Applied analysis of hydrocarbons and non-polar contaminants in marine oil spills

Dennis Bilbao AlemanyDennis Bilbao Alemany 20242024 Applied analysis of hydrocarbons and non-polar contaminants in marine oil spills Dennis Bilbao Alemany 2024 Applied analysis of hydrocarbons and non-polar contaminants in marine oil spills Departamento de química analítica Applied analysis of hydrocarbons and non-polar contaminants in marine oil spills Dennis Bilbao Alemany 2024 Memoria para optar al grado de Doctor en Química en el Master de Medio Ambiente y Recursos Marinos. Dirigida por: Dr. Ailette Prieto Sobrino Dr. Nestor Etxebarria Loizate (cc) 2024 Dennis Bilbao Alemany (cc by-nc 4.0) I Índice Capítulo 1: Introducción 1.1. Transporte marítimo y petróleo .......................................................................................... 3 1.2. Caracterización del petróleo ................................................................................................ 10 1.3. Ensayos de toxicidad ............................................................................................................ 17 1.4. Muestreo y dosificación pasiva ........................................................................................... 20 1.5. Otros contaminantes orgánicos .......................................................................................... 23 1.5.1. Presencia en el medio ambiente ................................................................................ 25 1.5.2. Legislación y Normativa Ambiental ............................................................................ 33 1.5.3. Nuevas tecnologías en el tratamiento de aguas residuales ...................................... 35 1.6. Referencias ........................................................................................................................... 37 Capítulo 2: Objetivos y contexto 2.1. Objetivos y Contexto ......................................................................................................... 53 2.2. Referencias ....................................................................................................................... 56 Chapter 3: Chemical characterization of oil and Water Accommodated Fraction (WAF) at different temperatures 3.1. Introduction ....................................................................................................................... 61 3.2.Experimental Section ......................................................................................................... 63 3.2.1. Chemicals and materials ............................................................................................. 63 3.2.2. Oil analysis ................................................................................................................... 64 3.2.3. WAF production and characterization ....................................................................... 65 3.2.4. Gas chromatographic methods .................................................................................. 68 3.2.5. Direct analysis .............................................................................................................. 68 3.2.6. Solid phase microextraction ....................................................................................... 68 3.2.7. Stir-bar sorptive extraction ......................................................................................... 69 3.2.8. Suspect screening ........................................................................................................ 69 3.3.Results and discussion ..................................................................................................... 70 3.3.1 Oil analysis .................................................................................................................... 70 3.3.2 WAF formation kinetics ................................................................................................ 73 3.3.3 Assessment of the WAF composition .......................................................................... 76 3.3.4 WAF stability at room temperature ............................................................................ 81 3.3.5 .Suspect Screening ....................................................................................................... 83 3.4. Conclusions ........................................................................................................................ 90 3.5 References .......................................................................................................................... 91 II Chapter 4: PAHs toxicity at aqueous solubility using passive dosing and sea urchin embryos 4.1. Introduction ....................................................................................................................... 99 4.2. Experimental section ......................................................................................................... 102 4.2.1. Chemicals and materials ............................................................................................. 102 4.2.2. O-rings cleaning .......................................................................................................... 103 4.2.3. Loading PDMS silicone O-rings ................................................................................... 103 4.2.4. O-rings as passive dosser and chemical analysis ....................................................... 104 4.2.5. Sea urchin embryo toxicity (SET) testing .................................................................... 105 4.3. Results and discussion ....................................................................................................... 106 4.3.1. Loading of the O-rings ................................................................................................. 106 4.3.2. Stability of the WAFs ................................................................................................... 110 4.3.3. Toxicity assessment ..................................................................................................... 112 4.4. Conclusions ........................................................................................................................ 115 4.5. References ......................................................................................................................... 115 Chapter 5: Quantification and distribution of PAHs in mussels in Arctic regions 5.1. Introduction ....................................................................................................................... 123 5.2. Experimental section ......................................................................................................... 124 5.2.1. Chemicals and reagents .............................................................................................. 124 5.2.2. Sampling and sample pretreatment ........................................................................... 125 5.2.3. Analytical protocol....................................................................................................... 126 5.2.4. Instrumental analysis .................................................................................................. 127 5.3. Results and discussion ....................................................................................................... 127 5.3.1. Method performance and quality control/quality assurance (QC/QA) .................... 127 5.3.2. Analysis in real samples ............................................................................................... 129 5.4 Conclusions. ........................................................................................................................ 135 5.5 References. ......................................................................................................................... 136 Chapter 6:Development of an analytical method for the simultaneous determination of 50 semi-volatile organic contaminants in wastewaters 6.1. Introduction ....................................................................................................................... 141 6.2. Chemicals and reagents .................................................................................................... 143 6.3. Sampling and sample pre-treatment ............................................................................... 146 III 6.4. Analytical protocol ............................................................................................................. 147 6.5. Instrumental analysis ........................................................................................................ 149 6.5.1. Target analysis ............................................................................................................. 149 6.5.2. Suspect screening ........................................................................................................ 150 6.5.3. Analytical method validation ...................................................................................... 150 6.6. Results and discussion ....................................................................................................... 152 6.6.1. Optimisation of the extraction step ........................................................................... 152 6.6.2. Method validation ....................................................................................................... 157 6.6.3. Real samples analysis: target and suspect screening ................................................ 162 6.7. Conclusions ........................................................................................................................ 167 6.8. References ......................................................................................................................... 168 Capítulo 7: Conclusiones 7.1. Conclusiones ...................................................................................................................... 175 Introdución 1 Capítulo 1 8 Figura 1.2. Escala temporal de los efectos que se producen en vertidos de petróleos (image taken from https://www.seekpng.com/ipng/u2e6r5a9r5a9y3q8_fate-of-oil-at-sea-and-modelled- processes/. Como se observa, en las primeras horas del vertido, los fenómenos físico-químicos mayoritarios son la evaporación, disolución y dispersión. El efecto de la disolución empieza a perder importancia después de la primera hora, y toma el relevo la dispersión, cuyo mayor impacto se produce en las primeras diez horas. Los fenómenos más tardíos y menos duraderos son la sedimentación y emulsión, que aparecen a partir de las 10 horas. Finalmente, el proceso que más tarda en aparecer y ser relevante, es la biodegradación, ya que para que se produzca es necesario que la mezcla de hidrocarburos se haya dispersado y emulsionado. Los procesos representados, no constituyen un modelo estándar si no una simplificación de los procesos que ocurren. Cada vertido debe de ser evaluado de forma singular teniendo en cuenta Introducción 9 los factores propios. Esto dificulta una respuesta general e inmediata, por lo que cada vertido de petróleo constituye un reto para su control y tratamiento. En este sentido existen múltiples modelos de predicción, como “Oil Spill Contingency and Response” (OSCAR ) desarrollado por el SINTEF (Reed et al. 1995) o “Spill Impact Model Application Package/Oil Modeling Application Package”(SIMAP/OILMAP) (Spaulding et al. 2017) . Por ejemplo, un crudo con una baja gravedad API (veáse sección 1.2), según las siglas del American Petroleum Institute, será ligero y sufrirá un proceso de evaporación más acusado mientras que su disolución será menor en comparación con un crudo más pesado (Dhaka and Chattopadhyay 2021). Pero, por ejemplo, probablemente dependiendo de las condiciones climáticas, este crudo puede ser que se disuelva más en un clima tormentoso, debido al efecto de las olas y del viento, en comparación con un clima tranquilo sin este tipo de fenómenos. Actualmente, las respuestas ante un vertido de petróleo han evolucionado (ver Tabla 1.1), y se aplican diferentes métodos, como es el caso de la incineración in-situ (in-situ burning), la extracción física del petróleo, el uso de dispersantes o de agentes biológicos, entre otros. La incineración in-situ se basa en quemar el vertido en el lugar del incidente y se suele aplicar en vertidos producidos en aguas abiertas. Este tipo de acciones requiere que la mancha de petróleo este fuertemente concentrada en una zona concreta para proceder a su incineración. Para la compactación en un área confinada se pueden utilizar medios físicos o medios químicos. Los primeros son habitualmente brazos resistentes al fuego que, mediante empuje y dirigidos por barcos, confinan el petróleo en un espacio predefinido (Rojas-Alva et al. 2020). Otro método físico es la extracción del petróleo que se lleva a cabo mediante el uso de “Skimmers” o desnatadores, que succionan el petróleo de la superficie y lo almacenan en tanques. Existen múltiples tipos de bombas, circulares, de superficie, que son utilizadas en casi todos los desastres ocurridos, Capítulo 1 10 pero su uso es limitado y requiere de condiciones atmosféricas favorables, ausencia de viento y de corrientes (Etkin and Nedwed 2021). Entre los métodos químicos empleados destaca el uso de agentes surfactantes. Este tipo de compuestos se derraman a lo largo del perímetro de la mancha de petróleo, y hace que disminuya la tensión superficial del agua colindante (Buist et al. 2013) de forma que la propia agua ejerce una fuerza de contracción que permite la compactación de la mancha de petróleo. Actualmente existen dos productos comerciales aceptados por la Agencia de Protección Ambiental de Estados Unidos (U.S. Environmental Protection Agency 2012; U.S. Environmental Protection Agency 2016): TS6535 (mezcla de 65 % sodio monolaurato y 35 % 2 etil butanol en volumen (Fritt-Rasmussen et al. 2017)) y OP40 (copolímero de polidimetilsiloxano patentado (Fritt-Rasmussen et al. 2017)). Evidentemente, la temperatura del agua, el tipo de petróleo y demás factores, afectan al tratamiento con agentes químicos. Según los últimos estudios (Fritt-Rasmussen and Brandvik 2011; Bullock et al. 2017), una vez la mancha del petróleo es concentrada, este tipo de tratamiento consigue una gran disminución de la cantidad de petróleo además de ser un método rápido y efectivo. Como se puede observar en la Tabla 1.1, dichas acciones son ampliamente utilizadas en la mayoría de los derrames de petróleo. 