Occurrence of emerging pollutants in waste electrical and electronic equipment (WEEE) and sewage sludge from Norway

Facultad de Ciencias Trabajo Fin de Grado Grado en Química Occurrence of emerging pollutants in waste electrical and electronic equipment (WEEE) and sewage sludge from Norway Author: Daniel Gutiérrez Martín Tutor/s: Rebeca López Serna Alexandros Asimakopoulos Universidad de Valladolid Daniel Gutiérrez Martín Acknowledgments. First of all, I would like to express my very sincere gratitude to my supervisors Rebeca López Serna (UVa) and Alexandros Asimakopoulos (NTNU). Moreover, I would like to extend my great appreciation to Gabriela Castro Varela (NTNU). Rebeca, gracias por todo el apoyo, la libertad cuando me hacía falta y la rápida respuesta ante cualquier duda o sugerencia. En todos los meetings que hemos tenido, he aprendido mucho y he podido desarrollar mi pensamiento crítico. Espero que en los próximos años pueda seguir aprendiendo mucho más de ti, y obtener así una muy buena formación como químico. Alexandros, thank you for receiving me in your laboratory as an Erasmus+ student, giving me all that was needed to develop several projects, the faith you put in me and the independence I got working in your lab. Your positivism, enthusiastic encouragement and guidance were very important for me. We still have some projects to finish, and I am very glad for it. Gabi, gracias por ayudarme en cada etapa del TFG, por aconsejarme y enseñarme. Aprecio mucho todo tu conocimiento, entusiasmo y paciencia. Tu labor ha sido imprescindible para poder completar el TFG. Tienes un futuro tan brillante como merecido. Y en lo social, a pesar de los -20 °C en la calle y las escasas 6 h de luz, mi llegada a Noruega fue mucho más fácil gracias a ti. Thank you to the Norwegian Research Council (NRC) for funding this thesis, under the international project ‘’SLUDGEEFFECT: Life cycle from removing hazardous substances and plastic through thermal treatment’’ (Project Number: 302371; 2020-2023). Me gustaría también reconocer a Susana V. González y Patricia Aguilar. Susana, gracias por toda tu ayuda en el ‘‘MSlab’’, y por responderme siempre a cualquier pregunta. Patri, estuviste desde mis primeros pasos en Noruega, y me ayudaste en la parte social pero también académica. Tenemos proyectos que continuar, y me hace mucha ilusión. ¡Suerte con el PhD! Moreover, I would like to thank all the friends that I made during these months. Specially to Fer, Olga, Stefano, Martina, Jessica, Fra, Carmen… After several hours in the lab or writing, sometimes more than expected, it was cool to meet such nice people just to talk, hang out or cook some croquetas. You are also part of my thesis. Y finalmente, a mi familia. Alejarse varios meses no siempre es fácil, pero sentirme apoyado en cada decisión y etapa de la vida lo facilita todo. Ohana. Universidad de Valladolid Daniel Gutiérrez Martín Index List of abbreviations ................................................................................................................................. 1 List of Tables. ........................................................................................................................................... 5 List of Figures........................................................................................................................................... 6 Graphical abstract ................................................................................................................................... 7 Abstract ................................................................................................................................................... 7 Resumen .................................................................................................................................................. 9 1. Introduction. ................................................................................................................................. 11 1.1 Emerging contaminants. ......................................................................................................................... 11 1.2 Justification and objectives. .................................................................................................................... 11 1.3 Benzotriazoles and benzothiazoles. ......................................................................................................... 12 1.3.1 Benzotriazoles ........................................................................................................................... 13 Applications ............................................................................................................................. 13 Studied BTRs ............................................................................................................................ 13 1.3.2 Benzothiazoles. .......................................................................................................................... 14 Applications. ............................................................................................................................ 14 Studied BTHs. ........................................................................................................................... 14 1.3.3 Background................................................................................................................................ 17 1.3.4 Health and toxicology................................................................................................................. 18 1.3.5 Production ................................................................................................................................. 19 1.4 Environmental samples. .......................................................................................................................... 19 WEEE ................................................................................................................................................. 20 Definition. ................................................................................................................................ 20 Composition, classification and disposal. ................................................................................... 20 Production. .............................................................................................................................. 21 Regulation. ............................................................................................................................... 22 Sewage sludge .................................................................................................................................... 22 Definition ................................................................................................................................. 22 STPs ......................................................................................................................................... 23 Production, composition, and disposal ...................................................................................... 25 Environmental distribution ................................................................................................................. 26 1.5 Theoretical background and method selection. ....................................................................................... 27 Sample preparation ............................................................................................................................ 27 Instrumental analysis .......................................................................................................................... 29 1.6 Working plan .......................................................................................................................................... 30 2. Material and methods. ................................................................................................................. 32 2.1 Chemicals and materials. ........................................................................................................................ 32 2.2 Sampling and sample information. .......................................................................................................... 33 WEEE ................................................................................................................................................. 33 Sludge ................................................................................................................................................ 35 2.3 Sample preparation. ............................................................................................................................... 38 Universidad de Valladolid Daniel Gutiérrez Martín WEEE ................................................................................................................................................. 38 Sludge: dissolved phase ...................................................................................................................... 39 Sludge: solid and particulate matter .................................................................................................... 39 2.4 Instrumental conditions .......................................................................................................................... 39 2.5 Calculations for method validation and quantification and data analysis. ................................................. 41 2.6 Calculation of distribution coefficients. .................................................................................................... 43 2.7 Quality assurance and quality control. ..................................................................................................... 43 3. Results and discussion................................................................................................................... 45 3.1 UHPLC-MS/MS – Instrumental method validation .................................................................................... 45 3.2 Overall method validation ....................................................................................................................... 47 3.3 Occurrence of BTRs and BTHs in WEEE .................................................................................................... 49 3.4 Occurrence of BTHs and BTRs in sewage sludge. ...................................................................................... 52 3.5 Evolution of BTRs and BTHs in sewage sludge treatment. ......................................................................... 55 3.6 Mass loading of BTHs and BTRs. .............................................................................................................. 59 3.7 Sludge-water distribution coefficients (Kd). .............................................................................................. 60 4. Conclusions................................................................................................................................... 62 References. ............................................................................................................................................ 