Evaluación de los niveles de disruptores endocrinos en muestras procedentes de estaciones depuradoras de aguas residuales de la isla de Gran Canaria mediante la utilización de sistemas cromatográficos acoplados

UNIVERSIDAD*DE*LAS*PALMAS*DE*GRAN*CANARIA* * * TESIS*DOCTORAL* * EVALUACIÓN*DE*LOS*NIVELES*DE*DISRUPTORES* ENDOCRINOS*EN*MUESTRAS*PROCEDENTES*DE* ESTACIONES*DEPURADORAS*DE*AGUAS*RESIDUALES* DE*LA*ISLA*DE*GRAN*CANARIA*MEDIANTE*LA* UTILIZACIÓN*DE*SISTEMAS*CROMATOGRÁFICOS* ACOPLADOS* * Assessment*of*EndocrineADisrupting*Compounds*levels*in* Wastewater*Treatment*Plant*Samples*from*the*Island*of*Gran* Canaria*by*using*Hyphenated*Chromatographic*Techniques* * Tanausú*Vega*Morales* * LAS*PALMAS*DE*GRAN*CANARIA* ESPAÑA* 2012* * Agradecimientos. . ! .Al. escribir. estas. líneas. se. me. vienen. a. la. cabeza. muchas. personas. que,. de. una. manera.u.otra,.han.contribuido.a.que.esta.Tesis.Doctoral.se.haya.hecho.realidad..A. todos.ellos,.gracias.. . .En.primer.lugar.no.podría.nombrar.a.otras.personas.que.no.fuesen.mis.padres... Cada.una.de.las.palabras.que.conforman.este.manuscrito.han.sido.escritas.gracias.a. su.esfuerzo.y.dedicación..Su.afán. por. darme. una. buena. educación,. por. formarme. como.persona,.por.darme.lo.que.fuera.necesario.aún.excediendo.sus.posibilidades,. sobrepasa.los.límites.del.amor.y.el.cariño.. . .Mis. hermanos. también. han. jugado. un. papel. fundamental. durante. estos. últimos. cuatro.años..Siempre.dándome.aliento.cuando.lo.necesitaba,..ánimo.en.los.momentos. difíciles..Muchas.gracias.por.estar.siempre.ahí.cuando.me.han.hecho.falta,.no.tengo. palabras.para.expresar.mi.gratitud.para.con.ustedes... . ..También.quiero.agradecer.el.cariño.que.me.han.transmitido.mis.compañeros.de. Laboratorio,.a.los. cuales.considero.como.verdaderos.amigos.. A.Borja.me.gustaría. agradecerle. las. horas. que. se. ha. pasado. filtrando. muestras,. preparando. patrones,. ayudándome.en.lo.que.fuera.necesario..A.Rayco.y.Cristina.les.debo.dar.las.gracias.no. solo.por.su.ayuda.en.el.laboratorio,.sino.por.todos.los.momentos.que.hemos.vivido. juntos.fuera.del.trabajo,.por.su.trabajo.como.editores.y.buscadores.profesionales.de. documentos,.y.por.esas.riquísimas.tartas.de.cumpleaños..A.Carlos,.Adrián.y.Jana,.les. tengo. que. agradecer. su. eterna. disponibilidad,. la. positividad. que. transmiten,. la. maravillosa. atmósfera. que. ha. existido. en. nuestro. laboratorio. se. debe. en. gran. medida. a. ustedes.. A. Álvaro,. Miriam. y. Cristina. Mahugo,. por. ayudarme. a. dar. mis. primero.pasos.en.este.mundo,.por.estar.siempre.receptivos.y.solventarme.cualquier. duda.que.surgiera... . A.mis.suegros,.Loli.y. Juan,.por. haberme.tratado. como.a.un.hijo.desde.el.primer. momento,. por. ser. parte. de. mi. familia,. por. esos. deliciosos. almuerzos,. ¡y. por. esas. grandísimas.partidas.de.parchís!.. . A.Sarah,.mi.Sarah,.por.que.esta.Tesis.Doctoral.es.tan.mía.como.suya..Ella.ha.sido. calma.en.la.tormenta,.luz.en.las.tinieblas,.orden.en.el.caos..Sin.ella.el.resultado.de.lo. que.aquí.se.presenta.sería.totalmente.distinto.. . .A.Dagmar. Solichová.y.Pter.Solich,. por.todas. las.facilidades.que.me.dieron.para. realizar. la. estancia. en. Hradec. Králové.. Su. apoyo. tanto. a. nivel. científico. cómo. humano.me.ha.hecho.crecer.como.persona.en.todos.los.sentidos.. . Al. Ministerio. de. Educación. por. la. beca. FPU. (AP2007Y02312). del. Programa. Nacional.de.Formación.del.Profesorado.Universitario,.sin.cuyos.fondos.habría.sido. imposible.realizar.las.investigaciones.presentadas.en.este.trabajo.. . Y.por.