1.2. Caracterización del petróleo Petroleum, traducido literalmente del latín, aceite de roca, es un líquido generalmente de color negro, oleaginoso, inflamable, con una composición elemental carbono, hidrógeno, azufre, nitrógeno y oxígeno, y que puede contener distintos metales a nivel de trazas. La composición molecular se caracteriza por mezclas de hidrocarburos alifáticos, lineales y cíclicos y aromáticos, cuya proporción dependerá de cada petróleo. El petróleo, como el carbón, es fruto de la transformación química de la Introducción 11 materia orgánica en ambientes anóxicos y en condiciones de temperatura y presión que hacen viable su transformación en este líquido viscoso (Santos et al. 2014; Wang et al. 2016). La variedad de compuestos que componen el petróleo es ingente. La clasificación, debido a su simplicidad, que más se ha extendido es en función de su densidad, o en términos petrológicos, grados API. La densidad del petróleo depende directamente de su composición. Según este tipo de clasificación existen 4 tipos de petróleos (Yang et al. 2017): (i) petróleos ligeros, aquellos cuyo grado API se encuentra entre 35-45; (ii) medio, cuando el rango obtenido se encuentra entre 25-35; (iii) pesado, con valores de API entre 10-25 y (iv) muy pesado, cuando es menor de 10 grados API. • Petróleos ligeros: se componen en su gran mayoría de parafinas, hidrocarburos alifáticos lineales saturados con estructura análoga al metano (CH4). En caso de presentar ramificaciones son conocidos como isoparafinas. También se compone de naftenos. Debido a su composición es de los más preciados ya que su aprovechamiento es mayor que las alternativas por su bajo contenido en compuestos pesados. • Petróleos de densidad media: En este caso se compone de las mismas familias de los ligeros, pero su composición en hidrocarburos aromáticos, ceras y alquitrán es mayor. • Petróleos pesados y super-pesados: son petróleos con una pequeña fracción de parafinas y naftas y mayor en hidrocarburos aromáticos, alquitrán o asfaltenos. Además de los compuestos mencionados anteriormente, en el petróleo también se detectan otros compuestos minoritarios como los siguientes: • Compuestos oxigenados: son moléculas que contienen uno o varios átomos de oxígeno. Capítulo 1 12 • Compuestos nitrogenados: son moléculas que contienen uno o varios átomos de nitrógeno. • Compuestos sulfurados: compuestos orgánicos que contienen uno o varios átomos de azufre. • Compuestos organometálicos: Compuestos orgánicos que contienen algún metal en su estructura. • Metales: es habitual encontrar metales en el petróleo como cobalto, hierro, níquel, cromo o vanadio. Dada la naturaleza compleja del petróleo, la caracterización química exhaustiva no ha sido abordada hasta hace pocas décadas debido a los recientes avances de la instrumentación analítica. De forma general, se pueden destacar los sistemas cromatográficos, de espectrometría de masas (EM) y la resonancia magnética nuclear (RMN) (Krupcík et al. 2004; Perini et al. 2012; Ye et al. 2020). Esto ha ocasionado un aumento en la publicación de nuevos estudios relacionados con el petróleo y su análisis, ya que se pasan de de 5,000 en 2010 a más de 19,000 en 2021 (Servier 2021). Sin duda, la técnica más ampliamente utilizada ha sido la cromatografía de gases (GC), inicialmente acoplada a un detector de ionización en llama (FID), éste último detector se está viendo actualmente desplazado por la espectrometría de masas tanto de baja (low resolution mass spectrometry, LRMS) como de alta resolución (high resolution mass spectrometry, HRMS) (Abdel-Shafy and Mansour 2016). Con el objeto de optimizar la separación cromatográfica y la resolución instrumental, suele ser conveniente fraccionar el petróleo de forma previa. Tal y como lo describen Zhendi Wang y colaboradores (Wang et al. 1994), los petróleos ligeros se pueden fraccionar mediante una columna rellena de sílica-gel y de este modo se pueden separar los hidrocarburos alifáticos y los aromáticos. Introducción 13 Las recuperaciones obtenidas en dicho estudio estaban por encima del 95 % para los alcanos y por encima del 85 % para los compuestos aromáticos. Otro método más exhaustivo es la separación secuencial previa en compuestos saturados, aromáticos, resinas y asfaltenos (SARA) como se observa en la Figura 1.3 (Ashoori et al. 2017; Garaniya et al. 2018; Khemka et al. 2021; Neves and Santos 2021). Este fraccionamiento, parte del mismo fundamento que el estudio comentado anteriormente. En este caso se lleva a cabo un fraccionamiento del petróleo en 4 etapas. En la primera se produce la precipitación de los asfaltenos (fracción más pesada y con mayores puntos de ebullición del petróleo), tras diluir la muestra en npentano. Posteriormente, tras filtrar la fracción soluble, se trata la fracción soluble en pentano con sílica-gel y se evapora todo el disolvente sobrante mediante un rotavapor. Una vez seco el residuo, se introduce la sílica-gel en la columna cromatográfica y se realiza la elución secuencial mediante los siguientes disolventes: i) n-pentano para la elución de los compuestos saturados; ii) tolueno para la fracción aromática y iii) una mezcla de tolueno-metanol para las resinas más polares. De este modo se pueden diferenciar las familias de los compuestos que componen el petróleo lo que permite la realización de análisis y ensayos independientes con cada una de ellas. Una tendencia general en la biodegradación de hidrocarburos presentes en los petróleos es el orden de degradación de n-alcanos> compuestos monoaromáticos (incluido benceno, tolueno, etilbenceno y xilenos conocidos como BTEX)> alcanos ramificados y cíclicos> compuestos aromáticos policíclicos (disminución de la degradación con un mayor número de anillos aromáticos), y una correlación negativa entre la degradación y el grado de sustitución de los grupos alquilo. Los nalquiltoluenos no se incluyen comúnmente en la huella química de los derrames de petróleo, pero se han sugerido en la bibliografía (Kristensen et al. 2015) como parte de un análisis complementario de Capítulo 1 14 los n-alcanos en la evaluación preliminar de la contaminación por hidrocarburos. Figura 1.3. Esquema general de separación por familias de las fracciones del petróleo mediante la elución secuencial en columna cromatográfica y la aplicación de distintos disolventes. Modificación de Garaniya (Garaniya et al. 2018). El análisis de alquiltoluenos puede proporcionar información adicional sobre las tasas y patrones de biodegradación en la etapa inicial de un derrame de petróleo, así como en la identificación de la fuente (Wang et al. 2004; Douglas et al. 2007; Wang et al. 2007). Para la determinación de la huella de un vertido, los compuestos que están tomando mayor importancia son los marcadores biológicos o biomarcadores (Arekhi et al. 2021). Estos compuestos comprenden familias muy variadas de compuestos orgánicos entre los que se pueden encontrar terpenos o esteranos triaromáticos. Una vez se produce la fosilización de la materia orgánica, éstos compuestos sufren una serie de cambios químicos dando lugar a los denominados biomarcadores, compuestos que una vez formados, presentan una gran estabilidad química. Estos se encuentran presentes tanto en el crudo, en rocas o sedimentos (Salat et al. 2021). A diferencia de otros Introducción 15 compuestos y sus precursores orgánicos (habitualmente suelen ser esteroles, terpenoides, etc.) son fácilmente detectables en mezclas de hidrocarburos en niveles de miligramo por kilogramo e inferiores, lo cual le confiere a este tipo de compuestos un gran interés analítico (Wang et al. 2016; Salat et al. 2021). Los terpenos se componen de unidades de isopreno (2-metil-1,3-butadieno). La unión de dos o más unidades de isopreno componen las familias de los terpenoides. Esta familia fue descubierta en el siglo XIX por el premio nobel Otto Wallach (Christmann 2010), determinando la unidad estructural básica de los terpenoides (el isopreno) y finalmente la familia de estos. Los terpenoides conforman una familia de biomarcadores muy interesante pudiendo formar compuestos cíclicos o lineales y distinguiéndose 8 familias en función del número de unidades de isopreno que pueden presentar, desde el hemiterpano (una única unidad de isopreno) hasta politerpano (presenta 9 o más unidades de isopreno). De hecho, algunos estudios (Arekhi et al. 2021), han demostrado la estabilidad de algunos biomarcadores pesados pertenecientes a las familias de esteranos y terpenos. Los compuestos que sufrieron cambios a lo largo de los 10 años de estudio resultaron ser exclusivamente los compuestos con menor peso molecular pertenecientes a la familia de tricicloterpanos y esteranos. Un ejemplo práctico para el uso de la huella dactilar en los análisis de petróleo, es la investigación que llevó a cabo Wang y colaboradores en el año 2002 (Wang et al. 2004). En abril de ese año se produjo un vertido en el rio Detroit, frontera natural que separa Estados Unidos de Canadá. Se tomaron 11 muestras, de las cuales 3 fueron enviadas al laboratorio de los investigadores mencionados. Estos tomaron las muestras y realizaron el fraccionamiento de las mismas con el mismo método mencionado previamente (Wang et al. 1994). Una vez separado el petróleo en dos fracciones, los hidrocarburos alifáticos y aromáticos se analizaron tanto mediante GC-FID como GC-MS. Mediante Capítulo 1 16 la cuantificación de biomarcadores y compuestos policíclicos aromáticos (PAHs) y comparando ratios entre compuestos, llegaron a la conclusión de que los tres puntos analizados mostraban contaminación por el mismo vertido de petróleo y presentaba uno de ellos un mayor envejecimiento del petróleo por las condiciones climáticas y geográficas. Son múltiples los esfuerzos realizados tanto por las administraciones públicas como por los organismos privados en la investigación relacionada con los vertidos de petróleo, desde la identificación de la fuente de origen o métodos de toma de decisión a la evaluación de la toxicidad del vertido. Fruto de estos esfuerzos son las bases de datos mundiales sobre productos petrolíferos. Algunas de las más importantes son Environment Canada (Whiticar et al. 1992), que es una base de datos elaborada por el Departamento de Medio Ambiente de Canadá, y que incluye diversas propiedades tanto físico-químicas como de comportamiento tras un vertido (dispersabilidad, etc.). Por otro lado, Concawe, la división de compañías petroleras que operan en Europa, ha publicado múltiples informes donde se detallan productos crudos originarios de Oriente Medio y Europa, metodologías para la evaluación de la toxicidad de productos derivados del petróleo o para su análisis mediante micro-extracción en fase sólida (SPME)-GC (King et al. 2001; Comber et al. 2011; Comber et al. 2016). Otras instituciones como es el caso de la National Oceanic and Atmospheric Administration (NOAA), desarrolló en 1994 el modelo ADIOS (Automated Data Inquiry for Oil Spills) (National Oceanic and Atmospheric Administration 1994) para la toma de decisiones ante un vertido a partir de bases de datos de los petróleos que incluye. Este programa ha sido actualizado desde entonces hasta la versión actual (2016). Paralelamente, existen multitud de institutos de investigación que trabajan en el análisis e identificación de petróleos y vertidos de petróleo o en la evaluación de la toxicidad de los petróleos, ya que esto depende mucho de la localización donde se produce, como ha sido Introducción 17 mencionado. Actualmente, los institutos de investigación más relevantes en este sentido son el SINTEF (SINTEF 1950) situado en Noruega o el Cedre (CEDRE 1978) en Francia, ambos referencias mundiales en la investigación del petróleo. 