64 Appendices ............................................................................................................................................... I Appendix A. Supplementary Tables ................................................................................................................. II Appendix B. Supplementary Figures. ............................................................................................................. VII Universidad de Valladolid Daniel Gutiérrez Martín 1 List of abbreviations A 2-ABTH – 2-amino-benzothiazole 5-ABTR – 5-amino-benzotriazole ABS – Acrylonitrile, Butadiene Styrene plastic APCI – Atmospheric pressure chemical ionization AR% – Absolute recovery AREP – Advanced Recycling, Recovery, and Reuse B BTH – Benzothiazole BTHs – Benzothiazoles BTR – 1H-benzotriazole BTR-COOH – benzotriazole-5-carboxylic acid BTRs – Benzotriazoles C 2-Cl-BTH – 2-chlorobenzothiazole 5-Cl-BTR – 5-chlorobenzotriazole CLEVER – Closed Loop Emotionally Valuable E-Waste Recovery D DL – Disposable Liner E ECHA – European Chemicals Agency ECOSAR – Ecological Structure Activity Relationships ED – Endocrine disruptor EEE – Electronic and electric equipment EIP – European Innovation Partnership EPA – Environmental Protection Agency EPs – Emerging Pollutants ESI – Electrospray Ionization ESI+ – Electrospray Ionization in positive mode E-waste – Electronic waste F FAO – Food and Agriculture Organization FTIR – Fourier-transform infrared spectroscopy G GC – Gas chromatography GPC – Gel permeation chromatography H HPLC – High-Performance Liquid Chromatography Universidad de Valladolid Daniel Gutiérrez Martín 2 I ISWA – International Solid Waste Association ITU – International Telecommunication Union L LC – Liquid chromatography LLE – Liquid-liquid extraction LOD – Limit of detection logP – Logarithm of octanol-water distribution coefficient LOQ – Limit of quantification LSE – Liquid-Solid Extraction M 2-M-BTH – 2-morpholin-4-yl- benzothiazole 2-Me-BTH – 2-methylbenzothiazole 2-MeS-BTH – 2-methylthio- benzothiazole 4-Me-BTR – 4-methylbenzotriazole 5-Me-BTR – 5-methylbenzotriazole m/z – mass/charge ratio MAE – microwave-assisted extraction ME% – Matrix effect MobileRec – Collection, Disassembly and Recycling of Mobile Telecommunication Equipment) MR% – Method recovery MRM – Multiple Reaction Monitory MS – Mass Spectrometry MS/MS – Tandem Mass Spectrometry MSPD – Matrix solid-phase dispersion N NORMAN – Network of reference laboratories, research centres and related organisations for monitoring of emerging environmental substances NP – Normal phase NSES – National Strategy for Electronic Stewardship NTNU – Norwegian University of Science and Technology NTP – National Toxicology Program O 1-OH-BTR – 1-hydroxybenzotriazole 2-OH-BTH – 2-hydroxy-benzothiazole OECD – Organisation for Economic Cooperation and Development P PE – Polyethylene Universidad de Valladolid Daniel Gutiérrez Martín 3 PET – Polyethylene terephthalate PFCs – Perfluorinated compounds PFPE – Perfluoropolyether PHWW - Pressurized hot-water extraction PLE - pressurized liquid extraction PNEC – The Predicted No Effect Concentration PP – Polypropylene PS - Polystyrene PUR – Polyurethane PVC - Polyvinyl chloride Q Q1 – Transition of quantification Q2 – Transition of confirmation QqQ – Triple quadrupole QSAR – Quantitative structure-activity relationship QTOF – Quadrupole-Time of Flight QuEChERS - Quick, Easy, Cheap, Effective, Rugged and Safe R RT – Retention time RAS – Return activated sludge RMR% – Relative method recovery RP – Reverse phase S 2-S-BTH – 2-mercaptobenzothiazole 2-SCNMeS-BTH – 2-thiocyanomethyl- thiobenzothiazole SDA – Small Domestic Appliances SPE – Solid-Phase Extraction SRMs – Secondary raw materials StEP – Solving the E-waste Problem STP – Sewage Treatment Plant T TA – Target analyte TTR – tolyltriazole U UAE – Ultrasonic-assisted extraction UHPLC – Ultra High-Performance Liquid Chromatography UNCRD – United Nations Centre for Regional Development UNU – United Nations University W WAS – Waste activated sludge Universidad de Valladolid Daniel Gutiérrez Martín 4 WEEE – Waste electrical and electronic equipment WW – Wastewater WWTP – Wastewater treatment plant X XTR – Xyliltriazole  ∆E - Energy gap ΣBTHs - Summatory of benzothiazole concentrations ΣBTRs - Summatory of benzotriazole concentrations Universidad de Valladolid Daniel Gutiérrez Martín 11 1. Introduction. 1.1 Emerging contaminants. According to the NORMAN network (Network of reference laboratories, research centres and related organisations for monitoring of emerging environmental substances), emerging pollutants (EPs) can be defined as ‘’pollutants that are currently not included in routine monitoring programmes at the European level and which may be candidates for future regulation, depending on research on their (eco)toxicity, potential health effects and public perception and on monitoring data regarding their occurrence in the various environmental compartments’’ [1][2]. In 2016, the NORMAN network updated a list with more than 950 non-regulated substances which have already been detected in the environment. They are classified in families i.e., flame retardants, pharmaceuticals, industrial and household additives, pesticides and their derivatives, personal care products, endocrine disrupting compounds, biocides, surfactants as well as drugs of abuse, steroids and hormones, gasoline additives, nanomaterials, and swimming pool disinfection by-products, among others [1][3]. The study of these compounds and their health and environmental effects is required to establish suitable regulations to control their worldwide spread. 1.2 Justification and objectives. It has been widely proven that EPs constitute a risk to the environment and human health [3][4]. Therefore, the concentration levels in the environment must be limited to avoid this impact. In this project, samples of sludge from STPs and waste electrical and electronic equipment (WEEE) were analyzed. Regarding to the sewage sludge, it is paramount to monitor the presence and concentrations of EPs in STPs to assess their removal efficiency. In Europe, 50% of the produced sludge in 2019 was applied as fertilizer in agriculture, while 28% was incinerated and 22% landfilled. In the particular case of Norway, 82% of the produced sludge was used in agriculture and only 1% was incinerated [5][6]. Hence, residues from fertilizers produced from sewage sludge may contaminate aquifers and/or reach to human food, while Universidad de Valladolid Daniel Gutiérrez Martín 12 sludge incineration contributes to the climate change, and dumping constitutes an environmental management issue. Moreover, WEEE entails increasing concern too, as its disposal may introduce hazardous substances into the environment, which in combination with its high production level, could produce adverse effects to humans, wildlife and the environment [7]. The main objective of this project was to study the occurrence of different EPs in WEEE, and sludge samples collected from different recycling facilities and STPs located in Norway. In addition, the effect of different thermal treatments applied in the facilities was evaluated for the removal of the pollutants. In this context, the application of one single sample preparation was assessed for the simultaneous extraction of several families of compounds, such as parabens, phthalates, perfluorinated compounds (PFCs), bisphenols, benzophenones, benzotriazoles (BTRs) and benzothiazoles (BTHs). In addition, it is worth pointing out that the same protocol was applied to both types of samples, sewage sludge and WEEE. Such method versatility would enable a faster and more cost-effective analysis. Finally, 6 BTRs and 5 BTHs were identified and quantified for both target matrices. The determination and quantification of the other families are not included in this manuscript. However, to the best of our knowledge, this is the first study focused on the determination of BTRs and BTHs in WEEE solid samples. Once again, as far as we known, this is the first study which monitored the occurrence of 2-M-BTH and 2-Me- BTH in sludge, and which reported distribution coefficients onto sludge for 1-OH-BTR, 5-Cl- BTR and 2-M-BTH. 1.3 Benzotriazoles and benzothiazoles. Benzotriazoles (BTRs) and benzothiazoles (BTHs) are highly produced anthropogenic chemicals commonly used as corrosion inhibitors and complexing agents in a wide variety of products and industrial applications [8]. Both families have in common the presence of a nitrogen heterocyclic ring in their structure, and its structure and applications are explained below. Universidad de Valladolid Daniel Gutiérrez Martín 13 1.3.1 Benzotriazoles The original compound of benzotriazoles, and the one that confers the name to the family, is 1-H-benzotriazole (BTR) which consists of a benzene fused to a 1,2,3 triazole ring containing 3 atoms of nitrogen. It exists in two tautomeric forms (Figure 1). At room temperature, 1a is the predominant form, where the hydrogen linked to nitrogen migrate easily between the nonconsecutive nitrogen atoms conferring weak acid-base properties to the molecule [9]. Figure 1. Tautomeric forms of BTR. Applications The presence of BTR and their derivates in the environment is a consequence of their high stability and their wide use in household and industrial applications. BTRs can establish a stable coordination in copper surface, thus they have been used as corrosion inhibitors since the end of 40s [9]. In addition, they are also currently used as flame inhibitors, in de-icing and anti-icing fluids, as ultraviolet light stabilizers in plastics, as silver protectors in dishwashing agents, as antifogging agents, as pigments and in dry cleaning equipment, among other applications [10] [11] [12]. Studied BTRs A total of 8 BTRs (Figure 2) were included in the present study. In particular; BTR, XTR (or 5,6 dymethyl-1H-benzotriazole), 5-ABTR, 5-Cl-BTR, 1-OH-BTR, BTR-COOH, 4-methylbenzotriazole (4-Me-BTR) and 5-methylbenzotriazole (5-Me-BTR). The mixture of 4-Me-BTR and 5-Me-BTR is commonly known as TTR [13]. Universidad de Valladolid Daniel Gutiérrez Martín 14 Figure 2. Benzotriazole (BTR) and its derivates. 1.3.2 Benzothiazoles. Regarding to benzothiazoles, the simplest compound is formed by a benzene nucleus fused to a 1,3-thiazole ring, which consists of a five-ring structure containing a nitrogen and a sulfur atom. Their derivates are the result of adding diverse functional groups, which are generically expressed as ‘R’ in Figure 3. Figure 3. Generic molecular formula for BTH family where 'R' refers to different chemical groups. Applications. BTHs are widely used as rubber products to accelerate vulcanization and to enhance mechanical strength and abrasion resistance [14]. They are also used as corrosion inhibitors, herbicides, antialgal agents, slimicides in paper and pulp industry, photosensitizers, constituents of azo dyes, in de-icing/anti-icing fluids, antitumor reagents, fungicides in lumber and leather production [15], in pharmaceutical synthesis [16] among other applications. Studied BTHs. A total of 9 BTHs (Figure 4) were included in the present study. In particular; BTH, 2-ABTH, 2- S-BTH, 2-MeS-BTH, 2-OH-BTH, 2-M-BTH, 2-Cl-BTH, 2-SCNMeS-BTH and 2-Me-BTH. Universidad de Valladolid Daniel Gutiérrez Martín 15 Figure 4. Benzothiazole (BTH) and its derivates. Table 1 summarizes some of the BTRs and BTHs physico-chemical properties. 