último.quiero.darle.las.gracias.a.mis.tutores,.Zoraida.y.José.Juan..Tenerlos.a. ustedes. guiando. esta. Tesis. es. motivo. de. orgullo.. Muchas. gracias. por. las. infinitas. horas.empleadas.en.mi.formación.. . Índice'general' ! ! I! ! Índice'general' CAPÍTULO!I.!INTRODUCCIÓN.!!!!!!!!!!!!!!!!!!!!1! ! !! I.1.!Compuestos!disruptores!endocrinos.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!3! ! !I.2.! Aplicación! de! técnicas! acopladas! a! la! determinación! de! compuestos! disruptores! endocrinos.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!19! ! I.2.1.!Alquilfenoles!polietoxilados.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!21! ! I.2.1.1.!Determinación!de!alquilfenoles!polietoxilados!y!sus!productos! de!degradación!en!muestras!líquidas!y!sólidas.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!21! ! ! ! I.2.2.!Hormonas!esteroideas.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!39! ! ! ! ! ! I.2.2.1.!Técnicas!de!extracción.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!48! ! ! ! ! ! ! ! ! ! I.2.2.1.1.!Muestras!líquidas.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!49! ! ! ! ! ! I.2.2.1.2.!Muestras!sólidas.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!50! ! ! ! ! ! ! I.2.2.2.!Técnicas!de!separación!y!sistemas!de!detección.!!!!!!!!!!!!!!!!!!!!!!!!51! ! ! ! I.2.3.!BisfenolQa.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!55! ! ! ! ! ! I.2.3.1.!Técnicas!de!extracción.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!63! ! ! ! ! ! Índice'general' ! ! II! ! ! ! ! I.2.3.1.1.!Muestras!líquidas.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!63! ! ! ! ! ! I.2.3.1.2.!Muestras!sólidas.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!65! ! ! ! ! ! ! I.2.3.2.!Técnicas!de!separación!y!sistemas!de!detección.!!!!!!!!!!!!!!!!!!!!!!!!66! ! !I.3.!Tratamiento!de!datos.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!69! ! ! ! ! ! I.3.1.!Análisis!estadístico.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!70!! ! ! ! ! I.3.1.1.!Análisis!de!los!resultados!en!muestras!reales.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!72! ! ! ! ! ! ! ! I.3.1.2.!Diseño!factorial.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!73! ! ! ! 1.3.2.!!Evaluación!de!la!actividad!disruptora!endocrina.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!76! ! !I.4.!Referencias.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!79! ! CAPÍTULO!II.!OBJETIVOS.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!107! ! CAPÍTULO!III.!PARTE!EXPERIMENTAL!Y!RESULTADOS.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!113! ! III.1.!Determinación!de!alquilfenoles!polietoxilados,!bisphenolQa,!17αQethinylestradiol,! 17β#estradiol!y!sus!metabolitos!en!muestras!de!aguas!residuales!mediante!SPE!y!LCQ MS/MS.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!115! ! III.2.!Determinación!simultánea!de!compuestos!disruptores!endocrinos!en!muestras!de! aguas!residuales!mediante!cromatografía!líquida!de!alta!resolución!con!detección!por! fluorescencia.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!129! ! Índice'general' ! ! III! III.3.! Determinación! y! evaluación! de! compuestos! mimetizantes! del! estradiol! en! las! fases! disuelta! y! particulada! de! muestras! de! estaciones! depuradoras! de! aguas! residuales.