepressant to treat depression, anxiety, and other conditions. Like many other pharmaceuticals, AMI and its by-products are incompletely removed during wastewater treatment and therefore they are released to rivers, estuaries and coastal waters. The presence of this kind of compounds in the water environment may involve a negative impact on non-target aquatic organisms at relatively low concentrations. However, the knowledge of AMI effects on aquatic organisms in the environment is scarce. Thus, the objective of this work is to determine the effects of environmentally-relevant concentrations of AMI on biological responses at biochemical and cellular levels in marine teleost. Gilt-head seabream (S. aurata) were exposed to AMI for 7 days at 0.2 mu g/L in an open flow system and a battery of biomarkers were investigated: acetylcholinesterase, catalase, superoxide dismutase, glutathione S-transferase, cytochrome C oxidase, P450 CYP1A1 ethoxyresorufin (O) dealkylation, and lysosomal biomarkers. Biomarkers were integrated as IBR/n (biological response index). Overall, it can be concluded that AMI exposure at environmentally-relevant concentration induces significant biological responses to stress in marine teleost, especially in lysosomal biomarkers. However, further research is needed about the effects of AMI and other pharmaceuticals on biomarkers in nontargeted species, to raise the knowledge about the toxicity of this type of emerging pollutants.The authors are indebted to Ainhoa Camille for helping us with her invaluable technical support. This work was financially supported by the Ministry of Economy and Competitiveness through the project CTM2017-84763-C3-1-R and the Basque Government (IT-742-13 & IT1213-19). H. Ziarrusta is grateful to the Spanish Ministry predoctoral fellowship and L. Mijangos to the University of the Basque Country for her postdoctoral fellowship

SETApp: A machine learning and image analysis based application to automate the sea urchin embryo test

[EN] Since countless xenobiotic compounds are being found in the environment, ecotoxicology faces an astounding challenge in identifying toxicants. The combination of high-throughput in vivo/in vitro bioassays with high-resolution chemical analysis is an effective way to elucidate the cause-effect relationship. However, these combined strategies imply an enormous workload that can hinder their implementation in routine analysis. The purpose of this study was to develop a new high throughput screening method that could be used as a predictive expert system that automatically quantifies the size increase and malformation of the larvae and, thus, eases the application of the sea urchin embryo test in complex toxicant identification pipelines such as effect-directed analysis. For this task, a training set of 242 images was used to calibrate the size-increase and malformation level of the larvae. Two classification models based on partial least squares discriminant analysis (PLS-DA) were built and compared. Moreover, Hierarchical PLS-DA shows a high proficiency in classifying the larvae, achieving a prediction accuracy of 84 % in validation. The scripts built along the work were compiled in a user-friendly standalone app (SETApp) freely accessible at https://github.com/UPV-EHU-IBeA/SETApp. The SETApp was tested in a real case scenario to fulfill the tedious requirements of a WWTP effect-directed analysis.Authors gratefully 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 and the Basque Government through the financial support as a consolidated group of the Basque Research System (IT1213–19). Iker Alvarez 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

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)

Testing Wastewater Treatment Plant Effluent Effects on Microbial and Detritivore Performance: a Combined Field and Laboratory Experiment