16 Table 1. Target analyte (TA) physico-chemical properties. Name Abbreviature CAS Formula M.W. (Da)a pKa b LogP c Benzothiazole BTH 95-16-9 C7H5NS 135.186 0.85±0.10 2.169 2-Mercaptobenzothiazole 2-S-BTH 149-30-4 C7H5NS2 167.251 9.80±0.20d 2.862 2-Hydroxybenzothiazole 2-OH-BTH 934-34-9 C7H5NOS 151.186 10.41±0.20 d 2.353 2-Aminobenzothiazole 2-ABTH 136-95-8 C7H6N2S 150.201 3.94±0.10 e 2.002 2-(Methylthio)benzothiazole 2-MeS-BTH 615-22-5 C8H7NS2 181.278 1.22±0.10 e 3.225 2-Morpholin-4-yl-benzothiazole 2-M-BTH 4225-26-7 C11H12N2OS 220.291 3.19±0.10 e 2.71 2-chlorobenzothiazole 2-Cl-BTH 615-20-3 C7H4ClNS 169.631 -0.23±0.10 e 2.814 2-(Thiocyanomethylthio)benzothiazole 2-SCNMeS-BTH 21564-17-0 C9H6N2S3 238.352 -0.09±0.10 e 3.118 2-Methylbenzothiazole 2-Me-BTH 120-75-2 C8H7NS 149.213 1.65±0.10 e 2.716 1H-Benzotriazole BTR 95-14-7 C6H5N3 119.124 8.38±0.10d 1.167 4-Methyl-1H-benzotriazole 4-Me-BTR 29878-31-7 C7H7N3 133.151 8.74±0.40 d 1.714 5,6-Dimethyl-1H-benzotriazole monohydrate XTR 4184-79-6 C8H9N3 147.177 8.92±0.40 d 2.261 Benzotriazole-5-carboxylic acid BTR-COOH 23814-12-2 C7H5N3O2 163.133 3.47±0.30 d 1.048 5-Chlorobenzotriazole 5-Cl-BTR 94-97-3 C6H4ClN3 153.569 7.46±0.40 d 1.811 1-hydroxybenzotriazole 1-OH-BTR 2592-95-2 C6H5N3O 135.123 7.39±0.58 d 0.1095 5-Aminobenzotriazole 5-ABTR 3325 11 9 C6H6N4 134.139 9.61±0.40 d 0.2499 5-Methylbenzotriazole 5-Me-BTR 136-85-6 C7H7N3 133.151 8.74±0.40 d 1.714 aSource: Chemspider.com. bSource: Scifinder. cPredicted by ECOSAR 2.0 (EPA). dStrongest acid pKa. eStrongest basic pKa. Universidad de Valladolid Daniel Gutiérrez Martín 17 1.3.3 Background BTRs are stable substances versus acids, alkalis, oxidation and reduction [17]. Consequently, they are expected to persist in water and, in general, in any environmental compartment [18]. Likewise, BTHs have been consistently identified in environmental waters in several studies as well [19][20]. Hence, recent studies reported median concentrations of BTRs and BTHs of 15.6 ng L-1 and 406 ng L-1, respectively, in tap water from 51 cities in China [21]. In addition, TTR was found in concentrations up to µg L-1, in effluent and influent wastewaters and sewage sludge [10][22][23][24]. Moreover, previous studies showed that treatments at the STPs are not efficiently removing these compounds from the wastewater, and as a result, they are continuously released into continental waters such as rivers and lakes, among others [10][23][25][26][27][28]. BTR and TTR [29][30][31] are the predominant BTRs in rivers, lakes and natural sea waters, as well as BTH [19]. In ground waters, BTR and TTR have been found in around 50% of the studied samples, in concentrations up to 1,032 and 516 ng L-1, respectively [32]. Additionally, some analysis of BTRs and BTHs in human samples such as urine, amniotic fluid or adipose tissue have been published in the literature. Hence, the reported median concentrations (µg L-1) in human urine (minimum/maximum) are summarized as follows: 1- OH-BTH (0.21/2.45); BTR (0.06/6.4); XTR (0.04/3.2); TTR (0.03/3.3); BTH (1.222/14.1); 2-OH- BTH (0.26/9.2); 2-MeS-BTH (0.24/0.33) and 2-ABTH (0.01/2.1) [11][16][33][34][35][36]. Additionally, BTH was found in amniotic fluid at median concentrations of 0.61 µg L-1, and TTR, 5-Cl-BTH and 2-OH-BTH at 0.026 µg L-1, 0.022 µg L-1 and 0.3 µg L-1, respectively [36]. Some studies have informed about the presence of 2-OH-BTH as the main BTH in adipose tissue, followed by the BTRs: TTR and XTR [37]. Regarding biota, BTHs have been observed in mollusks with concentrations between 229- 13,800 ng g-1 (dry weight), where BTH contributed with an 83.0%. Accumulated concentrations of BTRs ranged between 7.19 and 332 ng g-1, with a 78.5% predominance of XTR, 5-Me-BTR and 1-H-BTR [38]. In addition, BTR has been found in average concentrations from 40 to 75 ng g−1 in different species of fish [39] Finally, BTRs and BTHs have been monitored in indoor and outdoor dust, and 2-OH-BTH and XTR dominated in many cases [14][8][40]. Furthermore, a study of indoor dust in e-waste from a dismantling area in Qingyuan (China) showed accumulated concentrations of BTRs and BTHs Universidad de Valladolid Daniel Gutiérrez Martín 18 of 3,830 ng g-1 and 2,070 ng g-1, respectively, which are considerably higher than the concentrations found in other related dust studies [41]. 1.3.4 Health and toxicology. The National Program of the U.S. published in 1978 a bioassay of BTR for possible carcinogenicity, but the results did not show a clear evidence [42]. Later, the Committee of the Health Council of the Netherlands observed that BTR is an eye and a skin irritant, producing mutagenic effects in Salmonella typhimurium and in Escherichia Coli, and was categorized as injurious substance in case of inhalation or oral ingestion. It has been also classified as a suspected human carcinogen in previous studies [17][22]. Some other sources assured that BTHs could cause negative effects on the kidney and liver, and it is related to dermatitis and eye, skin, and respiratory irritation [43][44]. Furthermore, it is involved in microorganism mutagenicity, human cancerogenic [33] and aquatic toxicity [43]. It has also been observed that BTRs and BTHs are also toxic to luminescent bacteria, plants, and aquatic animals [45] and may have endocrine disruption properties [33]. Endocrine disruptors (EDs) are chemicals which alters the endocrine system and cause harmful effects in an organism or to its descent. There are indicators that BTR has endocrine disruptor properties which have arisen concern in the scientific community, but more research is still needed [46][47]. The predicted no effect concentration (PNEC) is defined as the concentration for which no adverse impact is expected in a particular species [48]. According to the NORMAN network, it is presented the lowest value among the PNECs predicted by QSAR for different species in freshwater (Daphnid magna and Selenastrum capricornutum) (Table 2) [1]. Additionally, the median lethal dose (LD50) for a substance is the dose required to kill half of the members in a tested population after a specified test duration. It is commonly predicted for fish and daphnia by the Ecological Structure Activity Relationships (ECOSAR). These parameters have been estimated for the target analytes (TA) in the present study and they are shown in Table 2. Universidad de Valladolid Daniel Gutiérrez Martín 19 Table 2. Toxicity parameters: Predicted LD50 (mg L-1) in fish and Daphnid (ECOSAR model - EPA) and lowest PNEC (µg L-1) for aquatic species. BTH 2-S-BTH 2-OH-BTH 2-ABTH 2-MeS-BTH LC50 Fish (mg L-1) 78.3 1.57 3.79 16.2 11.8 LC50 Daphnid (mg L-1) 45.2 0.336 7.22 1.56 7.52 Lowest PNEC (µg L-1) 30 0.76 14 1 1 2-M-BTH 2-Cl-BTH 2-SCNMeS-BTH 2-Me-BTH BTR LC50 Fish (mg L-1) 41.7 25.9 0.024 27.9 40.7 LC50 Daphnid (mg L-1) 25.3 15.9 0.028 16.9 244 Lowest PNEC (µg L-1) 14 1.14 0.38 1.92 7.77 XTR BTR-COOH 5-Cl-BTR 1-OH-BTR 5-ABTR LC50 Fish 11.3 655 21.8 195 160 LC50 Daphnid (mg L-1) 37.6 4180 92.3 2060 1,530 Lowest PNEC (µg L-1) 4.94 16.9 3.57 - a - a aNot found. 1.3.5 Production Currently, the global production of BTRs reaches 9,000 tonnes year-1 approximately [49]. The Environment and Climate Change Canada Health has recently published a ‘Draft Screening Assessment of the Benzotriazoles and Benzothiazoles Group’ including the total import of TTR and BTR in 2014 or 2015, reporting between 10,000 and 100,000 Kg of each compound per year [50]. The US National Toxicology Program (NTP) regularly summarizes the production of BTH from different sources. Hence, in 1993, United States produced between 4,500 and 45,000 Kg, and from August 1995 to October 1996, the import of BTH reached 203.9 Kg [51]. According to the European Chemicals Agency (ECHA), BTH is annually manufactured and imported to the European Economic Area between 10 to 100 tonnes [52]. 1.4 Environmental samples. In the present study, the occurrence of BTRs and BTHs was investigated in sewage sludge and WEEE samples collected in different areas of Norway. Information about the composition, regulation, production, and origin of these matrices is shown below. Universidad de Valladolid Daniel Gutiérrez Martín 20 WEEE Definition According to the European Union Law [53] electric or electronic equipment (EEE) can be defined as ‘‘equipment which is dependent on electric currents or electromagnetic fields in order to work properly, and equipment for the generation, transfer and measurement of such currents and fields which is designed for use with a voltage rating not exceeding 1,000 volts for alternating current and 1,500 volts for direct current’’. The same system of rules defines the waste electrical and electronic equipment (WEEE) as ‘‘electrical or electronic equipment which is waste within the meaning of Article 3(1) of Directive 2008/98/EC, including all components, subassemblies and consumables which are part of the product at the time of discarding’’. And within the Article 3(1) of Directive 2008/98/EC, waste is defined as ‘‘any substance or object which the holder discards or intends or is required to discard.’’ Similarly, electronic waste (e-waste) can be defined as any discarded device containing electronic circuits, such as transistors, capacitors, resistors, etc. They include domestic appliances (refrigerators, stoves, etc) and other electric apparatus, even if they are mainly made of plastics or steel [7]. However, in the recycling industries, the different components of e-waste are usually separated into fractions such as plastics, cables, metals, batteries, … Even if WEEE and e-waste present slight differences (i.e., e-waste always contains electronic circuits and WEEE includes all components), it is usually used indistinctly in the literature. Composition, classification and disposal It has been estimated that around 8% of the total waste corresponds to WEEE [54]. The components of WEEE may contain dangerous substances for the environment and health, but also precious materials. Steel and iron, non-flame retarded plastics and other metals constitutes the most important groups of materials and the separation of the components is highly expensive [7]. As a consequence, recycling is complicated but necessary. A classification of WEEE has been accomplished by the European Union, resulting into the following categories[55]:  Huge domestic electric appliances  Small domestic electric appliances  Information technology equipment Universidad de Valladolid Daniel Gutiérrez Martín 27 industries increase the amount of these kind of residues in the environment. Figure 9 shows the environmental distribution of EPs (applied to WEEE and sludge from STPs). Figure 9. Environmental distribution of emerging contaminants. Applied to WEEE and sludge from STPs. 1.5 Theoretical background and method selection. Many analytical methodologies for the analysis of EPs in different environmental matrices have already been developed by several research labs around the world. In all of them, matrix effect has entailed a challenge to face, as interferes in the analysis performance, especially in complex matrices such as sewage sludge and WEEE. Thus, a pre-treatment step is commonly carried out prior to the instrumental analysis. The most common techniques for related sample preparation and instrumental analysis in sewage sludge are reviewed below. However, due to its novelty, little information is available in the scientific literature in the case of WEEE. Additionally, the main advantages and disadvantages are presented. Sample preparation Sample preparation is the most important step during the development of the analytical methodology as it is responsible for removing undesirable compounds from the matrix which may interfere with the target analytes during the analysis. The first pretreatment step may Universidad de Valladolid Daniel Gutiérrez Martín 28 consist of a water removal. Centrifugation, decantation, air-drying and heating are options frequently used, but sample lyophilization is probably the most common to avoid uncontrolled evaporations or degradations [79]. Then, a liquid-solid extraction (LSE) usually takes place. LSE