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!133! ! III.4.! Determinación! de! varios! compuestos! mimetizadores! del! estradiol! en! lodos! de! estaciones!depuradoras!de!aguas!residuales!mediante!la!combinación!de!la!extracción! asistida!por!microondas!y!la!cromatografía!líquida!con!detección!de!masas!de!triple! cuadrupolo.!!!!!!!!!!!!!!!!155! ! III.5.!Desarrollo!y!optimización!de!una!metodología!analítica!basada!en!la!extracción!en! fase!sólida!tipo!“on-line”!acoplada!a!la!cromatografía!líquida!de!ultraQresolución!con! espectrometría! de! masas! de! triple! cuadrupolo! para! la! determinación! simultánea! de! compuestos!disruptores!endocrinos!en!muestras!de!aguas!residuales.!!!!!!!!!!!!!!!!!!!!!!167! ! III.6.!Extracción!asistida!por!microondas!combinada!con!extracción!en!fase!sólida!en! modo! “On-Line”! y! UHPLCQMS/MS! para! la! determinación! de! compuestos! disruptores! endocrinos!en!lodos!de!estaciones!depuradoras!de!aguas!residuales.!!!!!!!!!!!!!!!!!!!!183! ! III.7.!Evaluación!de!la!actividad!disruptora!endocrina!en!las!fases!disuelta!y!sólida!de! muestras! de! estaciones! depuradoras! de! aguas! residuales! procedentes! de! la! isla! de! Gran!Canaria!(España).!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!211! ! CAPÍTULO!IV.!CONCLUSIONES.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!243! ! ANEXOS.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!251! I. Acrónimos!!!!!!!!!!!!!!!253! II. Lista!de!figuras!!!!!!!!!!!!!!!257! III. Lista!de!tablas!!!!!!!!!!!!!!!265! IV. Lista!de!publicaciones!obtenidas!de!la!presente!Tesis!Doctoral! ! !!!!!!!275! V. Comunicaciones!a!congresos!!!!!!!!!!!!!!!!!!!!279! Capítulo)I:)Introducción) ! 6!I.1.)Compuestos)disruptores)endocrinos! ! !Estas!células!son!capaces!de!leer!las!instrucciones!y!de!ejecutarlas,!ya!sea!fabricando! proteínas!o!segregando!otra!hormona,!originando!una!gran!cantidad!de!respuestas!y! funciones!del!organismo!como:! - Mantenimiento!de!la!homeostasis.! - Regulación!del!crecimiento,!desarrollo!y!reproducción.! - Reacción!ante!estímulos!externos.! - Producción,!utilización!y!almacenamiento!de!energía.! !Teniendo! estos! principios! como! base,! ! los! mecanismos! de! acción! de! los! EDCs! se! pueden! dividir,! generalmente,! en! cuatro! formas! distintas! de! actuación,! las! cuales! vienen!explicadas!en!el!siguiente!gráfico1:! ! ! ! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 1!Extraído!del!“Curso!de!introducción!a!los!Disruptores!Endocrinos”.!Realizado!por!el!Instituto!Sindical!de! Trabajo,!Ambiente!y!Salud!(ISTAS).! En una situación normal, la hormona se une al receptor celular que desencadena la reacción esperada, al nivel esperado. ! CÉLULA Receptor celular Hormona Reacción REACCIÓN NORMAL Capítulo)I:)Introducción) ! I.1.)Compuestos)disruptores)endocrinos)7) ) ! ! ! !Aunque! en! la! actualidad! existe! cierto! grado! de! controversia! acerca! del! mecanismo! exacto!mediante!el!cual!actúan!los!EDCs!capaces!de!mimetizar!al!estradiol!endógeno! [11],!una!de!las!teorías!mas!ampliamente!aceptada!se!fundamenta!en!el!hecho!de!que,! las! células! receptoras! o! diana! del! estrógeno! (hERα),! poseen! una! zona! de! unión! a! la! hormona! más! grande! y! flexible! que! la! requerida! por! la! hormona! natural! (17β_ REACCIÓN BLOQUEADA CÉLULA Receptor celular Disruptor endocrino Hormona Al mimetizar a la hormona natural el disruptor endocrino puede unirse al receptor e interferir en la reacción, bloqueándola. REACCIÓN EXCESIVA CÉLULA Reacción Disruptor endocrino Receptor celular Reacción Los disruptores endocrinos también pueden unirse al receptor y generar una reacción más potente de la normal y en el momento inadecuado. REACCIÓN INSUFICIENTE CÉLULA Reacción Disruptor endocrino Receptor celular Reacción Los disruptores endocrinos también pueden unirse al receptor y generar una reacción más débil de la normal y en el momento inadecuado. Capítulo)I:)Introducción) ! 