The amount of pollutants and nutrients entering rivers via point sources is increasing along with human population and activity. Although wastewater treatment plants (WWTPs) greatly reduce pollutant loads into the environment, excess nutrient loading is a problem in many streams. Using a Community and Ecosystem Function (CEF) approach, we quantified the effects of WWTP effluent on the performance of microbes and detritivores associated to organic matter decomposition, a key ecosystem process. We measured organic matter breakdown rates, respiration rates and exo-enzymatic activities of aquatic microbes. We also measured food consumption and growth rates and RNA to body-mass ratios (RNA:BM) of a dominant amphipod Echinogammarus berilloni. We predicted responses to follow a subsidy-stress pattern and differences between treatments to increase over time. To examine temporal effects of effluent, we performed a laboratory microcosm experiment under a range of effluent concentrations (0, 20, 40, 60, 80 and 100%), taking samples over time (days 8, 15 and 30; 4 and 10 replicates to assess microbe and detritivore performance respectively, per treatment and day). This experiment was combined with a field in situ Before-After Control-Impact Paired (BACIP) experiment whereby we added WWTP effluent poured (10 L s(-1) during 20-40 min every 2 h) into a stream and collected microbial and detritivore samples at days 8 and 15 (5 and 15 replicates to assess the microbe and detritivore performance respectively, per period, reach and sampling day). Responses were clearer in the laboratory experiment, where the effluent caused a general subsidy response. Field measures did not show any significant response, probably because of the high dilution of the effluent in stream water (average of 1.6%). None of the measured variables in any of the experiments followed the predicted subsidy-stress response. Microbial breakdown, respiration rates, exo-enzymatic activities and invertebrate RNA:BM increased with effluent concentrations. Differences in microbial respiration and exo-enzymatic activities among effluent treatments increased with incubation time, whereas microbial breakdown rates and RNA:BM were consistent over time. At the end of the laboratory experiment, microbial respiration rates increased 156% and RN:BM 115% at 100% effluent concentration. Detritivore consumption and growth rates increased asymptotically, and both responses increased with by incubation time. Our results indicate that WWTP effluent stimulates microbial activities and alters detritivore performance, and stream water dilution may mitigate these effects.This work has been supported by the EU7th Framework Programme Funding under Grant agreement no. 603629-ENV-2013-6.2.1-Globaqua. We also acknowledge financial support in terms of pre doctoral grants from the University of the Basque Country UPV/EHU (L. Solagaistua) and the Basque Government (I. de Guzman, L. Mijangos). The manuscript benefited greatly from the valuable comments of John Kominoski and two anonymous referees. Also SGIker technical and human support (UPV/EHU, MICINN, GV/EJ, ESF) is gratefully acknowledged

Itsas ingurumenerako mikrokutsatzaile organiko hidrofobikoen presentzia eta eraginak aztertzeko estrategien taxutzea

The efects of hydrophobic microcontaminants such as alkylphenols, organophosphorus compounds, organochloride pesticides, phthalates and musk fragances were studied in mussels. The proposed analytical strategy is based on the combination of passive sampling studies, bioconcentration measurements of micropollutants in mussels, environmental NMR metabolomics of two tissues (gonad, muscle) and hemolymph of mussels and histological analysis. By means of this work, it was verified the feasibility of this strategy in controlled conditions with intent to use it in future environmental studies. The micropollutants mixture was responsible of early spawning in mussels. In fact, the most significant metabolic changes were observed in the same day that spawning took place.; Lan honetan, ingurumen analisirako metodologia planteamendu berri bat proposatu da. Horretarako, kimikariek eta biologoek baldintza kontrolatuetako esposizioa egiteko elkarlanean jardun genuen. Esperimentu honetan, muskuiluak eta lagin-biltze pasiboko tresnak hainbat mikrokutsatzaile hidrofoboren eraginpean jarri ziren. Batetik, kutsatzaileen kontzentrazioak neurtu ziren, lagin-biltze puntualen zein lagin-biltze pasiboen bitartez. Bestetik, muskuiluetan kutsatzaileen kontzentrazioak neurtu ziren. Biologoek, euren aldetik, muskuiluen azterketa histopatologikoa egin zuten. Azkenik, NMR-n oinarritutako metabolomika erabili zen muskuiluek kutsatzaileen aurrean ematen zuten erantzuna aztertzeko. Lau estrategien konbinazioarekin, kausa (kokteleko mikrokutsatzaileen kontzentrazioak eta muskuiluek metatutakoa) eta eragindako efektuak (histologia azterketetan behatutakoak) hobeto uler ditzakegu. Kutsatzaileek eragiten dituzten aldaketak ulertzeko eta ondorioak azaltzeko metabolomika baliabide erabilgarria dela ondorioztatu genuen. Kutsatzaileen koktelak errute goiztiarra eragin zien muskuiluei; izan ere, NMR metabolomika erabiliz, aldaketa metaboliko nabarienak errute-egunean ikusi ziren