is not analyte-selective, and thus, a subsequent clean-up step is usually required [80]. LSE makes use of an appropriate solvent to extract the target compounds from a solid matrix. Temperate as well as type and volume solvent(s) have been reported to play a role in the analyte extraction selectivity [80]. Some of the most common extraction techniques used in the analysis of EPs in sewage sludge are the followings: soxhlet (or the automated soxhlet), UAE (ultrasonic-assisted extraction), MAE (microwave-assisted extraction), PLE (pressurized liquid extraction), MSPD (matrix solidphase dispersion), PHWE (pressurized hot-water extraction) or QuEChERS (quick, easy, cheap, effective, rugged and safe) [81]. In the case of BTRs and BTHs, PLE, UAE, QuEChERS, PHWE or other conventional methods have been reported to be the most suitable for sewage sludge [82]. Solid phase extraction (SPE), gel permeation chromatography (GPC) or liquid-liquid extraction (LLE) are clean-up techniques which have been used after the extraction step described above. They are usually efficient in the removal of undesirable substances such as lipids, surfactants, etc. present in the sewage sludge matrix. SPE is a chromatographic extractive technique and has been reported to be the most commonly used to achieve this purpose in the sample extract [81]. Usually, it is performed in a cartridge with a solid phase (sorbent) whose properties and quantity determine the efficiency of the extraction. Due to the polarity of BTHs and BTRs, polymeric balanced polar/non-polar sorbents are used [82]. An initial cartridge activation and conditioning is required to eliminate impurities and optimize conditions in the cartridge (Figure 10). Usually, it is carried out by passing the same solvent that constitutes the sample. Then, the sample is passed through the sorbent which ideally retains the target analytes but not the interferences. After the selective retention, a cartridge rinsing and drying is usually conducted. Then, the elution of compounds is performed with an appropriate volume of a selected solvent. This clean-up protocol contributes to an extract concentration too, which is beneficial when working with low concentrated analytes, as it might be the case [80][83]. Universidad de Valladolid Daniel Gutiérrez Martín 29 Figure 10. Common steps in SPE. Instrumental analysis Liquid chromatography (LC) or gas chromatography (GC) coupled with mass spectrometry (MS) has been the predominant instrumental analysis techniques in the last years for analysis of EPs. GC requires analyte volatilization, which is not easy to achieve in the usually polar EPs without thermal degradation taking place. Derivatization may solve this limitation in some cases, but it entails additional sample manipulation. Thus, LC has been preferred over GC because of its versatility. [81][82] In LC, the analytes are separated because of their different affinity between the mobile and the stationary phases, this latter usually supported in a column. Partition is the most common mechanism used in this analysis, and the analytes achieve separation by differential solution in the stationary and mobile phase, depending on their polarity and the chromatographic phase polarities. Analytes should hold polarities amidst the mobile and the stationary phase polarities, which will be far apart. Partition LC may be applied in two different modes, reverse phase (RP) and normal phase (NP), depending on the mobile phase polarity (and therefore stationary phase one too). Despite NP should be preferred for polar compounds, RP stationary phases usually deliver more precise chromatograms and therefore, RP is usually the preferred mode when possible. High-performance liquid chromatography (HPLC) enables an efficient separation by using packed columns with particle sizes of around 5 µm and high-pressure pumps. Additionally, smaller stationary-phase-particle sizes increase the peak resolution and the analyte sensitivity in the chromatograms. Nevertheless, it comes with an increasing instrument pressure. The development of UHPLC systems overcame this limitation, enabling the use of stationary phase particles <2 µm, which allowed the reduction of the time of the analysis as well as the requirement of solvent volumes [84]. Universidad de Valladolid Daniel Gutiérrez Martín 30 Both LC and GC are separation techniques and need to be coupled to a detection instrumental technique to transduce a physical property related with the analyte concentration into an electric signal. Spectroscopic techniques such as UV-Vis or fluorescence were proposed as valid options at first. However, both sensitivity and selectivity are improved when mass spectrometry (MS) is used instead. In fact, mass spectrometry is the main detection technique in environmental samples, where the analytes are present at low concentration in very complex matrices [82]. In particular, in target analysis, tandem mass spectrometry (MS/MS) with a triple quadrupole analyzer is normally used in multiple reaction monitoring (MRM) mode. This consists of selecting a parent compound per analyte in the first quadrupole, which is fragmented in the collision cell and then, select a daughter ion to quantify. Both quadrupoles allows the transmission of a very narrow window of masses around the parent and daughter ion’s, so it is very specific and sensitive [85]. In environmental samples, at least an extra qualification transition is usually needed to confirm. However, one of the main drawbacks of this approach is that it is completely blind to any other substances present in the sample. Other proposals include the detection of other molecules apart from the target analytes as they perform full mass scan by using high-resolution mass spectrometry with a quadrupoletime of flight spectrometer (QTOF) or an Orbitrap, for instance. In contrast, in these conditions, sensitivity and dynamic range decrease. Regardless, LC-MS/MS is heavily affected by matrix effect, both ion suppression and enhancement, and entails a challenge, especially when analysing traces in complex environmental samples such as sewage, sludge, etc. [82][81]. To produce ionization, first and for most electrospray ionization (ESI) but also atmospheric pressure chemical ionization (APCI) are typically used in both positive and negative ion mode [81]. ESI consist of applying a high voltage at atmospheric pressure onto an aerosol made of the mobile phase dissolving the analytes after chromatographic. 1.6 Working plan The experimental analytical work was accomplished in the Norwegian University of Science and Technology (NTNU) under an Erasmus+ scholarship program. Firstly, an evaluation of the sample pretreatment was assessed. Once the optimal conditions were selected, the whole method was validated. Finally, the developed methodology, based on UAE-LSE and SPE followed by UHPLC-ESI-MS/MS, was applied to real samples of WEEE and Universidad de Valladolid Daniel Gutiérrez Martín 31 sewage sludge. Subsequent data processing of the resulting chromatograms was carried out and statistical treatment followed. During the whole period, databases such as SciFinder and Scopes were checked for scientific literature. Finally, besides this present manuscript, at least one related scientific paper is expected to be published too. Universidad de Valladolid Daniel Gutiérrez Martín 32 2. Material and methods. 2.1 Chemicals and materials. Analytical standards (purity) of 2-MeS-BTH (97%), 2-S-BTH (97%), 2-OH-BTH (98%), XTR (≥99%), BTH (≥97.0%), BTR-COOH (99%), 2-Me-BTH (99%), 2-ABTH (97%), 2-Cl-BTH (99%), 1- OH-BTR hydrate (≥97.0%), 5-Cl-BTR (99%), 5-Me-BTR (98%), 2-M-BTH (AldrichCPR grade), 5- ABTR (AldrichCPR grade) were purchased from Sigma Aldrich (Steinheim, Germany). 2-SCNMeS- BTH (98%) was purchased from Advanced ChemBlocks (Burlingame, USA). Labelled compounds used as internal standards (IS), 1H-Benzotriazole-(ring-d4) solution (10 µg mL-1 in acetone) (BTR-d4) was obtained from Sigma Aldrich and 5-methyl-1H-1,2,3-benzotriazole-d6 (4,6,7-d3 methyl-d3) solution (100 µg mL-1 in methanol) (5-Me-BTR-d6) was purchased from Chiron (Trondheim, Norway). A standard solution mixture (1 mg L-1) of all target analytes was prepared in methanol and stored at -20 °C for a maximum of a month. The same concentration was prepared for a solution of IS. The following solvents (purity) were purchased in VWR chemicals (BDH PROLABO®, Fontenaysous-Bois, France): methanol (≥99.8%) (MeOH) and acetonitrile (ACN) (≥99.9%) of LC-MS grade. Formic acid (FA) (≥96%) and hydrochloric acid (HCl) were acquired from Sigma Aldrich (Steinheim, Germany). Deionised water (Milli-Q grade water) was in-house obtained by a purification system (MILLIPORE S.A., Molsheim France). Glass microfiber filters GF/F diameter 47 mm (WhatmanTM, Middlesex, U.K.) with 0.45 µm of pore size. They were used to filter some sludge samples and to accumulate particulate matter from these samples. An ALPHA 1-4 LD plus freeze dryer (Martin Christ; Osterode, Germany) was employed for dewatering solid sludge and particulate matter samples. An automated solvent evaporation system (TurboVap® LV) from Biotage (Charlotte, U.S.A.), a Centrifuge 5810 (Eppendorf, Hamburg, Germany), a 3510 Ultrasonic Cleaner from Branson (Danbury, USA), a Vortex shaker (VWR, Bruchsal, Germany) and VWR® centrifuge tubes of 15 and 50 mL volume, relative centrifuge force (RCF) 12,500 g were used during the sample pretreatment. Universidad de Valladolid Daniel Gutiérrez Martín 33 For the clean-up step, a 24-port model Visiprep-DL Solid Phase Extraction vacuum manifold was purchased from Supelco (Bellefonte, PA, USA), as well as disposable flow control valve liners for the VisiprepTM. SPE cartridges, StrataTM-X 33 µm Polymeric Reversed Phase 200 mg / 6 mL were obtained from Phenomenex (Aschaffenburg, Germany). 2.2 Sampling and sample information. WEEE A total of 15 samples of WEEE and plastic materials were collected in Norway from different facilities. They were coded as R1 to R9 and Q1 to Q6. Each sample and the process of collection is described below and summarized in Table 3. R1-R9 samples were secondary raw materials (SRMs) arising from electronic waste collected in Norway and sieved after collection (<2 mm). SRMs consist of recycled materials that can be injected again into the economy [86]. The sampling date was December 2020. These SRMs are separated into different fractions which are described in Table 3. R1, R2 and R3 consist of small domestic appliances (SDA) which were separated according to the main kind of plastic they are made of i.e., acrylonitrile butadiene styrene (ABS), polypropylene (PP) polyethylene (PE) or polystyrene (PS). R4, R5 and R6 were the plastic components of different temperature exchange equipments (e.g., fridges). R7 was the reject fraction of SDA. Likewise, R8 is the reject fraction of R4, R5 and R6. On the other hand, R9 was an agglomerate of polyurethane (PUR) principally. Q1 consisted of a mixture of PVC (polyvinyl chloride) and other plastics, collected from a Norwegian WEEE recycler in 2017. Q3 was polyethylene terephthalate (PET) plastic from drinking water bottles, and Q4 is ground PE and PP from bottle caps, both collected in October 2020. An innovating process was tested for biomass valorisation and calorific fraction of municipal solid waste. A mixture of Q3 and Q4, in proportions 9:1, was introduced in a hopper and then pyrolyzed (800 °C). The generated gas was, then, condensed as an oil and water. The resulting solid phase from the pyrolysis was cooled and collected as a sample (Q2). The plastics Q5 and Q6 were manually removed from the