1.3. Ensayos de toxicidad Uno de los focos de interés más acuciantes es la evaluación de los efectos tóxicos y del riesgo medioambiental de los efectos que producen los vertidos de petróleo. En este sentido, las características intrínsecas del petróleo han dificultado el establecimiento de una metodología uniforme para su evaluación y la comparación de los efectos entre diferentes vertidos. Para evaluar la toxicidad, se parte de preparación de la fracción acomodada del petróleo (Water accomodated fraction, WAF) poniendo en contacto petróleo y agua. Esta mezcla es agitada a baja energía (sin la aparición de vórtex), de forma que se favorece la suspensión de los compuestos en agua, pero se evita emulsión o aparición de microgotas en el agua (Cruz, Coelho, and Aurand 1997). Además del WAF, también se suele evaluar la toxicidad del denominado WAF optimizado químicamente (CE-WAF), ya que en este caso además de la utilización de vórtex en su preparación, se adiciona un dispersante (práctica aplicada en el tratamiento de vertidos) lo que ocasiona una suspensión coloidal del petróleo cambiando tanto la composición, apariencia, como la toxicidad del mismo. Actualmente, el método de producción de este tipo de medios se ha estandarizado gracias al propuesto por Singer y colaboradores (Singer et al. 2000). En la Tabla 2 se presentan las mejores condiciones según este estudio para la preparación de WAF y CE-WAF. Como se observa en la Tabla 1.2, las condiciones para preparar WAF y CE-WAF son distintas, tanto en relación con las condiciones de agitación como los tiempos de preparación, requiriendo Capítulo 1 24 Introducción 25 Otra de las posibles fuentes de contaminantes es la pérdida de carga en alta mar, que posteriormente y debido a las corrientes marinas, son arrastradas a la costa, tal y como ha pasado con los “pellets” de plástico que han inundado recientemente las costas de Galicia, Asturias, Cantabria(García 2024 Jan 9). Estas no son las únicas vías de entrada de los potenciales contaminantes al medio marino, que como se muestra a continuación, en función de la familia de compuestos referida, presenta una fuente distinta. El uso de las aguas costeras como zona de baño, aporte de agua dulce o presencia de fuentes de contaminantes antropogénicas ocasiona la contaminación de estas por una amplia variedad de compuestos químicos. Aunque han sido identificados en el crudo del petróleo más de 200 compuestos orgánicos (Wang et al. 2007), dentro del amplio número de sustancias orgánicas diferentes que existen, encontramos familias no relacionadas con el petróleo igualmente importantes a nivel medio ambiental. Dentro de las familias no relacionadas con el petróleo de carácter no-polar, encontramos una amplia variedad de compuestos orgánicos semi-volátiles (SVOCS), como es el caso de los filtros UV, los bifenilos policlorados (PCBs), entre otros. 1.5.1. Presencia en el medio ambiente Gracias a su estabilidad y semi-volatilidad, los SVOCS están considerados contaminantes ubicuos, ya que su transporte y deposición puede darse a largas distancias, por lo que se han detectado en una amplia variedad de matrices medioambientales (Riaz et al. 2021). Entre los SVOCS se encuentran los PAHs, los bifenilos policlorados, los esteres de ftalato (PAEs), los plaguicidas organoclorados como el dicloro difenil tricloetano (DDT), los productos de cuidado personal como las fragancias sintéticas, o los filtros ultravioletas, entre otras familias. A pesar de que Capítulo 1 26 el uso, fabricación y distribución de muchos de ellos se ha restringido, incluso prohibido a nivel europeo bajo la DIRECTIVA 79/117/CCE del Consejo (Parlamento Europeo 1983), dada su gran persistencia, actualmente se siguen detectando en distintos tipos de matrices medioambientales, como los sedimentos o muestras acuosas de diferente naturaleza (Lao et al. 2021; Wrobel and Mlynarczuk 2021). El hecho de que la vida media de muchos de estos compuestos sea superior a 10 años, les ha hecho ganarse la denominación de contaminantes orgánicos persistentes (POPs) suscitando un interés creciente, tanto por parte de la comunidad científica, como por parte de las diferentes administraciones reguladoras (Tang et al. 2020). Fruto de este interés internacional, son múltiples los estudios que han tratado de evaluar la presencia de estas familias de compuestos en una variedad de matrices acuosas medioambientales y sólidas como los sedimentos y los mejillones, tal y como se observa en la Tabla 1.4. Los bifenilos policlorados son un ejemplo de estos compuestos que han sido ampliamente investigados debido a su estructura aromática halogenada que presenta una gran estabilidad química y física. Un ejemplo claro de su persistencia es el estudio realizado por Tatiane y colaboradores (Combi et al. 2016). En su estudio, la concentración en sedimentos (histórico de la contaminación de un lugar) presentó mayor concentración de PCBs que en el sedimento superficial, lo que demuestra el efecto de la prohibición, en la década de los 70, del uso de este tipo de compuestos. Otro de los estudios realizados en la bahía de Bengala, en la costa de Bangladés, se centró en la evaluación de las aguas superficiales y en todos los puntos de muestreo se detectaron PCBs, indicando la amplia presencia en extensión que pueden alcanzar estos compuestos. Introducción 27 De los estudios revisados, las mayores concentraciones obtenidas en aguas para PCBs se obtuvieron en el estudio realizado en China por Yang y colaboradores (Yang et al. 2019), que reportaron entre 13-100 ng/L en aguas interiores. Otro de los factores que evidencia la importancia del estudio de estos compuestos, es su presencia en la Antártida, como lo demuestra el estudio realizado por Vecchiato y colaboradores (Vecchiato et al. 2015), evidenciando el transporte a largas distancias de estos compuestos. Por otro lado, las concentraciones más bajas en sedimentos se encontraron en zonas alejadas de la actividad humana, por ejemplo, en el Océano Índico, con concentraciones sorprendentemente inferiores a las encontradas en la Antártida (116-183 pg/g), como se observa en la Tabla 1.4. Un organismo que está considerado como centinela del estado de contaminación de los medios marinos costeros es el mejillón, debido a su capacidad de filtrar agua y acumular compuestos orgánicos en sus tejidos. Todo esto los hace organismos idóneos a la hora de evaluar la bioacumulación de los compuestos en el medioambiente y específicamente en este tipo de matrices (Beyer et al. 2017). Múltiples estudios se han realizado en este sentido (ver ejemplos en la Tabla 1.4). En un estudio realizado en California (Roldán-Wong et al. 2020), se obtuvieron altas concentraciones de PAHs en los mejillones analizados. Los compuestos con concentraciones más altas detectadas fueron los de bajo peso molecular como naftaleno o fenantreno. Por el contrario, se encontraban en menor concentración o incluso no eran detectados en los sedimentos. Esto es debido seguramente a la relativa fácil degradación que pueden sufrir estos compuestos en comparación a los de alto peso molecular, siendo degradados en el sedimento, pero manteniéndose inalterados cuando son acumulados por los bivalvos. Además, el hecho de ser mayoritarios los de menor peso molecular, demuestra el origen antropogénico de estos compuestos. Capítulo 1 28 Introducción 29 Capítulo 1 30 Otro estudio que también realiza la cuantificación de estos compuestos en mejillones en Groenlandia realizado por Poulsen y colaboradores (Poulsen et al. 2021) reportó resultados menores que en el realizado en California, pero encontró diferencias entre el puerto muestreado y los puntos de referencia, lo que evidencia la contaminación de los puertos frecuentados por pequeñas naves de pesca. Además, a pesar de que los PAHs de menor peso molecular están en mayor concentración en las muestras comentadas, la presencia de compuestos con mayor peso molecular ha sido relacionada con una mayor ecotoxicidad de estos compuestos (Roldán-Wong et al. 2020). Otra familia de SVOCs que merece ser destacada son los pesticidas. Dichos compuestos comprenden una amplia gama de familias con propiedades insecticidas, herbicidas o fungicidas. La síntesis del primer insecticida se realizó en los años 40, es el caso del dicloro difenil tricloroetano (mayormente conocido como DDT) para el control del tifus, la malaria y otras enfermedades, cuyo origen radicaba en insectos, extendiéndose su uso posteriormente (Chueycham et al. 2021). Desde entonces, la producción de esta gama de compuestos no ha dejado de crecer, aunque ha aumentado también la preocupación por la presencia y detección de los mismos, así como de sus potenciales efectos tóxicos tanto en el medio ambiente como en el ser humano (Rodríguez 2007). Actualmente se estima que un 40 % de los pesticidas utilizados son organoclorados (Gupta 2004). Como otros compuestos de baja polaridad, los plaguicidas organoclorados (también conocidos como OCPs), tienden a acumularse en fases orgánicas, como la fase lipídica de animales vivos, lo que ocasiona que se bioacumule a lo largo de la cadena trófica, que presentan efectos tóxicos en humanos muy variados, desde la disrupción endocrina (Holm et al. 2006), desarrollo de tumores (Cohn et al. 2007) a la afectación del sistema reproductivo (Song et al. 2016). Actualmente se estima que un millón de personas desarrolla alguna enfermedad crónica o muere a causa del uso y/o contactos con los Introducción 31 pesticidas (Jayaraj et al. 2016). Li y colaboradores (Li et al. 2018) reportaron la presencia de DDTs y hexaclorociclohexanos (HCHs). El β-HCH se detectó en todas las muestras analizadas. La concentracion máxima de HCHs fue de 156 ng/g y para los DDTs fue de 116 ng/g, tal y como se observa en la Tabla 1.4. Estos compuestos también han sido detectados en otras matrices como mejillones, tal y como reportaron Milun y colaboradores (Milun et al. 2016) (ver Tabla 1.4). Ellos encontraron en el mar Adriático concentraciones de hasta 8 ng/g, siendo el DDT el compuesto que presentaba niveles más elevados. Por el contrario, Herceg y colaboradores (Herceg-Romanić et al. 2014) detectaron los mismos contaminantes en menor concentración. Esta variación puede estar dada por la diferente tipología de los datos mostrados (muestra seca frente a muestra húmeda) y también debido al período de muestreo, abarcando en el caso de Milun y colaboradores los 9 años, mientras que en el caso de Herceg y colaboradores unos pocos meses. En los últimos años están suscitando especial interés los productos de cuidado personal (PCPs) (Wang et al. 2021). Debido a la gran variedad de características químicas y físicas que poseen, existen varias familias que comprenden estos compuestos, siendo las principales las fragancias sintéticas y los filtros solares, debido a su mayor utilización en una variedad de productos. En el caso de las fragancias, su clasificación depende de su estructura, definiéndose 4 grupos: fragancias de almizcle nitro, policíclico, macrocíclico y alicíclico (Vallecillos et al. 2015). Su estructura molecular se representa en la Figura 1.5. Capítulo 1 32 Figura 1.5. Estructuras tipo de las distintas familias ed fragancias de almizcle. De izquierda a derecha almizcle xileno (nitro), galaxolide (policíclico), civetona (macroclícico) y helvetolide (alicíclico). Actualmente se considera que las fragancias policíclicas representan el 90 % de las fragancias consumidas en Europa y Estados Unidos (Lee et al. 2014). Dadas las características estructurales de estos compuestos y su polaridad, han demostrado ser lipofílicas y persistentes en el medio ambiente, pudiendo bioacumularse a lo largo de toda la cadena trófica. Además algunos estudios reportan posibles efectos en la salud como la disrupción endocrina (Wong et al. 2019). Las fragancias de la familia de los nitro almizcles fueron las primeras en ser sintetizadas, pero debido a la presencia de grupos nitro en su estructura, generaron una considerable controversia con respecto a la idoneidad de su uso. Los filtros solares orgánicos son los más utilizados en la industria cosmética. Se caracterizan por su estructura