8!I.1.)Compuestos)disruptores)endocrinos! ! estradiol),!lo!cuál!convierte!a!dichos!receptores!en!sitios!altamente!vulnerables!como! punto! de! enlace! para! un! amplio! rango! de! interferencias! de! una! amplia! diversidad! estructural![12]!(Figura!I.1)2.!! ! Figura!I.1.!Modelo!de!acción!propuesto!para!el!estrógeno!natural!17β_estradiol!y!el!xenobiótico! bisfenol_a!(BPA)!en!células!pancreáticas!tipoβ.!! !Además,!teniendo!en!cuenta!la!notable!similitud!!de!los!receptores!estrogénicos!entre! los! diferentes! animales! vertebrados,! el! efecto! de! los! EDCs! sobre! las! funciones! endocrinas!se!extiende!a!una!gran!variedad!de!especies!animales!en!los!ecosistemas! [14].! ·∙)Efectos)sobre)la)salud)y)el)medioambiente) !En!la!actualidad!son!muchas!las!publicaciones!científicas!que!demuestran!los!efectos! negativos!de!los!EDCs!sobre!la!salud!de!los!seres!vivos,!los!cuales!parecen!producirse! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 2!Imagen!tomada!de!A.!Nadal!et!al.![13].! Capítulo)I:)Introducción) ! I.1.)Compuestos)disruptores)endocrinos)9) ) incluso!a!niveles!traza.!En!este!sentido,!la!preocupación!por!la!presencia!de!este!tipo! de! compuestos! químicos! en! el! medioambiente! no! es! nueva,! como! demuestran! los! artículos!de!Allen!y!Doisy![15]!a!principios!de!la!década!de!1920,!!o!el!de!Burlington!y! Linderman![16]!en!el!año!1950.!Sin!embargo,!el!grado!o!nivel!de!preocupación!se!ha! intensificado! notablemente! en! los! últimos! 10_15! años! debido! a! los! constantes! y! numerosos! informes! acerca! de! los! efectos! adversos! asociados! a! la! “salud! reproductiva”.! !Dicho! interés! ha! conducido! a! la! publicación! de! un! gran! numero! de! artículos! que! relacionan!la!presencia!de!este!tipo!de!compuestos!con!la!salud!reproductiva!en!los! seres! humanos,! como! por! ejemplo:! la! reducción! en! la! calidad! del! esperma! y! del! numero! de! espermatozoides! estimado! en! algunas! poblaciones! [17],! el! incremento! observado!en!el!número!de!casos!criptorquidia!e!hipoespadias![18],!o!el!aumento!del! numero!de!casos!de!cánceres!testiculares,!de!próstata!y!de!mama![19].! !Por! otra! parte,! los! efectos! de! los! EDCs!sobre! otros! vertebrados! en! los! ecosistemas! también!han!quedado!plasmados!en!la!bibliografía!científica.!A!modo!de!resumen,!se! han! descrito! una! gran! cantidad! de! alteraciones! en! un! amplio! rango! de! especies! animales,!como!por!ejemplo:!(a)!decrecimiento!en!la!fertilidad!de!algunas!especies!de! aves,!peces!y!mamíferos;!(b)!masculinización!y!feminización!(o!“imposex”)!en!algunas! especies! de! peces,! moluscos,! gasterópodos,! aves! y! mamíferos;! (c)! reducción! de! la! eficacia!en!los!procesos!de!incubación!en!ciertas!especies!de!tortugas,!aves!y!peces;!(d)! desórdenes! en! el! sistema! inmune! de! algunas! especies! de! aves! y! mamíferos;! o! (e)! alteraciones!en!las!funciones!tiroideas!en!aves!y!peces![20_24].! Capítulo)I:)Introducción) ! 