digested food waste in December 2020 and then, sieved. This could potentially be used as biofertilizer, but the presence of high amount of plastic entails a limitation. Q5 was mostly PE from green food waste bags collected in Oslo. Q6 Universidad de Valladolid Daniel Gutiérrez Martín 34 was mainly made of biodegradable plastic from food waste bags (more than 50%) and the rest of the packaging (mix of PP, PS, etc.). Table 3. WEEE samples. Code, category, mainly composition and image. Sample code Waste category Main plastic type Image R1 Small equipment ABS R2 Small equipment PP/PE R3 Small equipment PS R4 Temperature exchange equipment ABS R5 Temperature exchange equipment PE/PP R6 Temperature exchange equipment PS R7 Small equipment Mixed grinds R8 Temperature exchange equipment Mixed grinds R9 Temperature exchange equipment/large equipment PUR Q1 Norwegian WEEE recycler PVC and others Q2 Char from pyrolysis Q3/Q4 (9:1) PET/PE/PP Q3 Bottles PET Q4 Bottle caps PE/PP Q5 Plastic from food waste (bags) PE Q6 Plastic from food waste (bags and packaging) PE and others Samples Q2 to Q6 were included in the study due to a thermal analysis is being tested to see the removal efficiency of this EPs and to assess the thermochemical conversion of plastic. Universidad de Valladolid Daniel Gutiérrez Martín 35 Once the thermal analysis is probed with plastics from bottles, cap bottles and food waste plastics, it will be extended to WEEE samples due to the similarity of the materials. Additionally, this project included other families of EPs whose occurrence in these plastic samples (Q2 to Q6) was particularly interesting. Sludge Twenty-five grab samples from 3 different STPs in Norway have been collected in glass containers after different steps of the treatment. Then, when possible, samples were filtered through a glass microfiber filter GF/F with 0.45 µm of pore size under vacuum. The liquid fractions were kept at 4 °C and the filters were frozen before freeze-drying (-21 °C, 6 mbar) and conserved at room temperature until analysis. This last fraction is considered as the particulate phase. Those thick samples which filtration was not possible were directly frozen and freeze-dried. Once lyophilized, the samples were stored in aluminium containers at room temperature and in darkness until analysis. Information regarding each STP, sampling and conservation conditions are summarized below.  STP-1. Figure 11. Simplified scheme of the sludge treatment carries out in STP-1 and STP-2. Sampling points are specified with numbers 1, 2 and 3 for raw sludge, post-pasteurized sludge and digested sludge, respectively. STP-1 receives mainly industrial wastewater (up to 50%) as well as urban wastewater corresponding to 120,000 population equivalents. The influent wastewater undergoes flocculation (ClFeO4S and polyamine) and sedimentation. First sludge (after a first clarifier) and secondary sludge (after a second clarifier) are mixed. Afterwards, the sludge is thickened and then, follows a progressive heating to start the decomposition and submit a Universidad de Valladolid Daniel Gutiérrez Martín 36 pasteurization treatment ( 65 °C) for at least 1h. The resulting product is cooled up to 40 °C and digested under anaerobic and mesophilic conditions for at least 15 days. A reduction of a third of the sludge volume occurs, and biogas is produced, which is collected and used in energy production. After digestion, the sludge is dewatered by centrifugation and the resulting product is used as fertilizer to enrich agricultural soil. A simplified scheme is shown in Figure 11. A 3-days sampling season took place in November 2020. A sample from raw sludge, post-pasteurization sludge, and digested and dewatered sludge was collected each day in glass bottles and kept at 4 °C until analysis. Table 4 summarizes the samples collected from STP-1. Table 4. Sludge samples from STP-1. Sludge Phase Date Abbreviation Raw Particulate 11.11.2020 R_(1)_P_1 Raw Dissolved 11.11.2020 R_(1)_L_1 Raw Particulate 12.11.2020 R_(1)_P_2 Raw Dissolved 12.11.2020 R_(1)_L_2 Raw Particulate 13.11.2020 R_(1)_P_3 Raw Dissolved 13.11.2020 R_(1)_L_3 Post pasteurization Particulate 11.11.2020 P_(1)_P_1 Post pasteurization Dissolved 11.11.2020 P_(1)_L_1 Post pasteurization Particulate 12.11.2020 P_(1)_P_2 Post pasteurization Dissolved 12.11.2020 P_(1)_L_2 Post pasteurization Particulate 13.11.2020 P_(1)_P_3 Post pasteurization Dissolved 13.11.2020 P_(1)_L_3 Digested Solid 11.11.2020 D_(1)_S_1 Digested Solid 12.11.2020 D_(1)_S_2 Digested Solid 13.11.2020 D_(1)_S_3  STP-2 The same sampling strategy followed for STP-1 was performed in STP-2. Sludge is produced and treated similarly in STP-2 too (Figure 11) with small differences i.e., polyacrylamide is used as flocculant. It has been designed to have a capacity for 170,000 population equivalents. The sludge before and after decomposition reaches around 7,600 kg and 4,900 of dry matter per day, respectively, and 5,000 tonnes of dehydrated sludge is produced per year. Three samples from each sampling site in the STP-2 were collected in 3 consecutive days in December 2020. One litre of raw sludge (semi-liquid) and post-pasteurized sludge (semi- Universidad de Valladolid Daniel Gutiérrez Martín 43 assigned IS perform a satisfactory correction of the method deficiencies and the final method trueness is appropriate. Regarding data analysis, MassLynx and TargetLynx softwares (Waters, Milford, U.S.) were employed for the obtention of data from the LC-MS/MS and data treatment. Excel (Microsoft, 2010) was used to perform data analysis. 2.6 Calculation of distribution coefficients. The sorption of the different studied compounds onto the sludge was estimated by the calculation of the distribution coefficients (Kd) as shown in the equation (5): 𝐾𝑑=𝐶𝑠 𝐶𝑤 where Cs is the concentration of the compound in the particulate matter phase and Cw is the concentration of the compound in the dissolved phase. For quantification purposes, detected compounds with values under the LOQ have been substituted by the LOQ/2 [87]. Resulting Kd values indicated the tendency of each compound to adsorb into the solid phase. 2.7 Quality assurance and quality control. Quality assurance and quality control (QAQC) measures were required to avoid any potential contamination during sample preparation and analysis. Thus, all glass material was cleaned with distilled water (n=3) and rinsed with MeOH (n=3) before use. Any contamination coming from the laboratory or solvents was corrected with procedural blanks submitted to the same procedure as the samples and injected during the analysis of the real samples. All the samples were fortified with a mixture of labelled IS to account for any signal losses during sample preparation and instrumental analysis. Reagent blanks, i.e., blank samples without the matrix, spiked with IS (20 µg L-1), were also performed, and its signal (area) was subtracted from all the samples and blanks, assuming to be contamination and background noise in all samples within the batch. (5) Universidad de Valladolid Daniel Gutiérrez Martín 44 During the instrumental analysis, an external standard solution at a concentration of 10 µg L- 1 in MeOH:deionized water followed by a MeOH solvent were analyzed every 15 injections to determine the instrumental repeatability, possible carry over, as well as potential retention time drifting. Internal standards were used as reference during the QAQC tests too. Universidad de Valladolid Daniel Gutiérrez Martín 45 3. Results and discussion 3.1 UHPLC-MS/MS – Instrumental method validation Two MRM transitions per compound were recorded. The transition providing the highest response was selected for quantification purposes (Q1). Nevertheless, the second most intense transition (Q2) was selected for confirmation. This is usually needed in environmental samples due to the low concentrations and the possible effects of the matrix. The ratio between the area of Q1 and Q2 is the ion ratio and is used for confirmation purposes too (Table 8). The first peak which fulfil the requirements of ion ratio and linearity is considered as the instrumental limit of quantification (iLOQ), and instrumental limit of detection (iLOD) is calculated as iLOQ/3. Herein, iLOQs ranged from 0.05 to 5 µg L-1 in all TA (Table 8). Such low concentrations are possible due to the high sensitivity associated to UHPLC-ESI-MS/MS, and are absolutely paramount to efficiently determine EPs in environmental samples, as they are, both present and still harmful, at trace levels. Moreover, acidification prior analysis improved the retention in the chromatographic column resulting in better peak shape (Figure S1, Appendix B) and consequently, detection became easier for the lowest concentrations. Table 8. Instrumental limits of quantification and detection (µg L-1), RT and ion ratio. Compound iLOQ (µg L-1) iLOD (µg L-1) RTa Ion ratioa (RSD%) 1-OH-BTR 0.2 0.07 0.76 17.3 (5) 2-ABTH 0.1 0.03 0.91 84.5 (4) 2-Cl-BTH 5.0 1.67 2.86 43.1 (12) 2-M-BTH 0.05 0.02 2.90 94.3 (1) 2-Me-BTH 1.0 0.33 2.94 19.3 (29) 2-MeS-BTH 0.5 0.17 3.15 13.7 (29) 2-OH-BTH 2.0 0.67 2.70 92.5 (30) 2-S-BTH 0.2 0.07 2.87 54.1 (7) 2-SCNMeS-BTH 1.0 0.33 3.18 218 (1) 5-ABTR 0.1 0.03 0.29 28.3 (2) 5-Cl-BTR 0.05 0.02 2.67 18.0 (2) 5-Me-BTR-d6 - - 2.50 127 (2) BTH 5.0 0.33 2.78 36.7 (30) BTR 0.1 0.03 1.57 36.6 (4) BTR-COOH 2.0 0.67 1.18 73.0 (5) BTR-d4 - - 1.53 59.1 (4) TTR 0.05 0.02 2.51 41.8 (2) XTR 0.1 0.03 2.74 85.9 (1) aCalculated as average at three fortification levels (5, 10, 20 µg L-1) in external calibration curve. Universidad de Valladolid Daniel Gutiérrez Martín 46 The RT for each compound is presented in Table 8. The ratio between the RT of a compound and the RT of the IS is known as relative retention time (RRT). The RT of a compound may vary during the analysis because of the matrix but also due to the chromatographic parameters. As the RT of the IS should vary in a similar proportion as the RT of the analyte, drifting times will be compensated in the RRT. Ranges for the RRTs ± 0.1 were established in the external calibration curve and in the spikes (before and after extraction). Analytes in samples with RRTs out of that range were considered as non-detected. Precision was studied though the injection of a mixture of 10 µg L-1 standard along the sequence intra-day (repeatability) and inter-day (reproducibility) (Table 9). Linearity of the calibration curve was considered between the iLOQ and the maximum concentration which conserved the linearity. A coefficient of determination (R2) close to 1 was used to prove the linearity in this range. Excellent linearity was observed with R2 higher than 0.99 for all analytes except for 2-Cl-BTH, 2-OH-BTH and 2-SCNMeS-BTH (Table 9). Precision, linear range and R2 of the calibration curve are shown in Table 9. Linear equations and calibration curves of each compound are shown in Figures S2 – S17 in the Appendix B. Table 9. Precision and calibration parameters for BTHs and BTRs. Precision Linear range c R2 RSD% Intra-day a RSD% Inter-day b (µg L-1) 1-OH-BTR 17 13 0.2 - 20 0.9968 2-ABTH 17 16 0.1 - 20 0.9952 2-Cl-BTH 19 30 5.0 - 20 0.9745 2-M-BTH 6 26 0.05 - 20 0.9955 2-Me-BTH 15 24 1.00 - 20 0.9905 2-MeS-BTH 11 27 0.5 - 20 0.9933 2-OH-BTH 13 31 2 - 20 0.9859 2-S-BTH 15 26 0.2 - 20 0.9909 2-SCNMeS-BTH 13 23 1 - 20 0.9763 5-ABTR 15 28 0.1 - 20 0.9959 5-Cl-BTR 7 20 0.05 - 20 0.9971 BTH 21 23 5 - 100 0.9919 BTR 2 8 0.1 - 20 0.9967 BTR-COOH 22 16 2.0 - 20 0.9957 TTR 3 23 0.05 - 20 0.9960 XTR 6 16 0.1 - 20 0.9943 aN=7. b3 days, N=20 except BTH (2 days, N=12). cRange considered from iLOQ to the maximum value in the calibration with