molecular, que suele contener uno o varios anillos aromáticos con sustituciones hidrofóbicas para mejorar sus propiedades. Normalmente se suelen utilizar mezclas de filtros solares para la mejora de la protección de la radiación ultravioleta (Giokas et al. 2005). Dada la importancia que tiene el cuidado de la piel, el uso de filtros ultravioleta se ha masificado en los últimos años, siendo su principal vía de entrada al medio ambiente marino las actividades recreativas y las aguas residuales industriales o domésticas (Díaz-Cruz and Barceló 2015). Su uso generalizado, junto a su persistencia en el medio, ha ocasionado que múltiples estudios hayan Introducción 33 reportado su presencia en distintas matrices medioambientales como las aguas superficiales localizadas cerca de áreas metropolitanas (Tsui et al. 2014), y sedimentos (Huang et al. 2016). Son múltiples los estudios que han evaluado la toxicidad de estos compuestos de forma individual. Dado que la exposición real tiene lugar en presencia de una variedad de compuestos, el problema es estimar adecuadamente las interacciones que pueden tener lugar. Además, los derivados de las benzofenonas son los compuestos en los cuales se ha realizado mayor investigación al respecto (Huang et al. 2021). En este sentido hay estudios que relacionan la exposición a benzofenonas con una menarquia temprana, desarrollo tardío del sistema reproductor en niñas (Wolff et al. 2015), y desarrollo de diabetes (Ward et al. 2020), entre otras enfermedades. Debido a esto, dichos compuestos son evaluados en distintas muestras mediambientales. Por ejemplo, un estudio realizado al sur de China en aguas costeras por Tsui y colaboradores (Tsui et al. 2019) donde monitorizaron 11 filtros solares, 2 de ellos, 3-benzofenona (BP-3) y octocrileno, fueron reportados en todas las muestras analizadas. La concentración máxima obtenida fue de 145 ng/g. La amplia presencia de la 3- benzofenona usualmente está relacionada con su amplio uso en protectores solares personales. Para este compuesto, los estudios realizados en Portugal y en la costa española muestran grandes diferencias en los niveles de concentración, reportándose valores de 622 y 219 ng/g, respectivamente. 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Biomonitorización de PAHs y metales tras el vertido del Prestige. Procesos de bioacumulación y reparto de contaminantes. UPV/EHU. Navarro Villaverde, Patricia. (2008). Metodología para el análisis y validación de PAHs y metales en sedimentos y biota. UPV/EHU. Posada-Ureta, O., Olivares, M., Delgado, A., Prieto, A., Vallejo, A., Irazola, M., Paschke, A., & Etxebarria, N. (2017). Applicability of polydimethylsiloxane (PDMS) and polyethersulfone (PES) as passive samplers of more hydrophobic organic compounds in intertidal estuarine environments. Science of The Total Environment, 578, 392–398. https://doi.org/10.1016/J.SCITOTENV.2016.10.194 Posada-Ureta, O., Olivares, M., Zatón, L., Delgado, A., Prieto, A., Vallejo, A., Paschke, A., & Etxebarria, N. (2016). Uptake calibration of polymer-based passive samplers for monitoring priority and emerging organic non-polar pollutants in WWTP effluents. Analytical and Bioanalytical Chemistry, 408(12), 3165–3175. https://doi.org/10.1007/S00216-016-9381-7/METRICS Prieto Sobrino, Ailette (2008). Desarrollo de métodos analíticos de rutina para la determinación simultánea de contaminantes prioritarios en muestras medioambientales. Desarrollo de Métodos Analíticos de Rutina Para La Determinación Simultánea de Contaminantes Prioritarios En Muestras Medioambientales. Chemical characterization of oil and Water Accommodated Fraction (WAF) at different temperatures 3 Chemical characterization of oil and Water Accommodated Fraction (WAF) at different temperatures 61 3.1. Introduction Maritime transport through Arctic sea has increased along the last years due to the fast recession of the Arctic sea ice allowing the creation of new maritime ways (Lasserre et al. 2016). Therefore, the risk of oil spills is very concerning due to the exploitation of submarine resources and the delicate environmental features of this region. Moreover, the physical (low temperature, salinity, light intensity) and biological (fragility of marine species) conditions make remarkable oil spill’s persistence and its impacts (Kristensen et al. 2015; Brown et al. 2016). The ways to minimize, control or treat oil spills typically involves, among others, oil booms, skimmers and sorbents, which increases the cost (Nelson-Smith and Wardley-Smith 1984). The spray of surfactants to disperse more easily the oil slicks is one of the most immediate response to oils spills, as it has been described in the Deepwater Horizon incident (John et al. 2016). However, both the assumed stimulation of the microbiological biodegradation of the petroleum and the lack of impacts in higher trophic levels and communities are still under revision (Kleindienst et al. 2015). Oil contamination may cause serious problems to aquatic life, especially at the lowest level of the trophic chain (Ozhan and Bargu 2014). Most toxicological research of crude oil contamination has revealed that its toxicity is mainly caused by the presence of aromatic hydrocarbons (PAHs) in the water accommodated fraction (WAF) and the water soluble fraction (WSF). The main difference between these two fractions is that the former includes dispersed droplets and lacks the most volatile compounds (Forth et al. 2017; Bera et al. 2020). WAF is quite different from the parent oil and the concentration of components depends mainly on variables such as oil type, oil-water ratio, mixing time and exposure temperature(Faksness et al. 2008). Although the heaviest PAHs are potentially Chapter 3 62 more toxic, the lightest ones show a higher solubility in water leading to a higher bioavailability to marine organisms, and therefore, they can be considered as the responsible agents for most of the toxic effects. The most common procedure to obtain hydrocarbon saturate aqueous samples is the preparation of the water-accommodated fraction (WAF), which is typically carried out by mixing petroleum and seawater in 1:20 and 1:40 proportions during 24 hours (Faksness et al. 2008). However, according to the literature, certain controversy has arisen around the composition of this aqueous phase and, therefore, more elaborated alternatives have been proposed (Nordtug et al. 2011). Despite of the assessment of ecotoxicological effects of water accommodated fractions (Han et al. 2017)(Kim et al. 2013)(Williams et al. 2018) including the chemical characterization (Faksness et al. 2015; del Brio, Montagna, Lares, Parolo, Venturino, et al. 2018; Cariello Delunardo et al. 2019) it is hard to foresee the sort of environmental consequences that may take place after a spill, due to the high variety of oil types (heavy, diesel, light, among others), the susceptibilities of the different species and the resilience of the affected communities. In addition to this, the production of WAF for bioassay testing has shown a high controversy in the literature (Singer et al. 2000; Faksness et al. 2008; Forth et al. 2017) though operative standard procedures were agreed in most of the cases. In the framework of Grace project the suitability of different oil spill response method for the Artic environment have been tested(Jørgensen et al. 2019). In this sense, in the Plentzia Marine Station (PiE-UPV/EHU) several exposure and ecotoxicological studies based on the use of these WAF samples have been carried out (Blanco-Rayón et al. 2019; Katsumiti et al. 2019). Nonetheless, this Chemical characterization of oil and Water Accommodated Fraction (WAF) at different temperatures 63 work was accomplished in order to study the kinetic performance of the preparation of the WAF samples and to study their stability since it is a key to design and interpret further exposure experiments. In addition to this, it is particularly interesting to study the feasibility of PDMS sheets or discs both to monitor the WAFs and to use as passive samplers and dosing, as it has been pinpointed in the literature (Redman et al. 2014; Bera et al. 2018). In order to accomplish this work we designed a smaller vessel to prepare multiple WAF samples including PDMS sheets and PDMS-coated stir-bars (TwistersR). This way, the objectives of this work were to: (i) chemically characterize three different oils, Fuel Oil 180 (IFO 180), Naphthenic North Atlantic (NNA) and Marine Gas Oil (MGO), and (ii) evaluate the hydrocarbons composition of freshly prepared WAF and its dissipation or stability over time with and without dispersant presence (Finasol OSR52) at different temperatures (5, 10, 15, 20 and 25 °C). In addition, untargeted analysis by gas chromatography coupled to high resolution mass spectrometry (GC-QTOF) will be applied and the results will be compared with commercial libraries to obtain a tentative identification of their components in more depth. 3.2. Experimental Section 3.2.1. Chemicals and materials Mix standard solution of 18 Polycyclic Aromatic Hydrocarbon (PAHs) CRM47543: naphthalene (N), 1-methyl naphthalene (1-MN), 2-methylnaphthalene (2-MN), acenaphthylene (Acy), acenaphthene (Ace), fluorene (F), phenanthrene (P), anthracene (Ant), fluoranthene (Fl), pyrene (Pyr), benz[a]anthracene (BaAnt), chrysene (Chr), benzo[b]fluoranthene (BbF), benzo[k]fluoranthene (BkF), benzo[a]pyrene (BaP), indeno[1,2,3-cd]pyrene (IndPyr), dibenz[a,h]anthracene and (DBahAnt), Chapter 3 64 benzo[g,h,i]perylene (BghiP) was purchased from Supelco (Bellefonte,PA, USA). A mixture of 16 PAHs [Norwegian Standard (NS 9815: S-4008-100-T): phenanthrene, anthracene, fluoranthene, pyrene, 11H-benzo[a]fluorene (11BaF), 11H-benzo[b]fluorene (11BbF), benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, benzo[e]pyrene (BeP), indeno[1,2,3- cd]pyrene, dibenzo[a,h]anthracene, benzo[g,h,i]perylene and dibenzo[a,e]pyrene (DBaeP) was supplied by Chiron (Trondheim, Norway). A mixture of 5 deuterated compounds [Norwegian Standard (S-4124-200-T): naphthalene-d8, byphenyl-d10, phenanthrene-d10, pyrene-d10, benzo[a]anthracene-d12, benzo[a]pyrene-d10, benzo[ghi]perylene-d12 was adquired from Chiron. n- Alkano Mix (49452-U, C7-C40 Saturated Alkanes Standard) was purchased from Supelco. Methanol, hexane, toluene and dichloromethane (HPLC grade, 99.9 %) were purchased from Macron (Radnor, USA). Helium with 99.999 % purity was purchased from Messer (Barcelona, Spain). Silica gel was purchased from Supelco and anhydrous sodium sulphate from Probus (Barcelona, Spain). 3.2.2. Oil analysis Three oils were studied in this work, NNA, IFO 180 and MGO. IFO 180 oil is used in ships and energy production plants, MGO is most commonly used in medium and medium/high speed ships and, finally, NNA oil is obtained from North Sea of Norway and their name is referred to the platform in which was extracted. These three oils are characterized by their density (between 0.845 g/mL for NNA and 0.97 g/mL for IF0180) and their viscosity (from 3.45 mm2/s of NNA to 180 mm2/s of IFO 180). Finasol OSR52 was used as dispersant (Total Special Fluids, Paris, France), which is a third generation dispersant and is approved to be used by EPA (USA), MMO (UK) and CEDRE (France). Chemical characterization of oil and Water Accommodated Fraction (WAF) at different temperatures 65 For the characterization of the oils, the fractionation method described by Wang and Fingas was applied (Wang et al. 1994). Briefly, a column was plugged with Pyrex glass wool at the bottom, and then, was rinsed with 20 mL methanol, hexane and dichloromethane. Once the wool was dried, the column was packed with 3 g of activated silica gel, topped with about 0.5 cm of anhydrous sodium sulphate. Column conditioning was carried out using 20 mL of hexane, which was discarded. Just prior to exposure of the sodium sulphate layer to air, 200-400 µL aliquot of the oil diluted in hexane (approximately 100 mg/mL) were transferred onto the column using 3 mL of hexane and all the solvents discarded. Elution was carried out using 12 mL of hexane and 15 mL of benzene: hexane mixture (50:50, v:v) for aliphatic and aromatic hydrocarbons, respectively. Elution solvents were evaporated to dryness under N2 stream using a TurboVap LV Evaporator (Zymark, Hopkinton, USA) at 35 ˚C, and then, the dry residue reconstituted with 250 µL of hexane. Finally, extracts were analysed by gas chromatography coupled to mass spectrometer (GC-MS) (see section 3.2.4). 