10!I.1.)Compuestos)disruptores)endocrinos! ! !En! muchas! ocasiones,! estos! efectos! se! han! relacionado! con! niveles! traza! de! EDCs,! llegándose!a!registrar!actividad!endocrina!a!concentraciones!tan!bajas!como!1!ng!·∙!L_1! [25].! ·∙)Fuentes)de)contaminación) !Los! EDCs! pueden! entrar! en! el! medio! ambiente! a! través! de! una! gran! variedad! de! fuentes! de! contaminación.! Si! embargo,! la! gran! mayoría! de! las! mismas! se! hallan! focalizadas!en!puntos!localizados!(o!“hot)spots”)!de!descarga,!como!por!ejemplo!los! efluentes!de!las!estaciones!depuradoras!de!aguas!residuales!(EDARs),!o!las!aguas!de! escorrentía!procedentes!de!vertederos!o!de!suelos!dedicados!a!la!agricultura!intensiva.!! !!Hoy! en! día! se! estima! que! las! EDARs! representan! las! principales! fuentes! de! contaminación!de!EDCs![26_29],!dado!que!en!ellas!confluyen!diferentes!tipos!de!aguas! cuya!procedencia!puede!ser!tanto!de!origen!doméstico!como!industrial.!!! !Ambos! tipos! de! aguas! “brutas”! o! sin! tratar! pueden! aportar! diferentes! grupos!de! contaminantes!con!capacidad!disruptora!endocrina.!Por!un!lado,!las!aguas!domésticas! pueden!aportar!hormonas!naturales,!compuestos!farmacéuticos!estrogénicos!como!el! 17α_etinilestradiol,!detergentes!que!contengan!alquilfenoles!(APs),!desechos!plásticos! que! contengan! ésteres! de! ftalatos! o! bisfenoles,! etc.! Todas! estas! sustancias! pueden! llegar!a!las!EDARs!a!través!de!los!vertidos!de!aguas!grises!y!negras.!! !Por! el! otro,! las! aguas! procedentes! de! actividades! industriales! y! agrícolas! pueden! aportar!la!mayor!parte!de!compuestos!químicos!que!han!sido!catalogados!como!EDCs.! De! tal! forma,! los! vertidos! de! procesos! industriales! que! emplean! surfactantes,! Capítulo)I:)Introducción) ! I.1.)Compuestos)disruptores)endocrinos)11) ) plastificantes,! productos! agroquímicos,! o! pesticidas,! por! citar! algunos! ejemplos,! liberan!estas!sustancias!a!los!torrentes!de!agua!que!terminan!por!entrar!en!las!EDARs.!! !El! principal! problema! atribuible! a! las! EDARs! con! respecto! a! los! EDCs! reside! en! la! incapacidad! mostrada! por! la! mayoría! de! las! instalaciones! para! eliminar! este! tipo! de! contaminantes,!!de!manera!que,!en!la!mayoría!de!los!casos,!los!efluentes!de!las!plantas! de! tratamiento! suponen! una! entrada! continua! de! estos! compuestos! en! el! medio! ambiente! [30].! Este! fenómeno! es! especialmente! plausible! en! EDARs! carentes! de! tratamientos!terciarios.!! !Diferentes! tipos! de! fuentes! de! contaminación! de! EDCs! no! localizadas! han! sido! descritas!en!la!bibliografía,!las!cuales!se! encuentran! fundamentalmente!asociadas!al! uso!del!terreno!para!la!agricultura!y!la!ganadería.! !Dichas! fuentes! incluyen! a! las! emisiones! procedentes,! por! ejemplo,! de! industrias! lácteas!o!de!la!acuicultura![31].!En!el!caso!de!la!acuicultura,!se!ha!demostrado!que!el! desove!de!los!peces!a!nivel!local!puede!incrementar!notablemente!la!concentración!de! estrógenos! en! aguas! de! ríos! [32].! En! el! caso! de! las! granjas! de! ganado! e! industrias! lácteas,! también! se! ha! demostrado! que! pueden! ser! una! fuente! potencial! de! compuestos!estrogénicos!debido!al!alto!nivel!de!hormonas!esteroideas!presente!en!la! orina!y!en!el!estiércol![33,!34].!!! !A! todo! ello! hay! que! sumarle! el! uso! de! productos! químicos! tales! como! pesticidas! y!! fertilizantes,!sustancias!ampliamente!utilizadas!en!este!tipo!de!actividades!que!pueden! contener,! entre! otros! tipos! de! EDCs,! surfactantes! estrogénicos! (p.e.! nonilfenoles! polietoxilados)!en!su!formulación!química![35,!36].!!! Capítulo)I:)Introducción) ! 