linearity. Universidad de Valladolid Daniel Gutiérrez Martín 47 3.2 Overall method validation Obtained recoveries and matrix effects (except for BTH) are compiled in Table 10 and Table 11, for WEEE and sludge, respectively, at a 20 µg L-1 fortification level. Recoveries and matrix effects for the other fortification levels studied are presented in the Appendix A (Table S1-S4). The proposed methodology was suitable for the extraction of 11 analytes out of the 16 studied. Compounds which are not included in Tables 10 and 11 presented high RSD% or the extraction was not good enough. Hence, 2-Cl-BTH, 2-OH-BTH, 2-S-BTH, 2-SCNMeS-BTH and 5- ABTR were proposed to be ruled out of the analytical method in both matrices. AR%, ME%, MR% and RMR% are presented in Tables S5 and S6 for those compounds, as well as a tentative explanation about their lack of performance. Table 10. Absolute recoveries (AR%), matrix effects (ME%), method recovery (MR%) and relative method recovery (RMR%) for a fortification level of 20 µg L-1 in WEEE samples. Compound AR% (RSD%) ME% MR% (RSD%) RMR% (RSD%) 1-OH-BTR 78 (6) -4 75 (6) 100 (18) 2-ABTH 67 (2) -31 44 (3) 59 (10) 2-M-BTH 139 (6) -68 43 (7) 74 (13) 2-Me-BTH 80 (3) -35 49 (3) 82 (17) 2-MeS-BTH 53 (26) -7 44 (31) 74 (46) 5-Cl-BTR 104 (1) -50 52 (1) 69 (12) BTR 60 (8) 14 63 (9) 84 (21) BTR-COOH 99 (2) -25 74 (2) 99 (10) TTR 66 (3) -10 57 (3) 77 (15) XTR 101 (4) -51 49 (4) 65 (16) Table 11. Absolute recoveries (AR%), matrix effects (ME%), method recovery (MR%) and relative method recovery (RMR%) for a fortification level of 20 µg L-1 in sewage sludge samples. Compound AR% (RSD%) ME% MR% (RSD%) RMR (RSD%) 1-OH-BTR 76 (2) -4 70 (2) 90 (6) 2-ABTH 75 (2) -12 65 (2) 84 (6) 2-M-BTH 75 (2) -53 35 (2) 71 (5) 2-Me-BTH 61 (3) 3 64 (3) 129 (8) 2-MeS-BTH 42 (8) 29 24 (19) 49 (22) 5-Cl-BTR 72 (2) -42 41 (2) 53 (7) BTR 78 (3) 90 72 (7) 92 (4) BTR-COOH 88 (7) 30 105 (8) 135 (2) TTR 171 (11) 75 116 (29) 149 (31) XTR 89 (12) -20 40 (20) 51 (27) Universidad de Valladolid Daniel Gutiérrez Martín 48 Absolute recoveries remained above 59% for all the compounds with the exception of 2-MeS- BTH, which were below 53% in both matrices (Tables 10 and 11). On the other hand, AR% for 2-M-BTH in WEEE and TTR in sludge were above 100%. Even though some of these values are not in the range of 70-130%, they were really encouraging due to the number of compounds analyzed with the same sample preparation applied to two completely different matrices. Additionally, the lack of accuracy provided for some of the AR% values was overcome with a suitable correction with the ISs. Regarding ME%, in WEEE, most of the compounds suffered suppression (ME% < 0) during the ionization in the ESI source. Potential matrix effects during the ionization were taking into consideration during the quantification through the preparation of samples spiked before the sample preparation. The MR% for WEEE and sludge (fortification level: 20 µg L-1) ranged between 40 – 116% except for 2-M-BTH and 2-MeS-BTH in sludge. However, results of MR% showed that external calibration was not an option to quantify the concentration of these compounds (Figure S18). Regarding RMR%, values between 59-100% were obtained in WEEE at a fortification level of 20 µg L-1 with excellent RSD% except for 2-MeS-BTH. The range in sludge was wider, again, due to the complexity of the matrix. Sensitivity of the method was evaluated with the method limit of quantification (mLOQ) and method limit of detection (mLOD), calculated for a nominal mass of 0.1 g (Table 12). Table 12. Sensitivity of the method (ng g-1). Compound mLOQ mLOD Compound mLOQ mLOD Compound mLOQ mLOD 1-OH-BTR 2.0 0.67 2-OH-BTH 20 6.7 BTH 50 17 2-ABTH 1.0 0.33 2-S-BTH 2.0 0.67 BTR 1.0 0.33 2-Cl-BTH 50 17 2-SCNMeS-BTH 10 3.3 BTR-COOH 20 6.7 2-M-BTH 0.50 0.17 5-ABTR 1.0 0.33 TTR 0.5 0.17 2-Me-BTH 10 3.3 5-Cl-BTR 0.50 0.17 XTR 1.0 0.33 2-MeS-BTH 5.0 1.7 BTH was suspected to be degraded in the stock solutions, as it was not detected in any spiked sample at any fortification level. A newly fresh stock solutions was prepared confirmed it. Additionally, the presence of two peaks in the samples and the absence of spikes in the matrix for BTH aroused the necessity of determinate which one was the peak of BTH. It was proved Universidad de Valladolid Daniel Gutiérrez Martín 49 with the addition of BTH just before the analysis, in a composite sample which has undergone the complete protocol, at 3 fortification levels and its analysis (Figure S19). The diverse matrix and families of EPs made a complicated task to extract all the analytes and reduce the matrix effect with a same methodology. However, this method showed acceptable recoveries and matrix effects for a high number of compounds. 3.3 Occurrence of BTRs and BTHs in WEEE Fifteen WEEE samples were analyzed, and the concentrations determined for both families, BTRs and BTHs, are shown in Table 13. BTR and TTR were found in all the samples with concentrations ranged between 2.5 – 410 ng g-1 and <LOQ - 46 ng g-1, respectively. Regarding the BTHs, the detection rate (DR%) for 2-M-BTH and BTH reached a 100% and BTH was the one found at highest concentration ranging between 85 and 8,831 ng g-1 (Table 13). Nevertheless, 2-M-BTH presented lower concentrations (median: 4.4 ng g-1). 2-ABTH was found at concentrations between 1.0 and 429 ng g-1 in all samples except in Q3. On the other hand, BTR-COOH was detected in only 1 out of 15 samples, and under the limit of quantification. Regarding samples coded as R1-R9, which were collected in the same facility, R8 presented the highest concentrations of BTRs (ΣBTRs = 496 ng g-1) whose main contributor was BTR with a concentration of 302 ng g-1 (Table 13). Nonetheless, all the target compounds, apart from BTR-COOH and 2-MeS-BTH, were identified in this sample. On the other hand, the highest concentrations of BTHs in these samples were found in R3 and R7, presenting ΣBTHs > 1700 ng g-1. It is worth pointing out that the concentration of BTH was remarkably high in all the studied samples. This was probably due to the high production and use of this chemical during the production of WEEE. In addition, BTH is also a common by-product of many other BTHs non studied in this study, such as 2-OH-BTH. Samples R1, R2 and R3 were constituent of electronic equipment with domestic appliances. The three samples showed similarities in the pattern of detected BTRs and BTHs. However, BTH was quantified in R3 at a higher concentration. Sample R7 was the reject fraction carried out before the plastic separation in R1, R2 and R3, and generally presented a higher content of ΣBTRs and of ΣBTHs than in those, except for R3 due to its high content of BTH. A similar comparison may be done for R4, R5, R6 Universidad de Valladolid Daniel Gutiérrez Martín 50 and its rejected fraction, i.e., R8. In this case, similar occurrence of ΣBTHs was found for all these samples. Nevertheless, the content of BTRs in R8 was considerably higher. In regard to Q samples, Q3 and Q4 came from bottles and cap bottles, respectively, and the occurrence of BTRs and BTHs were predominant in the caps. Pyrolysis was suspected to reduce the content of 2-M-BTH, 2-MeS-BTH, BTH, BTR and TTR, as their concentrations were clearly higher in Q3 y Q4 than the ones found in Q2, which was the char resulting of the pyrolytic treatment of a mixture of Q3 and Q4 (9:1). In fact, based on those concentrations, removal efficiencies (RE%) ranging between 84 – 100%, could be attributed to this treatment (Table 13). These RE% were calculated as the difference of concentrations between the samples before pyrolysis and after pyrolysis, divided by the concentrations before pyrolysis. Other compounds such as 1-OH-BTR, BTR-COOH and XTR were non-detected in these samples. In fact, the reduction of the components in the char may be explained with an analysis of the aqueous and oil phase produced after condensation of the gas phase formed during the pyrolysis. Due to the polarity of the compounds, aqueous phase is susceptible to dissolve the analytes. Results evidenced the efficiency of the pyrolysis due to the reduction of these EPs in WEEE so its applicability may increase in the concerned industries. Generally, Q5 and Q6 presented similar concentration pattern concentrations with BTR, XTR and 2-M-BTH, standing out among the others. Both were principally food waste bags, which explains their similarity. In comparison to other studies published in the scientific literature, the occurrence of BTRs and BTHs observed in the present study was lower than the one reported by Wenzheng et Al. in indoor dust samples from an e-waste dismantling area in Qingyuan (China) [41] . However, XTR was founded at higher concentrations, and the occurrence of 1-OH-BTR was low in both studies, with concentrations generally under the LOQ. To the best of our knowledge, no data has been previously reported for the presence of BTHs and BTRs in WEEE samples. 51 Table 13. Obtained concentrations of BTRs and BTHs in WEEE samples (ng g-1). Sample 1-OH-BTR 5-Cl-BTR BTR BTR-COOH TTR XTR ΣBTRs e 2-ABTH 2-M-BTH 2-Me-BTH 2-MeS-BTH BTH ΣBTHs g R1 <LOQ d 2.9 30 n.d. 4.1 n.d. 37 8.9 18 n.d. n.d. 792 818.9 R2 <LOQ 0.6 16 n.d. 3.0 n.d. 19.6 7.7 4.4 n.d. 12 520 544.1 R3 n.d. e 1.9 25 n.d. 5.3 n.d. 32.2 28 8.7 n.d. n.d. 2,819 2,855.7 R4 n.d. 0.5 3.8 n.d. 3.6 <LOQ 7.9 1.0 2.2 n.d. n.d. 140 143.2 R5 n.d. n.d. 6.3 n.d. 1.5 n.d. 7.8 6.2 2.2 n.d. 26 170 204.4 R6 n.d. 0.6 3.2 n.d. <LOQ n.d. 3.8 3.0 0.7 n.d. n.d. 131 134.7 R7 n.d. 7.9 84 n.d. 15 9.2 116.1 13 60 38 289 1,333 1,733 R8 155 1.0 302 n.d. 2.6 35 495.6 7.5 55 22 n.d. 119 203.5 R9 n.d. <LOQ 33 n.d. 19 17 69 3.5 0.7 29 25 317 375.2 Q1 <LOQ n.d. 20 n.d. 18 n.d. 38 3.1 <LOQ n.d. 8.0 91 102.1 Q2 n.d. n.d. 2.5 n.d. 1.7 n.d. 4.2 3.5 <LOQ 16 <LOQ 85 104.5 Q3 n.d. n.d. 4.1 n.d. 7.3 n.d. 11.4 n.d. 41 22 6.3 120 189.3 Q4 n.d. 8.6 123 n.d. 46 n.d. 177.6 429 21 n.d. 414 8,831 9,695 Q5 n.d. <LOQ 338 <LOQ 7.6 9.9 355.5 5.7 3.4 n.d. 90 377 476.1 Q6 <LOQ 1.4 410 n.d. 8.7 10 430.1 6.5 3.4 n.d. 286 2,502 2,797.9 Median a - f 1.4 25 - 6.3 10 37 6.4 4.4 22 26 317 375 D.R.% b 33 73 100 6.7 100 40 100 93 100 33 67 100 100 RE% c n.d. 100 84 n.d. 85 n.d. 85 92 100 19 100 91 91 aCalculated for values above the LOQ. bDetection Rate. cRemoval efficiencies for the pyrolysis. dUnder the limit of quantification. dNon-detected. eTotal concentration of BTRs in sample. fNon-calculated. gTotal concentration of BTRs in sample. Universidad de Valladolid Daniel Gutiérrez Martín 52 3.4 Occurrence of BTHs and BTRs in sewage sludge. A previous study has reported the differences between the concentrations of BTRs and BTHs in primary sludge (after a first sedimentation) and secondary sludge (after a secondary sedimentation) showing the efficiency of the associated biological STP treatment [23]. Other study included the occurrence of these compounds along the sludge treatment [75]. However, data is generally scarce for BTHs and BTRs. The present study focused on the occurrence and evolution of these EPs along the sewage sludge treatment in 3 different STPs. Hence, a total of 25 sewage sludge samples from 3 different STPs in Norway were studied. Nine of these samples were first filtered so the occurrence of BTRs and BTHs was reported in dissolve and particulate phase. Obtained data is presented in Tables 14-16 for STP-1, STP-2 and STP-3, respectively. TTR and XTR have been detected in all the sewage sludge samples, and they contribute to a 39%, 51% and 87% of the total mass of BTRs in STP-1, STP-2 and STP-3, respectively. However, the contribution of BTR also reached a high percentage in STP-1 and STP-2 (56% and 48%, respectively) even if it presented lower detection rates (56% and 11%, respectively). A lower contribution for the total mass of BTRs was found in STP-3 (10%), despite BTR was always detected. On the other hand, BTR-COOH has been only detected in some samples and under the limit of quantification. The polarity and low LogP of this compound suggested a trend to dissolve into water, which may explain its low occurrence in sewage sludge. The contribution of 1-OH-BTR and 5-Cl-BTR was low, under a 5%, in all the STPs. 2-MeS-BTH has been the main compound in the family of BTHs in terms of detection rate, and along with BTH, they showed the highest concentrations in all the studied sewage sludge samples. Hence, 2-MeS-BTH contributed to the total BTHs mass with 66%, 47% and 52% for STP-1, STP-2 and STP-3, respectively. Even though BTH was not ubiquitous (18 of 25 samples), its contribution to the total BTHs mass reached similar levels than 2-MeS-BTH (49% and 46%) in STP-2 and STP-3. 