3.2.3. WAF production and characterization 3.2.3.1. Standard production of small scale WAF The production of WAF was standardized in the framework of Grace project (Jørgensen et al. 2019) but in this work we scaled the method to produce small quantities of WAF. Briefly, thoroughly cleaned 160 mL glass bottles (Auxlaper, Barcelona, Spain) were used as shown in Figure 3.1. The bottles were filled with 130 mL of filtered seawater (filtered with PTFE 0.45 µm filter) and 0.65 g of oil on the top. When the dispersant was used, 0.065 g of Finasol OSR52 and 0.65 g of the oil were previously mixed. In order to ease the removal of the WAF without any contact with the oil layer, a glass tube (10 cm long, 10 mm i.d. and 1 mm thick) was fixed to the mouth of the bottle with the help Chapter 3 72 Consequently, oil diluted samples were analysed directly and the obtained profiles are shown in Figure 3.3. As can be seen, these three profiles are clearly different: In all cases, lighter compounds such as methylated naphthalenes and naphthalene show the highest concentrations, this profile is also reported by other studies as (Rossi and Anderson 1976; Polli et al. 2020). In those studies, naphthalene was the major compound of 16 PAHs of evaluated compounds. On the one side, IFO oil shows higher levels of methylnaphthalenes than the other two oils and it is the only one showing significant levels of Ant. On the contrary, NNA is richer in N and IFO show a lower concentration. The profile of intermediate PAHs, like F, Fl, P and Pyr is also different but in this particular case the concentrations follow the same pattern (IFO>MGO>NNA). Figure 3.3. Distribution of PAHs in the three oils studied (measured by direct analysis). Chemical characterization of oil and Water Accommodated Fraction (WAF) at different temperatures 73 3.3.2. WAF formation kinetics We found interesting to monitor the release of the target PAHs from the oil slick to the aqueous media first, and to the PDMS samplers (a sheet and a stir-bar). As depicted in Figure 3.4, the kinetic study can be modeled following a three compartment distribution equilibria, i.e. oil, water and PDMS (including both the sheet and the stir-bar) and assuming that the headspace fraction is negligible for semivolatile PAHs under this experimental setup (a closed and small headspace). Under the constrains of this system (at time 0, Co(0) = Co, Cw(0) = Csheet(0) =Cstirbar(0)= 0, where Co is the concentration in oil, Cw in water, and Csheet in the PDMS sheet and Cstirbar in the PDMS stir-bar) the set of differential equations (i.e. eqs 1,2,3) can be written only considering a single PDMS phase. Figure 3.4. A three-compartment model to study the distribution kinetics of hydrocarbons: the oil drop, the water-soluble fraction and the sorbed fractions in the PDMS sheet and stir-bar 𝑑𝐶0 𝑑𝑡 = 𝑘1𝑎 ·𝐶𝑤−𝑘1𝑑 ·𝐶𝑜 (eq.1) 𝑑𝐶𝑝𝑠 𝑑𝑡 = 𝑘2𝑎 ·𝐶𝑤−𝑘2𝑑 ·𝐶𝑝𝑠 (eq. 2) Chapter 3 74 𝑑𝐶𝑤 𝑑𝑡 =𝑉𝑂 𝑉𝑤·𝑘1𝑑 ·𝐶𝑂+𝑉𝑝𝑠 𝑉𝑤·𝑘2𝑎 ·𝐶𝑝𝑠 −𝑉𝑂 𝑉𝑤·𝑘1𝑎 ·𝐶𝑤−𝑉𝑝𝑠 𝑉𝑤·𝑘2𝑎 ·𝐶𝑤 (eq. 3) Though we have found the analytical solution of this system (Korobov and Ochkov 2011), it is still rather cumbersome to solve, so we decided to simplify assuming that the oil drop is an infinity well of PAHs and, therefore, Co(t) = Co. Therefore, we can write the following equations where we have now considered the two PDMS phases: the sheet and the stirbar 𝐶𝑤(𝑡)= 𝐶𝑜·(𝑘1𝑎 𝑘1𝑑)·(𝑉𝑂 𝑉𝑤)·(1−𝑒𝑥𝑝(−𝑘1𝑎 ·𝑡)) (eq. 4) 𝐶𝑠ℎ𝑒𝑒𝑡(𝑡)= 𝐶𝑤(𝑡)·(𝑘2𝑑 𝑘2𝑎)·(𝑉𝑝𝑠 𝑉𝑤)·(1−𝑒𝑥𝑝(−𝑘2𝑑 ·𝑡)) (eq. 5) 𝐶𝑠𝑡𝑖𝑟𝑏𝑎𝑟(𝑡)= 𝐶𝑤(𝑡)·(𝑘𝑑3 𝑘3𝑎)·(𝑉𝑡𝑤 𝑉𝑤)·(1−𝑒𝑥𝑝(−𝑘3𝑑 ·𝑡)) (eq. 6) As shown in Figure 3.5, the concentration of phenanthrene in the three media is plotted and though the kinetic profiles are slightly different in water and in PDMS, the steady state in the aqueous phase is achieved sooner than in the stir-bar and PDMS sheet. Since we have experimental Cw(t), Csheet(t) and Cstirbar(t) values, the 4-6 equations were fitted with Solver (Excel spreadsheet) as shown for Phen and Fl in Figure 3.5 and the solveraid macro (Prikler 2009) providing the values for the kinetic constants and their uncertainties. The slightly higher values of the kinetic constants for the stir-bar than for the sheet can be attributed to the higher stirring rate of the former. Similar plots were also obtained with those PAHs (i.e. pyrene, benzo(a)fluorene, benzo(b)fluorene, benzo(a)anthracene and chrysene) that were detected in the three phases but the as the water solubilities were getting lower for heavier and more hydrophobic PAHs, the uncertainties Chemical characterization of oil and Water Accommodated Fraction (WAF) at different temperatures 75 estimated for some of the kinetic parameters were rising, and therefore, only the results obtained for Phen and Fl are reported. PAH 𝒌𝟏𝒂 𝒌𝟏𝒅 𝒌𝟐𝒂 𝒌𝟐𝒅 𝒌𝟑𝒂 𝒌𝟑𝒅 P 6.6±0.4 e-5 8.0±0.4 e-2 30 ± 2 7.1±0.1 e-4 411 ± 21 1.62±0.02 Fl 4.8±4.0 e-7 6±14.0e-2 70±500 1.1±1.6 e-2 880 ± 800 1.0±0.7 e-2 Figure 3.5. On top, kinetic curves of phenanthrene and fluoranthene transfer from oil to water, PDMS sheet and PDMS stir-bar and the values of the kinetic constants with the uncertainty (±s). Below, the fitted kinetic parameters, as writen in eqs. 4-6. Chapter 3 76 If we observed in detail when the steady state is achieved in the water phase, we can say that for most of the PAHs it is between 50-100 h of stirring. However, to see the same in the polymer phases we have to keep stirring beyond 200 h. Consequently, the WAF conditions implemented in all the works is slightly lower (40 h) than the suggested by these results. However, the gap of concentrations using the reference procedure and a longer one is less than 20%, which can be acceptable for most of the experiments. Conversely, previous studies suggested less than 48 hours of oil-water exposition time (Singer et al. 2000) though the time to steady state can vary from oil to oil (Faksness et al. 2008), being lower for the naphthenic ones than for paraffinic or asphaltenic ones, which would only need 48 hours to steady state. 3.3.3. Assessment of the WAF composition Different WAFs were prepared using the oils included in section 3.2 at different temperatures (5, 10, 15, 20 and 25 °C) with and without dispersant OSR52 Finasol. The effect of the dispersant and the temperature is shown in Figure 3.6 Chemical characterization of oil and Water Accommodated Fraction (WAF) at different temperatures 77 Figure 3.6. ΣPAH in the WAFs prepared at different temperatures with and without the dispersant. In the first case, we have plotted the ΣPAH at all the temperatures and we can easily see that NNA shows significantly higher concentrations and remarkably enhanced when the dispersant was used. As it was pointed before, the effect of the addition of the dispersant is clearly uneven since for NNA the addition enhances the fraction of PAHs dissolved at all the temperatures but for MGO mostly the opposite effect is observed at all the temperatures except for the higher one. As can be seen, the pattern of PAH distribution (see Figure 3.7) in all oils WAFs at all the temperatures is rather similar surely due to the limit of the solubilities. The most abundant PAHs are the lighter ones as methylnaphthalenes and related compounds, similar results were reported in other studies as (Faksness et al. 2008). In the opposite, the heavy PAHs are mostly at very low concentrations and in many times below the limits of detection (LODs), this also was reported by Chapter 3 78 Figure 3.7. Distribution of detected PAHs in IFO, MGO and NNA (LogC(ng/g)) oils for five different temperatures: 5, 10, 15, 20 and 25 °C Chemical characterization of oil and Water Accommodated Fraction (WAF) at different temperatures 79 (Faksness et al. 2008), estimated as mean values ± 3 times the standard deviation. The LODs ranged from 0,001 to 24 ng g-1 and between 1 and 230 ng g-1 for PDMS sheet and Twister respectively. The addition of dispersant does not show a systematic increase of the concentrations of any PAHs in none of the oils this results are against to other studies as (Tairova et al. 2019) where when Corexit 9500 dispersant was added to WAF production, obtained concentration of total hydrocarbon content (THC) was incremented on a range of 20-25 fold. In the case of IFO the addition of dispersant produces a significant increase of the concentrations of the heavier PAHs (BaAnt, BaP…) and a systematic increase of the concentration of the rest of the PAHs. In the case of MGO and for NNA the opposite trend is advisable. Regarding to the effect of the temperature, it is better to observe these variations with target PAHs instead that the sum of all of them, as shown in Figure 3.8 for phenanthrene and pyrene. In the case of bare oils, without the addition of dispersant, the plots of the experimental concentrations and the linear trend suggest a steady increase of the solubility with IFO and MGO and more scattered results with NNA. Those results are according to (Whitehouse 1984) in which study evaluate the effect of salinity and temperature on solubility on aromatic hydrocarbons, and their results shown a increases of solubility of these compounds when medium temperature rises. Chapter 3 80 Figure 3.8. Concentrations (ng/L) of Phenanthrene and Pyrene for different WAFs prepared without dispersant (on top) and with dispersant (below) and at different temperatures (5, 10, 15, 20 and 25 °C). (With dispersant) (With dispersant) (Without dispersant) (Without dispersant) Chemical characterization of oil and Water Accommodated Fraction (WAF) at different temperatures 81 Though it is beyond the scope of this work, the slopes of the linear fits of Phen and Pyr are rather constant, which would allow the prediction of the solubility of these compounds regardless the type of oil. Additionally, it is also worth saying that the increase of the temperature from 5 °C to 25°C would almost double the concentration of these PAHs, and, therefore, the potential risk of oil spills is dramatically enhanced in warmer seas and, eventually, by the rise of temperatures all over the world. The effect of the temperature when the oils were mixed up with the dispersant is completely different, as shown in Figure 3.8 for Phen and Pyr. According to these plots, only MGO would follow the pattern observed before without the addition of the dispersant. In the other two cases, the trend is the opposite and it is slightly more clearly seen with IFO than with NNA. 3.3.4. WAF stability at room temperature One of the key points of the WAFs is the stability because this would limit the scope and the suitability of different bioassays or toxicological tests. The stability of WAF was studied with and without dispersant with NNA oil. In Figure 3.9 we have plotted the decay of P in both bare WAF and WAF with dispersant, though the concentration with dispersant are two orders of magnitude higher, it is worth mentioning the parallel decay trend (without dispersant slope = -0.028 ± 0.002 and with dispersant slope = -0.0226 ± 0.0005). In practical terms, these values would suggest that the half-life of these WAFs would be roughly between 24 h and 30 h with and without dispersant, respectively. In contrast, other studies as (del Brio, Montagna, Lares, Parolo, and Venturino 2018), evaluate toxicity of WAF and also determinated dissipation rates of TPH, and they concluded that t½ was 71 ± 32 h significantly higher than obtained in this study, though less precise. Chapter 3 88 Table 3.3. Tentatively identified compounds in each analysed oil applying the parameters presented in Table 3.2. Compound name Formula Troll IFO Diesel Naphthalene, 2-ethyl- C12H12 x x Naphthalene, 2-methyl- C11H10 x Naphthalene, decahydro-2-methyl- C11H20 x x Nonacosane C29H60 x Nonadecane, 2-methyl- C20H42 x Nonadecane, 3-methyl- C20H42 x Nonane C9H20 x Nonane, 2,6-dimethyl- C11H24 x x Nonane, 2-methyl- C10H22 x Nonane, 3,7-dimethyl- C11H24 x x Octacosane C28H58 x Octadecane C18H38 x Octadecane, 3-methyl- C19H40 x Octadecane, 6-methyl- C19H40 x Octane, 2,6-dimethyl- C10H22 x x Octane, 3-methyl- C9H20 x o-Xylene C8H10 x x x Pentacosane C25H52 x x x Pentadecane, 3-methyl- C16H34 x Phenanthrene C14H10 x x x Phenanthrene, 2,3,5-trimethyl- C17H16 x Phenanthrene, 2,5-dimethyl- C16H14 x Phenanthrene, 2,7-dimethyl- C16H14 x x Phenanthrene, 2-methyl- C15H12 x x Phenanthrene, 3,6-dimethyl- C16H14 x x x Phenanthrene, 3-methyl- C15H12 x x x Phenanthrene, 4-methyl- C15H12 x x x p-Xylene C8H10 x x x Retene C18H18 x Tetracosane C24H50 x x x Tetradecane C14H30 x Chemical characterization of oil and Water Accommodated Fraction (WAF) at different temperatures 89 Table 3.3. Tentatively identified compounds in each analysed oil applying the parameters presented in Table 3.2. Compound name Formula Troll IFO Diesel Tetradecane, 3-methyl- C15H32 x Tetradecane, 4,11-dimethyl- C16H34 x x Tetradecane, 4-methyl- C15H32 x x Tricosane C23H48 x x x Tridecane, 2-methyl- C14H30 x Tridecane, 3-methyl- C14H30 x Tridecane, 4-methyl- C14H30 x x x Undecane C11H24 x Undecane, 2,6-dimethyl- C13H28 x x x Undecane, 3-methyl- C12H26 x x As evidenced by the Table 3.3, oils consist primarily of two groups of compounds: hydrocarbons and aromatic hydrocarbons. In this case, Diesel presents the most abundant of identified compounds; this is probably due to the origin of the diesel. Diesel, as a product of hightemperature distillation, contains the most "volatile" compounds, which are suitable for analysis by gas chromatography. In the case of IFO, a type of heavy fuel oil, it likely contains compounds that are too heavy to be analyzed by gas chromatography. Currently this technique has been little used for the identification of petrochemical compounds. A limited number of studies reviewed have employed this technique to analyze oils by applying standards for the identification and quantification of various compounds. In this regard, the compounds identified through suspect screening correlate with those found in studies analyzing PAHs and their alkylated derivatives. For instance, Yang et al. (Yang et al. 2017) conducted a comprehensive assessment of various PAHs and their alkylated derivatives, ranging from naphthalene to chrysene, Chapter 3 90 across a range of petroleum products including diesel, IFO-180, and distilled diesel and these compounds were found in all the samples analysed. They observed a notable concentration of alkylated PAHs, particularly of naphthalene and phenanthrene, attributed to their lighter molecular weight. A similar distribution was observed in the present study where a large number of light PAH isomers were detected. 