12!I.1.)Compuestos)disruptores)endocrinos! ! !!Ambas! fuentes! de! contaminación! procedentes! de! las! actividades! agrícolas,! la! excreción! de! hormonas! por! parte! de! organismos! vivos! y! el! uso! de! fertilizantes! y! pesticidas,!contribuyen!notablemente!a!las!fuentes!no!localizadas!(“non7point)source) runoff”)!indicadas!en!la!Figura!I.2.3! ! ! ! Figura!I.2.!Esquema!de!los!diferente!compartimentos!que!necesitan!ser!monitoreados!para!caracterizar! el!destino!y!transporte!de!los!EDCs!en!el!medioambiente.!! ·∙)Potenciales)mecanismos)de)transporte)) !El!destino!de!los!disruptores!endocrinos!en!el!medio!ambiente!está!altamente!ligado!a! las!propiedades!físico_químicas!de!los!mismos.!En!la!Tabla!I.1!se!adjuntan!algunas!de! las!propiedades!más!relevantes!de!los!compuestos!estudiados!en!esta!Tesis!Doctoral.! ! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 3!Figura!tomada!de!C.G.!Campbell!et!al.![37].! Capítulo)I:)Introducción) ! I.1.)Compuestos)disruptores)endocrinos)13) ) ! Tabla!I.1.!Propiedades!físico_químicas!de!varios!EDCs!estudiados!en!esta!Tesis!Doctoral.!a!Coeficientes!de! partición.!b!Solubilidad!a!20º!C.!c!Dato!no!encontrado!en!la!bibliografía.! ! Compounds! Chemical!structure! Log!KOW a! Solubility! (mg!L>1!)b! pKa! Norgestrel)(NOG)) ! 3.48! 35.84! _c! Diethylstilbestrol) (DES)) ! 5.64! 3.32! 9.73! Testosterone)(TE)) ! 3.27! 67.76! _c! 17α7ethynylestradiol) (EE)) ! 4.15! 4.8! 10.3! 17β7estradiol)(E2)) ! 3.94! 13.0! 10.71! Estriol)(E3)) ! 2.81! 13.0! 10.4! Estrone)(E1)) ! 3.43! 13.0! 10.3_10.8! Bisphenol)a)(BPA)) ! 3.32! 120.0! 9.6_11.3! Nonylphenol)(NP)) ! 4.48! 1.57! 10.28! Octylphenol)(OP)) ! 4.12! 12.6! 10.38! Capítulo)I:)Introducción) ! 14!I.1.)Compuestos)disruptores)endocrinos! ! !!Tal!y!como!se!observa!en!dicha!Tabla,!la!mayoría!de!los!compuestos!seleccionados! presentan! valores! de! coeficientes! de! partición! octanol_agua! (logKow)! relativamente! altos.! Este! hecho! nos! indica! que! estas! sustancias! van! a! presentar! una! elevada! tendencia! a! asociarse! formando! complejos! orgánicos,! o! a! adsorberse! al! material! particulado!presente!en!las!muestras!y!por!ende,!a!acumularse!en!los!sedimentos![37].! La!Figura!I.2.!muestras!alguna!posibles!trayectorias!que!siguen!los!EDCs!una!vez!que! alcanzan!el!medio!ambiente.!!! !Es! importante! resaltar! que! el! estudio! de! estos! contaminantes! en! la! fase! sólida! de! muestras!medioambientales!ha!ido!ganado!relevancia!en!la!comunidad!científica,!dado! que,! una! vez! que! estas! sustancias! se! han! asociado! a! la! fracción! sólida,! su! biodisponibilidad! en! el! medioambiente! aumenta! considerablemente,! pasando! a! formar!parte!de!la!biomasa!de!una!forma!más!directa.!! !Esta!naturaleza! generalmente! lipofílica! también! les! confiere!una! mayor! persistencia! en!el!medio!y!la!capacidad!de!bioacumularse!en!el!tejido!graso![38,!39].!Por!lo!tanto,! aunque!las!mayores!entradas!de!estos!contaminantes!al!medioambiente!proceden!de! efluentes! localizados,! existe! la! posibilidad! de! un! fenómeno! de! disrupción! endocrina! más!globalizado!a!través!del!paso!de!estos!contaminantes!de!unos!organismos!a!otros! mediante!de!la!cadena!trófica![40].! !!El! fenómeno! de! bioacumulación! de! los! EDCs! en! diferentes! organismos! ha! sido! demostrado!en!varias!ocasiones:!