2-ABTH was detected in 17 of 25 samples. However, only 7 samples presented values above the LOQ, and the concentrations were generally low. In previous studies, 2-ABTH has also been poorly detected in sludge samples and low concentrations were reported [10][23]. 2-M-BTH and 2-Me-BTH were present in lower concentrations despite their detection rates, in particular, 18/25 and 17/25, respectively. Universidad de Valladolid Daniel Gutiérrez Martín 59 3.6 Mass loading of BTHs and BTRs. Annually, high amounts of treated sludge from STPs are discharged into landfills or applied to soils with agricultural purposes. BTHs and BTRs mass loads per year have been estimated according to the concentrations determined in the present study and the sludge production data provided from the STPs. Hence, STP-1 produces 15 m3 of final sludge every day, which supposes around 58 Kg of BTRs and 493 Kg of BTHs every year. The individual contribution to those loads is distributed as 41 Kg y-1 of TTR, 12 Kg y-1 of XTR, 5 Kg y-1 of 5-Cl-BTR, 7 Kg y-1 of 2-ABTH, 4 Kg y-1 of 2-M-BTH, and 482 Kg y-1 of 2-MeS-BTH. Likewise, STP-2 yields the highest amount of BTHs with 2-MeS-BTH (326 Kg y-1) followed distantly by 2-ABTH (6 Kg y-1) and 2-M-BTH (3 Kg y-1), respectively. Even if the treated amount of sewage sludge is larger in STP-2, its BTHs load was slightly lower than in STP-1. However, the total amount of BTRs delivered through the STP-2 final sludge was estimated to be widely bigger, reaching 1,831 Kg per year. TTR contributed with 88% while XTR and 5-Cl-BTR did it with 11% and 1% of the total BTRs mass load, respectively. STP-3 treats the largest amount of sludge in comparation to the others STPs, and produces 38,000 tons of soil per year with agricultural purposes. Thus, 2,313 Kg of BTRs and 5,056 Kg of BTHs are discharged in the environment every year with this soil. In comparison with other 198 76 44 35 61 331 0 50 100 150 200 250 300 350 Sum of concentrations (µg L-1) STP-1 BTRs BTHs 1737 17 656 537 55 143 0 250 500 750 1000 1250 1500 1750 Sum of concentrations (µg L-1) STP-2 BTRs BTHs 3310 1251 183 1119 682 399 0 500 1000 1500 2000 2500 3000 3500 Sum of concentrations (ng g-1) STP-3 BTRs BTHs Figure 13. Evolution of the total concentrations of BTRs (blue) and BTHs (orange) in the sludge along each STP treatment. Concentration expressed in ng L-1 for STP-1 and STP-2, and ng g-1 for STP-3. Universidad de Valladolid Daniel Gutiérrez Martín 60 STPs, the mass load of BTRs was slightly higher than in STP-2 but there is a notorious increment in the total BTHs. Additionally, BTR is the compound which contributes the most to the BTRs mass load, with 1,718 Kg per year. In contract, this compound was not detected at the final sludge in the other STPs. TTR (200 Kg y-1), XTR (164 Kg ye-1) and 5-Cl-BTR (231 Kg y-1) were the other BTRs contributing to final soil load mass. Regarding BTHs, 2-MeS-BTH contributed with a 95% to the BTHs mass abundance in the soil, and the rest corresponded to 2-M-BTH. 3.7 Sludge-water distribution coefficients (Kd). The distribution of the target analytes between the sewage sludge solid and liquid phases was estimated when possible by calculating their Kd, which are summarized in Table 17. Hence, results of particulate phase were expressed as a mean of all three sampling days in ng g-1 (Table S7) and similar for results of dissolved phase (Tables 14 and 15) but in ng mL-1. Resulting Kd were compared to those previously reported on the literature. Similar to other studies [10][23][27], results were above 1. This fact indicates a general trend of distribution onto particulate matter for BTRs and BTHs. It should be noticed that despite the sampling protocol was carried out in 3 consecutive days, concentrations of each compound could widely vary among them due to, for instance, spikes in the industry discharges. TTR and XTR have been detected in almost all the studied samples. Calculated Kd values in raw sludge were significantly higher than those obtained in the pasteurized sludge. Aikaterini et al. reported Kd values in activated sludge lower than the ones found in the present study [27]. In base to these results, TTR and XTR seem to have a tendency to be adsorbed onto the particulate phase. To the best of our knowledge, this is the first study reporting estimated Kd values for 1-OH- BTR, 5-Cl-BTR and 2-M-BTH in sewage sludge samples (Table 17). These compounds have the trend to be adsorbed onto the particulate phase. However, concentrations were generally low, and more research is needed to clarify this fact. 2-MeS-BTH has been found in particulate matter with an average concentration of 207 ng g-1 for in STP-1 and STP-2 (Table S7). In addition, it was not present in the liquid phase. This is consistent to the fact that it presents the highest logP of all the studied BTHs and BTRs (Table 1). Similar results were reported by Asimakopoulos et al. in secondary sludge, indicating 2- MeS-BTH as the compound with the highest Kd, being absence in the dissolved phase too [10]. Universidad de Valladolid Daniel Gutiérrez Martín 61 Additionally, Athanasios et al. also pointed 2-MeS-BTH as providing the highest Kd among all the BTHs and BTRs in common between their and the present study. [23]. In STP-1, BTR was not found in the dissolved phase and BTH was mainly distributed in particulate phase. In STP-2, BTR was not-detected in either the particulate or dissolve phases and BTH was found principally in the dissolve phase. As each STP received influent wastewater from different areas, the organic matter content might differ between them. Organic matter content is related with the adsorption of other organic compounds onto it [92]. This could explain the wide disparity observed among the BTH Kd values in both STPs. However, data about the sludge-water distribution for BTRs and BTHs is still limited, and more research is needed. Table 15. Distribution coefficient values, Kd (in L Kg-1) for BTRs and BTHs in STP-1 and STP-2 samples. Kd is only applied for those samples that had been initially filtered i.e., raw sludge in STP-1 (Raw STP-1), and pasteurized sludge in STP-1 (Past. STP- 1) and in STP-2 (Past. STP-2). Concentration between LOQ and LOD were considered as LOQ/2 for Kd calculation purposes. Samples 1-OH- BTR 5-Cl- BTR BTR TTR XTR 2-M- BTH 2- MeS- BTH BTH Referen ce Raw STP-1 328 241 P 1809 331 ±332 260 P 337 Present study Past. STP-1 P a 332 P 616 ±554 48 ±16 P P P Present study Past. STP-2 141 181 ±69 N.C 64 ±6 23 ±23 152 ± 94 P L d Present study Activated sludge N.C. b N.C. 220 ±9 170 ±48 c 87 ±17 N.C. N.C. N.C. [27] Primary sludge N.C. N.C. 3.8 2.2 N.C. N.C. 19 2.1 [10] Secondary sludge N.C. N.C. 288 7.4 N.C. N.C. N.C. 148 [10] Primary sludge N.C. N.C. 7 ±3 0.8 ±0.2 N.C. N.C. 21 ±3 3 ±2 [23] Secondary sludge N.C. - 133 ±104 6 ±4 181 ±56 147 ±63 [23] aOnly detected in particulate phase. bNot calculated. cConsidering the 4-methyl-benzotriazole. dOnly detected in dissolve phase. Universidad de Valladolid Daniel Gutiérrez Martín 62 4. Conclusions A versatile and efficient methodology has been proposed for the analysis of more than 40 compounds from different families of EPs (parabens, phthalates, PFCs, bisphenols, benzophenones, BTRs and BTHs) in several complex solid environmental matrices. Focusing on benzotriazoles and benzothiazoles, the proposed methodology has been satisfactorily validated for the analysis of 11 compounds in WEEE and sewage sludge samples. Additionally, the occurrence of BTRs and BTHs in WEEE has been studied in 15 samples. To the best of our knowledge, this is the first study which focused on the analysis of these EPs in WEEE. Obtained concentrations showed a predominance of BTR and BTH which were present in all the samples with median concentrations of 25 and 317 ng g-1, respectively. Additionally, the thermal treatment conducted in Q2 demonstrated a dramatic reduction of their concentrations. Therefore, pyrolysis qualified to efficiently eliminate most of the target compounds in the final char. Further research is expected to assess whether this promising performance compensates for the high energy costs associated. Grab samples of sewage sludge collected in three different STPs have been analyzed after different steps of the treatment. Occurrence and evolution of individual compounds have been discussed, as well as their overall trends as BTRs and BTHs families. Some compounds were degraded (or transformed), other showed recalcitrant properties, and some of them even increase their concentration along the treatment. Different TPs have been proposed. However, the lack of information about transformation pathways and the absence of some BTRs and BTHs in the present study hindered the possibility of finding a clear explanation for each case. To the best of our knowledge, this is the first study reporting concentrations of 2- M-BTH and 2-Me-BTH in sewage sludge. Moreover, mass loads have been estimated, showing that high amounts of BTRs and BTHs are disposed every year into landfills or applied to agricultural soils, when the sewage sludge is used as fertilizer. In particular, an average between 58 and 2,313 Kg of BTRs, and 335 and 5,056 Kg of BTHs is released into the environment every year, because of the sludge produced in the STPs. Additionally, the coefficient of distribution (Kd) between particulate and dissolved phase has been calculated. Generally, these pollutants showed a clear tendency to be adsorbed onto the sludge, except for BTH. Again, to the best of our knowledge, this was the first time that kd values for 1-OH- BTR, 5-Cl-BTR and 2-M-BTH were reported. Universidad de Valladolid Daniel Gutiérrez Martín 63 In summary, a unique methodology has been successfully validated and applied for the analysis of 11 BTHs and BTRs in environmental matrices and WEEE. 