3.4. Conclusions According to this thorough study we can conclude that WAF samples are better prepared if we stir for at least 48 hours but this would not assure stable samples, since the concentration of the PAHs would be halved in approximately one day. Even if we add surfactants to get more stable emulsions, what is clearly observed is that the concentration is much higher but the stability is rather the same. Addition to this, we have also observed that the solubility of the PAH increase linearly with the temperature but when dispersant was added the trend was inversed for two of the oils studies. Consequently, in order to design or interpret exposure ecotoxicological bioassays the stability of the WAFs should be deeply considered. In order to assure constant exposures we should renew the WAFs at least once every 24 hours or, alternatively, to modify the experimental setup and use passive dosing systems as suggested in this work. Nonetheless, this alternative should be study in depth. The composition of oils is mainly by hydrocarbons and aromatic hydrocarbons. Diesel likely contains the highest number of identified compounds, primarily due to its origin and the hightemperature distillation process it undergoes. Consequently, it is composed mainly of more volatile Chemical characterization of oil and Water Accommodated Fraction (WAF) at different temperatures 91 compounds, in contrast to oils, which likely possess higher boiling points and compounds that cannot be analyzed by gas chromatography. 3.5. References Bera G, Doyle S, Passow U, Kamalanathan M, Wade TL, Sylvan JB, Sericano JL, Gold G, Quigg A, Knap AH. 2020. Biological response to dissolved versus dispersed oil. 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PAHs toxicity at aqueous solubility using passive dosing and sea urchin embryos 4 Chapter 4 104 When the O-rings were prepared directly with oils, they were introduced in the test tubes, oil added (0.9 or 1.75 g of oil per O-ring) and the mixture shacked during 24, 48 or 72 hours. In this case, two oils (high viscosity IFO 180 and low viscosity Troll oil) were tested. Then, O-rings were cleaned with Milli-Q water, dried using clean paper tissue in order to remove the oil residues and weight and desorbed (see section 4.2.4). 4.2.4. O-rings as passive dosser and chemical analysis Once O-rings loading methods were optimized, three O-rings were loaded at the best tested conditions with 3 oils, Diesel, Troll and IFO 180. O-rings were introduced in a vial with 10 mL of seawater in order to monitor the release of PAHs. After 5 hours of shaking, the presence of PAHs was evaluated in the seawater medium by solid phase microextraction (SPME) according to (Rianawati and Balasubramanian, 2009). Briefly, a 30 µm polydimethylsiloxane thickness fibre from Supelco was conditioned by heating in a GC injection port at 250 ˚C for 30 min, and SPME extraction was performed in vials with 20 mL of WAF and stirring at 250 rpm at 30 ˚C. The fibre was immersed during 50 minutes in the water sample with a soft agitation, then, it was desorbed on the injection port at 250 ˚C during 8 minutes. In the case of O-rings, they were chemically desorbed using 3 mL of ethyl acetate which were added to each O-ring and sonicated twice in an ultrasonic bath during 30 min. Extracts were collected and evaporated to dryness under N2 steam. Finally, the dry residues were reconstituted in 230 µL of hexane and 20 µL of internal standard were added. The extracts were stored at -20 °C until their analysis. Analysis was carried out using an Agilent 6890N gas chromatograph (GC) coupled to an Agilent PAHs toxicity at aqueous solubility using passive dosing and sea urchin embryos bioassay as complimentary tools 105 5975 N mass spectrometer (MS) (Agilent Technologies, Avondale, PA, USA). The detector was operated in selected ion monitoring (SIM) mode and a DB-5MS+DG chromatographic column (Agilent Technologies, 30 m x 0.25 mm, 0.25 μm) was used. Transfer line, ion source and quadrupole analyser temperatures were maintained at 300, 230 and 150 °C, respectively. He was used as carrier gas at 1.3 mL/min of flow and 2 µL were injected by splitless mode at 300 °C injection port temperature. The following temperature program was used for PAHs separation: 100 °C for 2 min; ramp at 16 °C /min to 240 °C and hold 2 min; ramp at 4 °C /min to 300 °C and hold 25 min. PAHs concentrations in the O-ring extracts were quantified with a nine-point external standard calibration curve (20-2440 ng/mL). Signal integration was done with HP Chemstation software (B.03.01, Agilent Technologies, Palo Alto, CA). 4.2.5. Sea urchin embryo toxicity (SET) testing Sea urchin 48-h embryo toxicity assay was carried out according to the International Council for the Exploration of the Sea (ICES, Beiras et al., 2012). Gametes were obtained from sexually mature sea urchins (Paracentrotus lividus) collected from a rocky shore in Armintza (43º26’01.1″N 2º53’56.1″W; Bay of Biscay) in spring (March-May) 2019 and maintained in aquaria at the Plentzia Marine Station (PiE). Seawater tanks were maintained at 15±1°C and natural photoperiod. Every two days sea urchins were fed with macroalgae and dregs were siphoned. Spawning and fertilisation were carried out as described by (DeMiguel-Jiménez et al., 2021). Within 30 min after fertilisation, the successfully fertilised eggs were transferred to glass vials containing 10 mL of the test solutions (50 embryos/mL), capped with Teflon lids. Toxicity assays were conducted in complete darkness at 20 °C. Gametes were obtained injecting 1 mL 0.5M KCl through the perioral membrane. Eggs were sieved through a nylon mesh (100 µm) and maintained in suspension with filtered sea water. Sperm was pippeted directly from the surface of males. Quality assurance was carried Chapter 4 106 out by checking gamete viability (egg roundness and sperm motility) and fertilisation rate (>90 % fertilised eggs) in an inverted microscope. Fertilisation was achieved by adding a few drops of sperm to the egg suspension. Fertilised eggs were transferred to glass vials containing 10 mL of the test solutions (50 embryo/mL; 3 replicates for each condition). After 48 h of exposure, larvae were fixed by adding a few drops of 37-38 % formaldehyde and observed in an inverted microscope (Nikon Eclipse Ti-2). Eggs were counted using a Sedgewick-rafter counting cell (Pyser Optics, Edenbridge, United Kingdom). Within 30 minutes, the fertilised egg suspension was distributed in glass vials (20 mL) containing a known volume of test sample (3 mL), assuring a final concentration of 40 eggs/mL. 4.3. Results and discussion 4.3.1. Loading of the O-rings In the case of the oil dissolved in methanol, three variables were analysed, i.e. the concentration of oil in methanol, the speed of the shaker and the shaking time. Linear regression methods (R script in RStudio 2023.06.0) were applied to a set of polynomic models and the best one was: log ∑𝑃𝐴𝐻 = 𝑏𝑜𝑖𝑙 ·𝑜𝑖𝑙𝑚𝑎𝑠𝑠 + 𝑏𝑟𝑝𝑚 · 𝑟𝑝𝑚 + 𝑏𝑜𝑖𝑙_𝑟𝑝𝑚 ·𝑜𝑖𝑙𝑚𝑎𝑠𝑠 · 𝑟𝑝𝑚 eq. 4.1 Residual standard error: 0.1969 on 6 degrees of freedom Multiple R-squared: 0.995, Adjusted R-squared: 0.9925 F-statistic: 397.1 on 3 and 6 DF, p-value: 2.749e-07 boil = 39±4; brpm = 0.0060 ± 0.0006; boil_rpm = -0.10 ±0.01 As also shown in Figure 4.1, the only significant variables were the concentration of oil in methanol and the shaking speed (Pr(>|t| <1E-4) but not the shaking time. PAHs toxicity at aqueous solubility using passive dosing and sea urchin embryos bioassay as complimentary tools 107 Figure 4.1. PAH loading in O-rings as a function of the oil mass dissolved in MeOH and the shaking speed (260 and 600 rpm). As a result, 0.05 g of oil per mL of MeOH, 600 rpm, and 48 hours were selected as the most suitable conditions for loading the O-rings, and they were subsequently used in further experiments. This pattern was also observed for 1-Methylnaphthalene and 2-Methylnaphthalene, the main PAHs found in the mixture. The methanol/O-ring mass ratio was then evaluated at different times (24-72 hours) and the results obtained as sum of PAHs concentrations were fitted to different models including square terms and interactions. The best model was: ⬚∑𝑃𝐴𝐻 = 𝑏0+ 𝑏1· 𝑡 + 𝑏2· 𝑡2 eq 4.2 Residual standard error: 1.69 on 2 degrees of freedom Multiple R-squared: 0.991, Adjusted R-squared: 0.982 F-statistic: 109.8 on 2 and 2 DF, p-value: 0.009025 b0 = 24.6±0.8, b1 = 23.1±1.7; b2 =-9.6±1.7 Chapter 4 108 showing that only time was the significant variable while the volume of methanol was not significant. In Figure 4.2 can be seen the fit to that function. Therefore, an intermediate oil/O-ring ratio of 3 was considered a good balance and regarding the shaking time, 50 hours seem to be enough to achieve the saturation of the O-ring. Figure 4.2. Sum of PAHs concentrations in the evaluation of methanol/O-ring ratio at different times (24, 48 and 72 h). In the case of the O-rings loaded directly with the oils, the ratio of oil per O-ring was studied during 24, 48 and 72 h. The best model was: ⬚∑𝑃𝐴𝐻 = 𝑏𝑚𝑎𝑠𝑠 · 𝑚𝑎𝑠𝑠 + 𝑏2· 𝑡2 eq 4.3 Residual standard error: 137.2 on 2 degrees of freedom Multiple R-squared: 0.969, Adjusted R-squared: 0.938 F-statistic: 31.3 on 2 and 2 DF, p-value: 0.031 bmass = 276±73 PAHs toxicity at aqueous solubility using passive dosing and sea urchin embryos bioassay as complimentary tools 109 and the results obtained in terms of sum of the PAHs concentrations are included in Figure 4.3. In this case, the only significant term was the mass of oil (prob(t) = 0.06) but not the shaking time. Figure 4.3. Sum of PAHs concentrations in the study of the ratio (0.9 versus 1.75 g of oil per O-ring) of oil per O-ring during 24, 48 and 72 h. There are multiple studies using this type of system for passive dosing of no polar organic compounds (Kow 3.3-6.4). The procedures of loading the rings are multiple, such as the evaporation of the solvent saturated with PAHs (Brown et al., 2001; Mayer et al., 1999), whose problem is the crystallisation of the compounds, the exposure of the passive dispensers in the contaminated medium itself, although it requires a long exposure time as well as presenting the problem of dependence on the medium and subject to the variation of the concentration in it (Claessens et al., 2015). The aforementioned phenomena decrease the loading of the compounds on the O-rings. The method used by Smith, for instance (Smith et al., 2010d), includes the partitioning of PAHs between methanol and the passive dispenser (231 mg PDMS O-rings). This approach ensures that the passive dispenser Chapter 4 110 is not affected by the swelling and can be easily washed away with water. In this study, the concentrations were obtained by applying the above method for 72 hours at 21 °C. Next, we will proceed by delicately cleaning the rings using a lint-free tissue. This important step aims to ensure that there are no traces of methanol whatsoever. The rings will then be fully immersed in Milli-Q water, effectively eliminating any remaining impurities. Finally, the rings were immersed in water to check the concentration obtained, which was 1.5 mg/L for phenanthrene and 30 mg/L for naphthalene, respectively. This study also reports the stability of all compounds, except for naphthalene, likely due to its high solubility and high volatility. In this study, the obtained concentrations are lower compared to previous studies. This can likely be attributed to the different volumes used (20 mL vs. 1 mL) and the different temperatures applied (20 °C vs. 37 °C). Moreover, the salting out effect has been reported in multiple studies, directly affecting the partitioning of the compounds in the medium (Whitehouse, 1984). 