(a)!Liebig!et!al.![41]!han!analizado!recientemente!la! bioacumulación! del! 17α_ethynylestradiol! (EE)! en! algunos! organismos! bentónicos,! observando!factores!de!bioconcentración!en!peso!seco!entre!254!y!646!veces;!(b)!Ahel! y!colaboradores![42]!también!encontraron!elevados!factores!de!bioconcentración!de! Capítulo)I:)Introducción) ! I.1.)Compuestos)disruptores)endocrinos)15) ) nonilfenol!y!sus!etoxímeros!en!algunas!especies!de!peces!(1_300)!y!de!algas!(más!de! 10.000).!No!obstante,!no!observaron!ninguna!biomagnificación!al!estudiar!la!presencia! de! estas! sustancias! en! sus! consumidores.! Este! fenómeno! ha! sido! posteriormente! confirmado! por! Hu! et! al.! [43],! quienes! tampoco! encontraron! evidencias! de! biomagnificación! para! el! NP! y! sus! etoxilados! (NPnEOs).! De! tal! forma,! aunque! la! biomagnificación!no!se!ha!demostrado!extensamente!en!la!bibliografía,!sí!existen!una! serie!de!evidencias!de!este!fenómeno!en!algunas!especies!acuáticas!de!los!niveles!altos! de!la!cadena!trófica![45,!46].! !Los!valores!de!solubilidad!mostrados!en!dicha!Tabla!también!sugieren!que!la!mayoría! de! los! EDCs! podrían! no! permanecer! durante! periodos! largos! en! la! fase! disuelta.! Sin! embargo,!todos!los!EDCs!que!se!muestran! en!dicha!tabla!han!sido!determinados! en! diferentes!muestras!de!agua!alrededor!de!todo!el!planeta![37].! !En! algunos! casos! se! han! encontrado! EDCs! incluso! en! muestras! procedentes! de! acuíferos!y!de!fuentes!de!aguas!minerales,!lo!cual!sugiere!algún!tipo!de!transporte!en! la!fracción!soluble![30].!Dentro!de!las!posibles!hipótesis!para!este!tipo!de!transporte!se! incluyen:! (a)! el! transporte! de! los! EDCs! en! forma! coloidal,! (b)! la! formación! de! metabolitos! o! precursores! más! solubles! que! los! compuestos! de! origen,! como! por! ejemplo! los! alquilfenoles! carboxilados,! (c)! el! incremento! de! la! solubilidad! de! los! compuestos!debido!a!un!elevado!pH!en!el!medio!(considerando!que!la!mayoría!de!los! EDCs!en!estudio!tienen!un!pKa!en!torno!a!10),!y!(d)!la!formación!de!micelas,!las!cuales! aumentarían!la!estabilidad!de!los!EDCs!en!la!fase!disuelta![46].! !La!inclusión!de!varios!EDCs!(NP,!OP,!BPA,!E2!y!EE)!en!la!fase!coloidal!(0.001_1!μm)!ha! sido!recientemente!estudiada!por!Liu!y!colaboradores![47].!En!dicho!estudio!se!analizó! Capítulo)I:)Introducción) ! 22!I.2.1.1.)T.)Vega)Morales)et)al./)Trends)Anal.)Chem.,)(2009))1186?1200! ! ! ! ! Determination of alkylphenol ethoxylates and their degradation products in liquid and solid samples T. Vega Morales, M.E. Torres Padro ´n, Z. Sosa Ferrera, J.J. Santana Rodrı´guez Alkylphenol polyethoxylates (APEOs) are a group of non-ionic surfactants used extensively in industrial, agricultural and domestic applications. Their metabolites, generated by the biotransformation of APEOs in wastewater-treatment plants and natural water bodies, are more toxic and more persistent than the parent compounds, can mimic natural hormones and disrupt endocrine functions by interacting with estrogen receptors. Since the discovery of the adverse effects of these pollutants on wildlife and human health, analytical methods have been developed to determine them in environmental matrices. This article is an overview of the current methods employed in trace analysis of APEOs and their degradation products. We compare the analytical techniques used