Análisis de psicoactivos en aguas, sedimentos y alimentos: de la epidemiología de alcantarilla a la forensía medioambiental

Licit and illicit drugs of abuse, pharmaceuticals and personal care products are emerging contaminants whose impact on ecosystems is still poorly understood. In the last decades there has been a growing interest in the study of these substances due to their high consumption and continuous discharges into environmental compartments, after their insufficient disposal in wastewater treatment plants (WWTPs). The origin and quantity of these pollutants are often related to the characteristics, health conditions and habits of the populations that pour into these WWTPs. Therefore, the study of these pollutants has a double aspect: the magnitude, origin and patterns of consumption can be evaluated epidemiologically through the range of compounds detected in the WWTP and environmentally by tracking and managing the hazard of these substances to ecosystems. Wastewater-based epidemiology analysis, also called sewage epidemiology, is an important tool for estimating local consumption through the study of mass flows of unchanged drugs of abuse or their metabolites. The EMCDDA (European Monitoring Centre for Drugs and Drug Addiction) recognizes this method as an alternative to official methods based on interviews, medical data and criminal statistics to establish the consumption of drugs of abuse. The adverse effects of drug consumption on human health make it possible to predict that the presence of these compounds in surface waters could have toxicological consequences on aquatic flora and biota. However, one of the tools, that has not yet been sufficiently developed to address all these studies, are the techniques for determining these compounds in different environmental matrices. In addition to the complexity of the matrix, the low concentrations and the instability of these compounds make their determination difficult. Therefore, General Objective has been to develop more reliable analytical methods to (i) determine the presence of studied drugs in different environmental matrices, (ii) establish patterns of consumption of these substances in Valencia and its metropolitan area, through sewage epidemiology and (iii) know the sources, levels, transport and fate of these substances in the environment applying environmental forensics approaches, and keeping in mind how could they affect to humans as ultimate goal. Specific Objectives of this Doctoral Thesis has been divided into the following aims: 1. Develop selective and sensitive analytical methodologies for identification and quantification based on liquid chromatography tandem mass spectrometry (LC-MS/MS) and ultra-high performance liquid chromatography hybrid quadrupole time-of-flight mass spectrometry (UHPLC-QqTOF-MS / MS) to determine selected substances. 2. Apply the developed methodologies in water analysis (influents and effluents of the WWTPs and surface waters), particulate material present in the influent of the wastewater, dehydrated sludge processed in the treatment of the treatment plants and sediments deposited by surface waters. 3. Establish human biomarkers capable of estimating the population size to which each WWTP supplies that offer reliable data and compare them with the traditional methods used so far for this purpose. 4. Determine the consumption of licit and illicit drugs at the population level by analyzing wastewater from the excretion of unchanged drugs or their metabolites. 5. Analyse the efficiency of treatment of wastewater in WWTPs, according to the percentage of elimination of drugs in effluents. 6. Verify the influence and effects of human pressure in a typical Mediterranean river basin through an integrated study of water contamination by drugs and its relation with other characteristics of water quality. 7. Estimate the bio- and photodegradation and transformation of the stereoisomers of a psychoactive substance (fluoxetine) in different matrices (surface water and active sludge from WWTP). 8. Evaluate the toxicity of certain drugs at different trophic levels by in silico and in vivo studies. 9. Estimate other possible routes of human exposure through food and their potential effects on population. In order to develop these objectives, the following Work Plan has been designed: Firstly, in order to define the aims clearly and to establish the limiting aspects of this topic, a thorough review of the literature published up to now has been carried out, which has allowed us to recognize the strengths and weaknesses to develop a future-oriented work. Drugs of abuse were selected attending to a consumption considered traditional or by being in the group of new psychoactive substances (NPS) —including both, illegal and those not yet subject to international control (known as legal highs)— marketed via the internet. Certain pharmaceuticals and personal care products were selected because of their high interest as emerging pollutants. Sampling campaigns were then designed in WWTPs of Pinedo I, Pinedo II and Quart-Benáger (influent, effluent and dehydrated active sludge) and in the Turia River basin (surface waters and sediments). During the work carried out at the University of Bath (England) the sampling campaigns of the activated sludge of the WWTP and the surface waters of the Avon River were designed. In addition, different foods were selected that could potentially contain some of the substances under study. Afterwards, analytical methods were developed and validated to determine licit and illicit drugs, pharmaceuticals and personal care products. These analytical methods are based on the following techniques: - Solid-phase extraction (SPE), solid-liquid extraction (SLE) and ultrasonic assisted extraction (UAE) - LC-MS/MS using both a triple quadrupole and quadrupole time-of-flight analysers. Finally, the developed methods were applied to the selected matrices where these compounds are susceptible