4.3.2. Stability of the WAFs After considering the loading procedure, which proposed directly loading the O-rings with oil as the optimal approach, we conducted a study to analyse the stability of the WAFs prepared using this method. In this case, as described before, three WAFs were prepared with one O-ring and the water concentration was monitored at each sample by SPME. The time profile of the WAFs is shown in Figure 4.4. PAHs toxicity at aqueous solubility using passive dosing and sea urchin embryos bioassay as complimentary tools 111 Figure 4.4. Stability evaluation of three different O-rings (A, B and C) during 68 hours (top) and a zoom on the top plateau (time < 60 h) (bottom). If we focus on the initial plateau, we can see that the concentration of the three WAFs was stable within the uncertainty of 2 times the standard deviation units. This plateau lasted up to 54 hours, as show in Figure 4.4 (bottom). Chapter 4 112 4.3.3. Toxicity assessment To assess the suitability of PAHs-loaded O-rings for bioassays, we have selected the sea urchin embryo test (SET). This test has been extensively studied at the Plentzia Marine Station (PiE) (Alvarez- Mora et al., 2022) and has been specifically employed to evaluate the toxicity of oils. In this case, given that the sea urchin embryos are exposed for a 48-hour period, utilizing preloaded O-rings seems to be a more suitable approach to conducting this bioassay, as opposed to using WAFs. In order to assess the suitability of this passive dosing approach, we conducted an initial test to evaluate the compatibility of the bare O-rings used in the assay with the O-rings that were loaded directly in oil. In Figure 4.5 we can see the comparison of the control samples (n=4) and the bare O- rings (n = 9). This comparison was carried out with the ggstatsplot library in R (Patil, 2021) and in this case, the parametric option (Student-t) was implemented since both sets of data were normally distributed according to the Shapiro-Wilk test (p. level > 0.1). As shown in Figure 4.5, the size difference (µm) between the controls and the bare O-rings was not significant, and therefore, we can assume that the O-ring do not show any toxic effect in this bioassay. In the same experiments, when oil-loaded O-rings were employed (data not shown) the larvae did not develop and therefore the toxic effect was 100 % lethal. In fact, the embryos were fully undeveloped. Consequently, we left aside this way of loading PAHs and tested the methanol diluted oil method. In Figures 4.6, we show the comparison of WAF 5 % and WAF 25 % with the methanol-diluted O-rings. As mentioned before, we carried out the comparison using the gg stats plot library in R and considering a parametric test. PAHs toxicity at aqueous solubility using passive dosing and sea urchin embryos bioassay as complimentary tools 113 Figure 4.5. Comparison of control set of samples and bare O-rings. As shown, the observed difference was not statistically significant (Student-t, p-value > 0.05). As shown in Figure 4.6 A, in the case of the WAF-5 % we can highlight two results. On the one side, the significant differences were observed with the control but the O-rings and WAFs of each oil were fully equivalent. However, though WAF samples are systematically lower than the O-rings, the differences were statistically negligible. When WAF-25 % samples were tested the results were very similar to the ones described before as can be seen in Figure 4.6 B. In this case the differences were also observed when the WAFs (IFO and MGO) or the O-rings (MGO) were compared to the controls. In this particular case, both WAFs and O-ring from MGO were considered significantly toxic when compared to the controls. As before, however, both O-rings and WAFs show equivalent patterns. Quantification and distribution of PAHs in mussels in Arctic regions 5 Quantification and distribution of PAHs in mussels in Arctic regions 123 5.1. Introduction Polycyclic aromatic compounds make up a range of organic compounds that are produced both naturally and anthropogenically. They are generated by the incomplete combustion of organic matter and released into the environment. These compounds are introduced into the environment in numerous ways, such as vehicle combustion, industrial emissions, oil spills, fires, volcanic eruptions, and domestic activities (Abdel-Shafy and Mansour, 2016). Because of their hydrophobic nature, these compounds tend to accumulate in sediments and biota, posing an environmental problem for both the ecosystem and humans. Water systems serve as a reservoir for these compounds, especially in coastal regions. This phenomenon can be attributed to the combined impact of human activities and the influx of rivers, which serve as channels for carrying these substances to the ocean. For that reason, some organisms are used as "sentinels" or bioindicators of pollution status. One of these widely used is the mussel, specifically the specie Mytilius (de Souza et al., 2024). As they rely on water filtration for their survival, they accumulate a wide range of organic compounds and minuscule suspended particles in their adipose tissue. Analysis of PAHs in mussels provides valuable spatio-temporal information and helps to identify potential sources of hydrocarbon contamination. In this sense, numerous studies have been conducted using mussels in monitoring programs, significantly contributing to our comprehension of PAH distribution dynamics and their presence in marine environments. Moreover, multiple studies have compared the presence and distribution of PAHs, revealing a significant variability. These works shed light on the unique impact of local anthropogenic activities, hydrological processes, and environmental conditions (Roldán-Wong et al., 2020; Wolska et al., 2012). One example is the study conducted by Baumard (Baumard et al., 1998) Chapter 5 124 in the Mediterranean Sea, where they detected PAH concentrations ranging from 25 to 80 ng/g in mussels collected at various sites. In contrast, at other sites, such as Santos estuary in Brazil, they reported higher PAHs levels ranging between 88 and 988 ng/g. (de Souza et al., 2024). These results emphasize the spatial heterogeneity of PAH pollution and highlight the need for region-specific management strategies to minimize environmental impacts. Research has revealed that mussels and other organisms from Arctic regions contain detectable levels of PAHs, although the concentrations are lower compared to more industrialized areas (Poulsen et al., 2021). For instance, recent studies (Poulsen et al., 2021) have revealed PAH concentrations in mussels collected from Greenland ranging from 28 to 311 ng/g d.w., indicating the far-reaching transport and deposition of PAH in polar regions. Arctic regions possess unique characteristics, rendering them extremely sensitive and vulnerable ecosystems to organic pollutants. Besides, the emergence of new shipping lanes may amplify the environmental risks associated with these regions. Within this context, the objective of this study was to conduct a thorough comparative analysis of the levels of PAHs in mussels collected from different sampling locations along the Norwegian coast, with a particular emphasis on the North Sea regions. Moreover, the elucidation of the spatial distribution, sources and ecological impacts of PAH pollution was also carried out. 5.2. Experimental section 5.2.1. Chemicals and reagents Mix standard solution of 18 Polycyclic Aromatic Hydrocarbons (PAHs) CRM47543: fluorene (F), phenanthrene (P), anthracene (Ant), fluoranthene (F), pyrene (Pyr), benz[a]anthracene (BaA), Quantification and distribution of PAHs in mussels in Arctic regions 125 chrysene (Chr), benzo[k]fluoranthene (BkF), benzo[a]pyrene (BaP), indeno[1,2,3-cd]pyrene (IPyr), dibenzo[a,h]anthracene (DBahA) and benzo[g,h,i]perylene (BghiP) was purchased from Supelco (Bellefonte,PA, USA). A mixture of 7deuterated compounds [Norwegian Standard (S-4124-200-T): naphthalene-d8, byphenyl-d10, phenanthrene-d10, pyrene-d10, benzo[a]anthracene-d12, benzo[a]pyrene-d10, benzo[ghi]perylene-d12 was adquired from Chiron (Trondheim, Norway). n- Alkano Mix (49452-U, C7-C40 Saturated Alkanes Standard) was purchased from Supelco. mussel tissue Standard Reference Material 2977 (Perna perna, edible brown mus-sel) were provided by the National Institute of Standards andTechnology (NIST, Gaithersburg, MD, USA). Methanol, hexane, isopropanol, ethyl acetate, toluene and dichloromethane (HPLC grade, 99.9 %) were purchased from Macron (Radnor, USA). Helium with 99.999 % purity was purchased from Messer (Barcelona, Spain). Silica gel used in the clean-up procedure was purchased from Supelco and anhydrous sodium sulphate from Probus (Barcelona, Spain). 5.2.2. Sampling and sample pretreatment The collection of mussels (Mytilus edulis) of two different sizes (small: 2–3 cm and large: 3.5– 4.5 cm) was conducted across different sites of the North Sea during the autumn of 2016 and 2017, as described in Table 5.1. The mussels were taken from the first meter of the lower intertidal zone and transported to the laboratory under ambient conditions. Whole mussel samples were frozen and subsequent chemical analysis of soft tissues was carried out. Chapter 5 126 Table 5.1. Country, location, and sampling site type of collected mussels during autumn of 2016 and 2017 (Benito et al., 2022). Country Location Latitude Longitude Sampling site type Germany Konigshafen 55.0423740 8.4491020 Mussel bed Germany Eckwarderhorne 53.5203010 8.2318160 Rock breakwater Scotland St. Andrews 56.3336020 -2.7761730 Rocky beach Scotland Leith 55.9773590 - 3.140404 Rocky beach Iceland Havfjordur 64.3581540 - 21..486458 Rocky beach Iceland Reykjavik (port) 64.1556050 -21.939218 Rock wall Norway Oslo (Malmøya) 59.8738960 10.7577430 Floating jetty Norway Oslo (Drøbak) 59.6158890 10.6520070 Rocky beach Norway Trondheim (port) 63.4426920 10.4254940 Pylons Norway Trondheim (WWTP) 63.4448670 10.3413310 Rocky beach Norway Trondheim (Rissa) 63.5617530 9.8997760 Pylons Norway Tromsø (port) 69.6541770 18.9684590 Concrete surface Norway Tromsø (aquarium) 69.6420890 18.9463900 Rocky beach Norway Svalbard (Longyearbyen) 78.2360810 15.6064820 Pylons 5.2.3. Analytical protocol The analysis of mussel tissues was based on Navarro et al.’s procedure (Navarro et al., 2009). Briefly, mussels between 0.25-0.5 g (freeze-dried samples) were accurately weighed using Falcon vessels, and deuterated PAHs solution of known concentration was added as surrogate together with Quantification and distribution of PAHs in mussels in Arctic regions 127 5 mL of acetone as extractant. PAHs extraction was performed using an ultrasound homogenizer SONOPULS HD 2070 (Bandelin electronic GMBH & Co. KG, Berlin, Germany) at 45 % power (maximum power 70 W) for 2 minutes. The extracts were then sonicated and filtered through Millex® HV PVDF 0.45 µm (Millipore, Carrigtwohill, Ireland) filters, and after the addition of nearly 1 mL of iso-octane were concentrated to ~0.5 mL, under a nitrogen stream using a TurboVap LV, Zymark, Barcelona, Spain. The extracts were subsequently cleaned-up using 5 g-Florisil cartridges (Sigma-Aldrich, San Luis, Misuri, USA) previously conditioned with n-hexane, and eluted with 25 mL of n-hexane:toluene 75:25 mixture. Subsequently, they were concentrated to dryness, redissolved in iso-octane and kept at −20 °C in the dark until the analysis via gas chromatography-mass spectrometry (GC-MS). 