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U.S. DEPARTMENT OF HEALTH, EDUCATION, Bioassay of 1H- Benzotriazole for Possible Carcinogenicity., 1978. https://ntp.niehs.nih.gov/data/tr/index.html. Universidad de Valladolid Daniel Gutiérrez Martín II Appendix A. Supplementary Tables Tables S1 – S4. Absolute recoveries (AR%), matrix effects (ME%), method recoveries (MR%) and relative method recoveries (RMR%) for 5 and 10 µg L-1 spikes are shown in Tables S1, S2 for WEEE. Tables S3, S4 compiles the results for sewage sludge. Table S1. Absolute recoveries (AR%), matrix effects (ME%), method recovery (MR%) and relative method recovery (RMR%) for a fortification level of 5 µg L-1 in WEEE samples. Compound AR% (RSD%) ME% MR% (RSD%) RMR% (RSD%) 1-OH-BTR 62 (44) -17 52 (44) 74 (36) 2-ABTH 72 (14) -29 42 (21) 60 (11) 2-M-BTH 58 (31) -42 26 (42) 46 (30) 2-Me-BTH 69 (20) -18 37 (31) 67 (18) 2-MeS-BTH 41 (18) 15 17 (59) 30 (65) 5-Cl-BTR 62 (29) -39 37 (29) 53 (19) BTR 120 (71) -15 84 (97) 120 (47) BTR-COOH 90 (23) -33 69 (23) 87 (13) TTR 72 (40) -27 44 (49) 63 (39) XTR 59 (34) -31 41 (34) 59 (24) Table S2. Absolute recoveries (AR%), matrix effects (ME%), method recovery (MR%) and relative method recovery (RMR%) for a fortification level of 10 µg L-1 in WEEE samples. Compound AR% (RSD%) ME% MR% (RSD%) RMR% (RSD%) 1-OH-BTR 69 (4) -5 66 (4) 111 (8) 2-ABTH 80 (6) -23 58 (7) 96 (10) 2-M-BTH 90 (26) -38 52 (28) 115 (13) 2-Me-BTH 74 (29) -23 48 (34) 107 (17) 2-MeS-BTH 50 (34) 11 42 (48) 94 (46) 5-Cl-BTR 68 (29) -37 43 (29) 71 (21) BTR 78 (27) -18 54 (34) 89 (27) BTR-COOH 82 (19) -16 69 (19) 115 (17) TTR 72 (26) -22 52 (28) 86 (19) XTR 72 (8) -40 43 (8) 72 (5) Universidad de Valladolid Daniel Gutiérrez Martín III Table S3. Absolute recoveries (AR%), matrix effects (ME%), method recovery (MR%) and relative method recovery (RMR%) for a fortification level of 5 µg L-1 in sewage sludge samples. Compound AR% (RSD%) ME% MR % (RSD%) RMR% (RSD%) 1-OH-BTR 50 (11) 14 42 (15) 54 (19) 2-ABTH 53 (25) 12 55 (27) 70 (25) 2-M-BTH 51 (16) -44 28 (17) 52 (22) 2-Me-BTH 42 (4) 41 60 (5) 114 (3) 2-MeS-BTH 62 (14) 160 17 (140) 33 (138) 5-Cl-BTR 53 (20) -30 35 (21) 45 (22) BTR 103 (4) 292 31 (85) 40 (46) BTR-COOH 73 (33) 79 85 (51) 109 (53) TTR 90 (23) 620 -198 (-75) -254 (-78) XTR 88 (31) 108 17 (331) 22 (385) Table S4. Absolute recoveries (AR%), matrix effects (ME%), method recovery (MR%) and relative method recovery (RMR%) for a fortification level of 10 µg L-1 in sewage sludge samples. Compound AR% (RSD%) ME% MR% (RSD%) RMR% (RSD%) 1-OH-BTR 70 (13) 6 67 (14) 85 (21) 2-ABTH 70 (10) 2 70 (11) 89 (17) 2-M-BTH 77 (2) -48 40 (2) 75 (5) 2-Me-BTH 57 (9) 13 66 (9) 125 (6) 2-MeS-BTH 47 (18) 85 19 (86) 36 (82) 5-Cl-BTR 70 (11) -37 43 (11) 56 (17) BTR 92 (10) 136 48 (45) 62 (52) BTR-COOH 81 (10) 70 116 (12) 149 (18) TTR 215 (17) 137 96 (89) 123 (89) XTR 85 (15) 22 35 (45) 44 (43) Universidad de Valladolid Daniel Gutiérrez Martín IV Tables S5-S6. AR%, R%, ME%, and MR% for the compounds that did not pass the validation test are shown in Tables S5 and S6 (WEEE and sludge, respectively). Table S5. Absolute recoveries (AR%), matrix effects (ME%), method recovery (MR%) and relative method recoveries (RMR%) for the compounds which are not semi-quantified. Values for 5, 10 and 20 fortification levels in WEEE. Compound Fortification level (µg L-1) AR% (RSD%) ME% MR% (RSD%) RMR% (RSD%) 5 12 (173) -134 -22 (-25) -39 (-12) 2-Cl-BTH 10 31 (140) -102 -4 (-303) -10 (-454) 20 112 (173) -110 -4 (-39) -6 (-47) 5 112 (4) 63 -524 (-27) -9333 (-28) 2-OH-BTH 10 48 (18) 460 -220 (-30) -494 (-38) 20 136 (13) -12 -16 (-189) -26 (-176) 5 17 (53) -166 -14 (-26) -25 (-33) 2-S-BTH 10 52 (144) -121 3 (517) 3 (379) 20 23 (53) -116 -3 (-11) -3 (-27) 5 89 (47) -246 367 (99) 653 (98) 2-SCNMeS-BTH 10 66 (24) -432 -5 (-1498) -12 (4627) 20 252 (47) -382 99 (122) 168 (112) 5 0.02 (146) -46 0.01 (146) 0.01 (144) 5-ABTR 10 0.01 (119) -40 0.01 (119) 0.01 (121) 20 0.09 (140) -62 0.03 (140) 0.05 (139) Table S6. Absolute recoveries (AR%), matrix effects (ME%), method recovery (MR%) and relative method recovery (RMR%) for the compounds which are not semi-quantified. Values for 5, 10 and 20 fortification level in sewage sludge. Compound Fortification level (µg L-1) AR% (RSD%) ME% MR % (RSD%) RMR % (RSD%) 5 160 (114) -80 19 (192) 36 (190) 2-Cl-BTH 10 37 (91) -71 4 (267) 7 (256) 20 6 (173) -78 -1 (-250) -2 (-281) 5 92 (16) 564 -163 (-99) -306 (-96) 2-OH-BTH 10 160 (7) 116 91 (56) 172 (54) 20 124 (5) 88 94 (18) 176 (21) 5 23 (41) -60 5 (88) 9 (87) 2-S-BTH 10 13 (20) -79 1 (80) 1 (77) 20 12 (55) -77 2 (81) 4 (77) 5 31 (8) -82 5 (9) 10 (15) 2-SCNMeS-BTH 10 12 (55) -71 12 (9) 23 (7) 20 38 (24) -73 10 (24) 21 (24) 5 6 (9) -35 0.1 (589) 0.1 (515) 5-ABTR 10 3 (39) -50 -0.2 (-311) -0.4 (-327) 20 2 (78) -53 0.2 (339) 0.4 (374) Universidad de Valladolid Daniel Gutiérrez Martín V Compounds showed in in Table S5 and S6 did not meet the quality requirement during validation and were ruled out from the analytical method. Linearity was investigated in the range from 0 – 20 µg L-1 considering an injection volume of 5 µL. Hence, corrected signal response (analyte peak area divided by IS peak area) was plotted versus spiked concentration. A linear model fitted along that range with determination coefficients above 0.9745. iLOQ levels were between 0.05 and 5 µg L-1. Therefore, sensitivity of the instrument was not an issue. 2-Cl-BTH and 2-S-BTH were not detected in the spikes (pre-extraction nor post-extraction then, the method was not suitable for this compound. In case of 2-SCNMeS-BTH and 2-OH- BTH, high contamination in the blanks was found, thus, their quantification was not possible either. 5-ABTR was not present in the spiked samples (before extraction), but good response was presented in the matrix matched (spikes post-extraction), which could be related with a very poor sample pretreatment recovery. pKa for this compound is quite high (9.61), thus, it might have remained retained in the cartridge. The elution volume should be further optimized for this analyte. Some methodological improvements could be proposed. Hence, a more exhaustive clean-up step in order to further reduce the interferences in the matrix, and thus, reduce the associated ME%. Regardless, a good compromise solution was accepted with this methodology, as it is able to quantitatively extract more than 40 compounds belonging to very diverse physicochemical featured families. VI Table S7. Determined BTRs and BTHs concentrations in particulate phase (ng g-1). BTRs BTHs Sample 1-OH-BTR 5-Cl-BTR BTR BTR-COOH TTR XTR 2-ABTH 2-M-BTH 2-Me-BTH 2-MeS-BTH BTH STP-1 R_(1)_P_1 n.d. a <LOQ b n.d. n.d. n.d. 177 <LOQ 25 n.d. 168 <LOQ R_(1)_P_2 51 2.1 5.6 n.d. 1.8 283 1.4 5.4 n.d. 151 540 R_(1)_P_3 238 <LOQ 4,881 n.d. 244 303 <LOQ 5.0 n.d. 152 n.d. P_(1)_P_1 n.d. 4.1 866 <LOQ 965 122 n.d. 8.3 66 281 907 P_(1)_P_2 n.d. 9.2 332 n.d. 486 113 n.d. <LOQ 100 239 1,104 P_(1)_P_3 16 2.1 242 n.d. 345 80 n.d. n.d. 65 204 747 STP-2 P_(2)_P_1 64 6.1 n.d. n.d. 26 43 n.d. 10 56 237 n.d. P_(2)_P_2 41 6.1 n.d. n.d. 32 42 n.d. 6.7 50 210 n.d. P_(2)_P_3 7.1 2.8 n.d. n.d. 97 97 <LOQ 12 59 217 n.d. aNon-detected. bUnder the limit of quantification. Universidad de Valladolid Daniel Gutiérrez Martín VII Appendix B. Supplementary Figures. Figure S1. Effect of the extract acidification (Formic acid, 0.1%, v/v) prior the UHPLC-MS/MS analysis. The resulting chromatographic peak after acidification was thinner and presented less tail as showed in Figure S1 for 2-Me-BTH (fortification level: 50 µg L-1) as an example. This improvement enabled a more reliable quantification and better limits of quantification. Figure S1. Differences between acidified (green) and non-acidified (red) standard of 50 µg L-1. Universidad de Valladolid Daniel Gutiérrez Martín VIII Figures S2-S17. Internal standard calibration curves for all the studied compounds in MeOH:Deionized water (1:1, v/v) are shown in Figures S2 – S17. Additionally, coefficient of determination (R2) and linear equation are included. Concentrations (µg L-1) are represented in abscissa axis. The ratio between the analyte (A) and the internal standard (AIS) areas are represented in Y-axis. y = 0.0592x - 0.0167 R² = 0.9952 0 0.5 1 1.5 0 5 10 15 20 A/AIS Concentration (µg L-1) 2-ABTH y = 0.1827x - 0.0285 R² = 0.9955 0 1 2 3 4 0 5 10 15 20 A/AIS Concentration (µg L-1) 2-M-BTH y = 0.0011x - 0.003 R² = 0.9745 0 0.005 0.01 0.015 0.02 510 15 20 A/AIS Concentration (µg L-1) 2-Cl-BTH y = 0.0184x - 0.0101 R² = 0.9905 0 0.1 0.2 0.3 0.4 0 5 10 15 20 A/AIS Concentration (µg L-1) 2-Me-BTH y = 0.0513x - 0.0158 R² = 0.9933 0 0.5 1 1.5 0 5 10 15 20 A/AIS Concentration (µg L-1) 2-MeS-BTH y = 0.0506x - 0.0146 R² = 0.9968 0 0.5 1 1.5 0 5 10 15 20 A/AIS Concentration (µg L-1) 1-OH-BTR Figure S2. Calibration curve, R2 and linear equation for 1-OH-BTR in MeOH/Deionized water 1:1 (v/v). Figure S3. Calibration curve, R2 and linear equation for 2-ABTH in MeOH/Deionized water 1:1 (v/v). Figure S4. Calibration curve, R2 and linear equation for 2-Cl-BTH in MeOH/Deionized water 1:1 (v/v). Figure S5. Calibration curve, R2 and linear equation for 2-M-BTH in MeOH/Deionized water 1:1 (v/v). Figure S6. Calibration curve, R2 and linear equation for 2-Me-BTH in MeOH/Deionized water 1:1 (v/v). Figure S7. Calibration curve, R2 and linear equation for 2-MeS-BTH in MeOH/Deionized water 1:1 (v/v). Universidad de Valladolid Daniel Gutiérrez Martín IX y = 0.0058x - 0.0013 R² = 0.9859 0 0.05 0.1 0.15 0 5 10 15 20 A/AIS Concentration (µg L-1) 2-OH-BTH y = 0.0185x - 0.0085 R² = 0.9909 0 0.1 0.2 0.3 0.4 0 5 10 15 20 A/AIS Concentration (µg L-1) 2-S-BTH y = 0.0266x + 0.033 R² = 0.9763 0 0.2 0.4 0.6 0.8 0 5 10 15 20 A/AIS Concentration (µg L-1) 2-SCNMeS-BTH y = 0.0823x - 0.0245 R² = 0.9959 0 0.5 1 1.5 2 0 5 10 15 20 A/AIS Concentration (µg L-1) 5-ABTR y = 0.0574x - 0.0125 R² = 0.9967 0 0.5 1 1.5 0 5 10 15 20 A/AIS Concentration (µg L-1) BTR y = 0.0046x - 0.0046 R² = 0.9957 0 0.02 0.04 0.06 0.08 0.1 0 5 10 15 20 A/AIS Concentration (µg L-1) BTR-COOH y = 0.0011x - 0.0015 R² = 0.9919 0.00 0.05 0.10 0.15 050 100 A/AIS Concentration (µg L-1) BTH y = 0.0537x - 0.0092 R² = 0.9971 0 0.5 1 1.5 0 5 10 15 20 A/AIS Concentration (µg L-1) 5-Cl-BTR Figure S8. Calibration curve, R2 and linear equation for 2-OH-BTH in MeOH/Deionized water 1:1 (v/v). Figure S9. Calibration curve, R2 and linear equation for 2-S-BTH in MeOH/Deionized water 1:1 (v/v). Figure S10. Calibration curve, R2 and linear equation for 2-SCNMeS-BTH in MeOH/Deionized water 1:1 (v/v). Figure S11. Calibration curve, R2 and linear equation for 5-ABTR in MeOH/Deionized water 1:1 (v/v). Figure S12. Calibration curve, R2 and linear equation for 5-Cl-BTR in MeOH/Deionized water 1:1 (v/v). Figure S13. Calibration curve, R2 and linear equation for BTH in MeOH/Deionized water 1:1 (v/v). Figure S14. Calibration curve, R2 and linear equation for BTR in MeOH/Deionized water 1:1 (v/v). Figure S15. Calibration curve, R2 and linear equation for BTR-COOH in MeOH/Deionized water 1:1 (v/v). Universidad de Valladolid Daniel Gutiérrez Martín X y = 0.1192x - 0.0207 R² = 0.996 0 1 2 3 0 5 10 15 20 A/AIS Concentration (µg L-1) TTR y = 0.1214x - 0.0345 R² = 0.9943 0 1 2 3 0 5 10 15 20 A/AIS Concentration (µg L-1) XTR Figure S16. Calibration curve, R2 and linear equation for TTR in MeOH/Deionized water 1:1 (v/v). Figure S17. Calibration curve, R2 and linear equation for XTR in MeOH/Deionized water 1:1 (v/v). Universidad de Valladolid Daniel Gutiérrez Martín XI Figure S18. Comparison among external calibration versus post and pre-extraction spikes. Matrix effect as well as the sample treatment affected the peak area produced for a same amount of analyte. This is presented in Figure S18 for TTR in WEEE matrix, where the external calibration curve showed the highest slope, followed by the post-extraction spikes and finally de pre-extraction spikes. Slope is related to sensitivity. This is consistent to the fact that external calibration samples did not contain matrix or undergo any analytical process. Signal in post-extraction spikes only registered signal withdrawal associated to ESI matrix effect. y = 18342x - 22041 R² = 0,997 y = 17581x - 41429 R² = 0,9918 y = 11238x - 20037 R² = 0,9962 0 75000 150000 225000 300000 375000 510 15 20 Intensity Concentration (µg L-1) TTR - WEEE External calibration Spikes post-extraction Spikes pre-extraction Figure S18. Comparation of the intensity for the external calibration, the spikes post-extraction and the spikes pre-extraction.