to be transformed, through several degradation processes, in a wide range of unknown substances, increasing the complexity of their determination. To close the cycle of environmental pollution and how these drugs can affect humans, we must consider that many of them come from natural plants, and that, in addition to their intentional consumption, can reach humans by other ways. This Doctoral Thesis has been structured into 5 chapters. Each chapter introduces the scientific publications carried out, which present the same internal structure: introduction, materials and methods, results and discussion, conclusions, references and complementary material. The First Chapter presents a general introduction of the role that mass spectrometry can play as a tool in so-called sewage epidemiology. The review of the published literature covers the estimation of the use of licit and illicit drugs of abuse, the population estimation using biomarkers, and the study of wide-scope analytical methods to establish the pattern of these substances and their transformation products. In addition, the future prospects of mass spectrometry in this area are established. This introduction has been structured as a review article: Scientific publication 1. Liquid chromatography-mass spectrometry as a tool for wastewater-based epidemiology (sent) Chapter 2 details the development and validation of analytical methods to determine the selected substances in different environmental matrices (water, sludge and particulate matter from WWTPs, and surface waters and sediments from the Turia River basin). LC-MS/MS and UHPLC-QqTOF-MS/MS based methods were selected to analyse the selected compounds. The results obtained in this chapter are structured into three scientific publications focused on the development of methods to identify and quantify these compounds. Scientific publication 2. Simultaneous determination of traditional and emerging illicit drugs in sediments, sludges and particulate matter Scientific publication 3. Analysis of psychoactive substances in water by information dependent acquisition on a hybrid quadrupole time-of-flight mass spectrometer Scientific publication 4. Universal method to determine acidic licit and illicit drugs and personal care products in water by liquid chromatography quadrupole time-of-flight In Chapter 3, the methodologies developed in the previous chapter are applied to the analysis of WWTPs influents and effluents to determine the incidence of drugs of abuse, estimate their consumption in the metropolitan area of Valencia and establish the removal efficiency of selected WWTPs. Furthermore, the toxicity of certain drugs at different trophic levels is evaluated through theoretical studies (ECOSAR). The consumption of drugs of abuse by the population supplied by these WWTPs is established from the data obtained from the influents by applying sewage epidemiology. An alternative method based on the analysis of biomarkers in the wastewater is also proposed to estimate this population. This chapter has been structured in three scientific publications, the first two focused on the estimation of drugs and alcohol, while the third focuses on assessing the methods to estimate the population served by the WWTP, both classical methods and recently proposed biomarkers are suitable alternatives to achieve this estimation. Scientific publication 5. Occurrence and removal of drugs of abuse in Wastewater Treatment Plants of Valencia (Spain) Scientific publication 6. Estimation of alcohol consumption during “Fallas” festivity in the wastewater of Valencia city (Spain) using ethyl sulfate as a biomarker Scientific publication 7. Estimating population size in wastewater-based epidemiology. Valencia metropolitan area as a case study. Chapter 4 addresses the environmental problems raised by these compounds through an integrated study of the generated pollution. In this section, a large number of aspects of this contamination are analyzed. The relationship between the presence of these compounds and the characteristics of the population is established through the use of a Geographic Information System (GIS). The relationship between the levels of these compounds and the quality parameters of these surface waters is determined by Pearson's correlations. This chapter presents the work carried out at the University of Bath, England, during a period in which the bio- and photodegradation and the enantioselective transformation of an antidepressant such as fluoxetine were estimated in the surface waters of the Avon River and activated sludge from the WWTP corresponding to that area. This study also evaluated the toxicity of fluoxetine at different trophic levels by in vivo studies. The final part of the chapter assesses the possible impact on humans due to the consumption of different food products that could contain cannabis or its metabolites. This last problem, derived from the feeding of livestock with hemp derivatives, is a hot topic that has worried the European Food Safety Authority (EFSA), up to the point of emphasizing in a statement the need for these studies. The chapter is organized into three scientific publications covering this subject (EFSA 2011). This chapter has been structured in three scientific publications. Scientific publication 8. Assessing drugs of abuse distribution in Turia River based on geographic information system and liquid chromatography mass spectrometry (sent) Scientific publication 9. Enantioselective transformation of fluoxetine in water and its ecotoxicological relevance (sent) Scientific publication 10. Analysis of cannabinoids by liquid chromatography-mass spectrometry in milk, liver and hemp seeds to ensure food safety Chapter 5 aims to provide an overview of the results and discussion of this Doctoral Thesis. Finally, the general conclusions are gathered and several annexes are included with complementary information (Index of Abbreviations, Index of Figures and Index of Tables).