5.2.4. Instrumental analysis The analysis of extracts was carried out with the gas chromatograph model 6890N, coupled to a Model 5975 MS detector (Agilent Technologies, California, USA). The detector was operated in selected ion monitoring (SIM) mode and a DB-5MS+DG chromatographic column (Agilent Technologies, 30 m x 0.25 mm, 0.25 μm) was used with He as carrier gas. Transfer line, ion source and quadrupole analyser temperatures were maintained at 300, 230 and 150 °C, respectively. He was used as carrier gas at 1.3 mL/min of flow and 2 µL were injected by splitless mode at 300 °C. The following temperature program was used: 100 °C for 2 min; ramp at 16 °C /min to 240 °C and hold 2 min; ramp at 4 °C/min to 300 °C and hold 25 min. 5.3. Results and discussion 5.3.1. Method performance and quality control/quality assurance (QC/QA) For external calibration, standard solutions were prepared by weighing in a concentration Chapter 5 128 range of 1 to 2000 ng/g, thus, instrumental limits of detection (LOD: 0.6-3 ng/g) were estimated as the lowest concentration detected in the three injection replicates (all the calibration levels were injected by triplicate), and in the case of instrumental quantification limits (LOQ: 2-10 ng/g), as the lowest concentration detected with a relative standard deviation (RSD) of less than 25 % and a closeness to the real concentration values between 75 – 125 %. Extractions were performed in triplicate to calculate precision in terms of procedural repeatability and relative standard deviations, RSDs<25 % were obtained in all the cases. Procedural blanks were also analysed by triplicate to check for potential cross-contamination or contamination through the process and not significant (areas of peaks in sample/blank ratio>10) analytes peak chromatographic areas were detected in any of them. Besides, the average signal (n=3) of the procedural blanks plus three or ten times its standard deviation was used to calculate the procedural limit of detection (LODpro: 2-9 ng/g) and procedural limit of quantification (LOQpro: 6-30 ng/g) respectively. All the samples were spiked at 1,000 ng/g concentration level with a surrogate mixture containing deuterated PAHs (see section 5.2.1) for absolute recoveries correction. A matrix-matched calibration approach between the analytes procedural LOQs and 949 ng/g level, was also built with the same objective (apparent recovery determination). All the compounds showed apparent recoveries between 40 % and 80 % using both approaches. Consequently, the surrogate correction approach was used in the quantitative analysis of all the mussel samples. For the verification of the method, the analysis of the Standard Reference Material 2977 was carried The obtained concentration values for reference material were in agreement with the certified values, as obtained Quantification and distribution of PAHs in mussels in Arctic regions 129 in paired t-test (p-value > 0.05) (Miller, 1993) . The matrix-matched calibration (949 ng/g) level was injected twice, at the beginning and at the end of the sequence, in order to examine possible signal drift. Hexane was injected every 5 samples injections along the sequence to check for carryover. No signal drift or carryover was observed throughout the sequence. 5.3.2. Analysis in real samples Table 5.2 shows all the results obtained on the mussels analysed. As can be seen, phenanthrene, fluorene, and pyrene were the compounds measured at higher frequencies, indicating them as ubiquitous contaminants. Since they are lighter PAHs they are less hydrophobic and more soluble in marine water than the heavier ones. The distribution of light and heavy PAHs in the soft tissues are plotted in Figure 5.1 together with the ratio Fluoranthene/ (Fluoranthene+Pyrene). As can be seen, the highest concentrations of PAHs are linked to the most polluted sites (i.e. small mussels from the port in Trondheim and mussels of both sizes from the port in Tromsø) and the lowest to the farm and reference sites. Additionally, higher concentrations of heavy PAHs (H-PAH, 4 or more aromatic rings) than the light ones (L-PAH, less than 4 aromatic rings) were observed in all the sites except in the farm in Trondheim, which belongs to the one with the lowest total concentration. Chapter 5 136 the samples studied in this work, the chemical scenario appears to be very simple and basically responds to implicit pollution level of the selected sites. When the cellular, biochemical and tissuelevel biomarkers were considered (Benito et al, 2022) the conclusions largely coincided with the chemical scenario depicted. The analysed mussels displayed varying degrees of contamination at all the sampling sites. The most commonly detected chemical compounds were phenanthrene, fluorene and pyrene. Although the compounds were detected in all of the samples, the PCA analysis efficiently distinguishes between the sampling points in contaminated and non-contaminated areas. Furthermore, no relationship between mussel size and degree of contamination has been found, and mussels of any size can be used for the assessment of the contamination. 5.5. References Abdel-Shafy, H.I., Mansour, M.S.M., 2016. A review on polycyclic aromatic hydrocarbons: Source, environmental impact, effect on human health and remediation. Egypt. J. Pet. https://doi.org/10.1016/j.ejpe.2015.03.011 Baumard, P., Budzinski, H., Michon, Q., Garrigues, P., Burgeot, T., Bellocq, J., 1998. Origin and Bioavailability of PAHs in the Mediterranean Sea from Mussel and Sediment Records. Estuar. Coast. Shelf Sci. 47, 77–90. https://doi.org/10.1006/ECSS.1998.0337 Benito, D., Paleček, D., Lekube, X., Izagirre, U., Marigómez, I., Zaldibar, B., Soto, M., 2022. Variability and distribution of parasites, pathologies and their effect on wild mussels (Mytilus sp) in different environments along a wide latitudinal span in the Northern Atlantic and Arctic Oceans. Mar. Environ. Res. 176, 105585. https://doi.org/10.1016/j.marenvres.2022.105585 de Souza, P.F., da Cunha, D.L., Daflon, S.D.A., Machado, A.R., Gaudie-Ley, L.W., de Mattos, J., da Fonseca, E.M., 2024. Bioaccumulation of PAHs in marine bivalves of the Santos Estuary (Brazil) associated with the evaluation of human consumption. Mar. Pollut. Bull. 199, 115900. https://doi.org/10.1016/J.MARPOLBUL.2023.115900 Quantification and distribution of PAHs in mussels in Arctic regions 137 Miller, J.C., 1993. Estadística para química analítica, 2a ed. ed. Addison-Wesley Iberoamericana, Wilmington, Delawarw. Navarro, P., Etxebarria, N., Arana, G., 2009. Development of a focused ultrasonic-assisted extraction of polycyclic aromatic hydrocarbons in marine sediment and mussel samples. Anal. Chim. Acta 648, 178–182. https://doi.org/10.1016/j.aca.2009.06.062 Poulsen, R., Gravert, T.K.O., Tartara, A., Bensen, H.K., Gunnarsen, K.C., Dicová, K., Nielsen, N.J., Christensen, J.H., 2021. A case study of PAH contamination using blue mussels as a bioindicator in a small Greenlandic fishing harbor. Mar. Pollut. Bull. 171, 112688. https://doi.org/10.1016/J.MARPOLBUL.2021.112688 Roldán-Wong, N.T., Kidd, K.A., Ceballos-Vázquez, B.P., Rivera-Camacho, A.R., Arellano-Martínez, M., 2020. Polycyclic aromatic hydrocarbons (PAHs) in mussels (Modiolus capax) from sites with increasing anthropogenic impact in La Paz Bay, Gulf of California. Reg. Stud. Mar. Sci. 33, 100948. https://doi.org/10.1016/J.RSMA.2019.100948 Wolska, L., Mechlińska, A., Rogowska, J., Namieśnik, J., 2012. Sources and fate of PAHs and PCBs in the marine environment. Crit. Rev. Environ. Sci. Technol. https://doi.org/10.1080/10643389.2011.556546 Development of an analytical method for the simultaneous determination of 50 semi-volatile organic contaminants in wastewaters 6 Development of an analytical method for the simultaneous determination of 50 141 6.1. Introduction The widespread use of everyday consumer products and industrial production has led to a rise in the emission of various contaminants into the environment (González-Gaya et al., 2021). Thus, a significant amount of priority and emerging pollutants, some of them considered persistent organic pollutants (POPs), are being released into the aquatic environment. These substances have detrimental effects on the ecosystem, causing habitat destruction, a decrease in biodiversity, and accumulation in the food chain, ultimately affecting many top predators (Lepom et al., 2009). Among these contaminants, we can highlight a diverse group of compounds called semi-volatile organic compounds (SVOCs) characterized by low water solubility, high lipophilicity, and high toxicity. Most SVOCs are non-polar compounds, which exhibit hazardous effects and low mobility in aqueous media leading to the accumulation in biological tissues and many other environmental compartments, such as sediments (Camacho-Jiménez et al., 2023). In this group, we can find several types of pollutants such as polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), phthalate esters, organochlorine pesticides, and other environmental endocrine disruptors (Li et al., 2018). In the last decade, wastewater treatment plants (WWTPs) have gained scientific attention as a relevant source of anthropogenic compounds to the environment along with industrial discharges, landfill leachates, agricultural runoffs, hospital effluents, aquaculture effluents, etc. Despite ongoing efforts to upgrade wastewater technologies to increase their efficiency and thus reduce the environmental impact of the released waters, many conventional treatments (e.g. activated sludge systems, advance oxidation procedures, Activated Sludge (CAS)) implemented in WWTPs were not designed to eliminate those compounds and thus, hundreds of compounds are continuously released Chapter 6 142 into the environment reaching aquatic ecosystems (Lindholm-Lehto et al., 2016; Mijangos et al., 2018). The presence of SVOCs in treated effluents from WWTPs is still a challenge for effective water quality management. These compounds may persist through conventional treatment processes, such as sedimentation, filtration, and disinfection, resulting in effluent discharges that still contain significant concentrations of contaminants. Additionally, there is concern about potential adverse effects on human health and aquatic ecosystems due to continuous exposure to these substances through drinking water and aquatic media. Research into advanced treatment technologies, such as advanced oxidation, activated carbon adsorption, and ultrafiltration membrane, has shown promise in effectively removing them from treated wastewater (Besha et al., 2017; Lin et al., 2012). However, a better understanding of removal mechanisms and long-term efficacy of these technologies is needed for their industrial-scale implementation. Within this context, the aim of this work was the optimisation and validation of an analytical method for the simultaneous analysis of 50 SVOCs (i.e. PAHs, PCBs, phthalate esters, photoinitiators, UV filters, musk fragrances and pesticides) in aqueous samples via gas chromatography coupled mass spectrometry (GC–MS) based on research group’s previous experience (Lopez-Herguedas et al., 2022; Ziarrusta et al., 2015). The factors affecting compounds’ preconcentration using solid-phase based extraction (i.e., elution and evaporation steps) were evaluated in order to improve the extraction yield. Once the analytical method was validated, the presence of the target contaminants and, thus, the effectiveness of the treatments, a CAS treatment, and an alternative secondary wastewater treatment based on MBR membranes, was evaluated with the effluent samples from the large WWTP of the Basque Country (Galindo, Biscay). Development of an analytical method for the simultaneous determination of 50 143 6.2. Chemicals and reagents The list of 50 target compounds, purchased from Sigma Aldrich (St. Louis, MO, USA), analysed in this work is provided in Table 6.1. The table includes information about the family they belong (PCBs, musk fragrances, insecticides, PAH and phthalate esters, etc), names, abbreviation, molecular formula and molecular weight, log Kow, supplier, solvent and surrogate used to correct the recovery for each analyte. Seven working solutions of a concentration range of 50–310 mg/g were prepared by weighing (Sartorius-Stedim, Barcelona, Spain) in isopropanol (99.8 %, Merck KGaA, Darmstadt, Germany) from the standard solutions included in Table 6.1. From those working solutions, a mixture solution containing all the analytes (concentration of 2 mg/kg) was prepared monthly for validation of the analytical procedure. All standard and working solutions were kept in the freezer at −20 °C until further use. The preconcentration and extraction of the samples was performed using home-made triphasic solid phase extraction (SPE) cartridges. Empty polypropylene cartridges (12 mL, Supelco, Bellefonte, PA, USA) were filled with polypropylene (PP) frits (Supelco) and 250 mg of the following sorbents: cationic exchange (Sepra ZT-WCX, 30 μm, 85 Å, Phenomenex, California, USA), anionic exchange (Sepra ZT-WAX, 30 μm, 85 Å, Phenomenex, California, USA) and reverse phase (Chromabond©HRX, 85 μm, 55–65 Å, Macherey-Nagel, Düren, Germany) in a 1:1:3 ratio, respectively. The solvents used during the SPE step were methanol (MeOH, HPLC grade, Macron Fine Chemicals, Avantor, Poland), ethyl acetate (EtOAc, HPLC grade, Macron Fine Chemicals, Avantor, Poland), ammonia (25 % NH3 , Sigma Aldrich), formic acid (FA, > 98 % purity, Panreac AppliChem, Barcelona, Spain), hexane (hex, HPLC grade, 95 %, Macron Fine Chemicals, Avantor, Poland) and toluene (tol, HPLC grade, 99.9 %, Lab-Scan Analytical Sciences, Gliwice, Poland). All standard solutions and extracts were shaken with a Vortex agitator (Reax top, Heidolph, Schwabach, Germany) before using. Chapter 6 144 Development of an analytical method for the simultaneous determination of 50 145