and we discuss potential advantages and disadvantages of the major detection and quantification techniques that have been coupled to liquid chromatography for the analysis of mixtures of APEOs in the past decade. ª2009 Elsevier Ltd. All rights reserved. Keywords: Alkylphenol ethoxylate (APEO); Degradation product (DP); Ethoxy unit (EO); Liquid chromatography (LC); Mass spectrometry (MS); Nonylphenol (NP); Nonylphenol ethoxylate (NPEO); Octylphenol (OP); Octylphenol ethoxylate (OPEO); Sample preparation 1. Introduction Alkylphenol polyethoxylates (APEOs) are a class of non-ionic surfactants that are used extensively as detergents, emulsifiers, wetting agents, and dispersing agents in industrial, agricultural and household applications. These compounds can be used in cleaning products, personal-care products, plastics, paints, textiles, resins, preservative coatings, pulp and paper, petroleum refining, pesticides and metal processing [1,2]. The two main kinds of APEOs are nonylphenol ethoxylates (NPEOs) and octylphenol ethoxylates (OPEOs), which represent approximately 80% and 20% of total APEO production, respectively [3].It is estimated that 60–65% of the total production of these compounds is incorporated into the aquatic environment [4], primarily through industrial and municipal wastewater discharges. The fate of APEOs in different environmental compartments (surface water, groundwater, sediment, soil and air) is controlled predominantly by their physicochemical properties that, in turn, influence their degradation. It is known that APEOs break down in wastewater-treatment plants (WWTPs), mainly during biological treatment, with a subsequent loss of ethoxy (EO) units [4,5]. This biotransformation leads to the formation of sub-products more toxic, more lipophilic, more estrogenic and more persistent than the parent substances [6,7]. The products formed in aerobic conditions included mono-, di-, and tri-ethoxylated NP and OP (NP 1–3 EO and OP 1–3 EO), and more polar short-chain and long-chain AP ethoxycarboxylate (APEC) and carboxylated AP ether carboxylate (CAPEC) derivatives [8,9]. However, the APs used as raw material in the synthesis of ethoxylated compounds, NP and OP, are formed only in anaerobic conditions [10]. Moreover, both APEOs, like their (bio)degradation products (DPs), are susceptible to be transformed into halogenated derivatives during chlorine disinfection in the presence of bromide ions [11]. These breakdown processes are also observed in natural water bodies [12,13]. In the past 20 years, several publications have reported that this class of T. Vega Morales, M.E. Torres Padro ´n, Z. Sosa Ferrera, J.J. Santana Rodrı ´guez* Department of Chemistry, Faculty of Marine Sciences, University of Las Palmas de Gran Canaria, 35017 Las Palmas de Gran Canaria, Spain 1186 0165-9936/$ - see front matter ª2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2009.07.011 * Corresponding author. Tel.: +34 928 452 915; Fax: +34 928 452 922; E-mail: [email protected] Trends Trends in Analytical Chemistry, Vol. 28, No. 10, 2009 23 compounds presents bioaccumulation in aquatic organism [14] and chronic toxicity [15], and can mimic natural hormones and disrupt endocrine functions by interacting with estrogen receptors [10,16,17]. The widespread use of APEOs, coupled with the characteristics listed above, have led to the incorporation of NP and OP in the list of 33 priority hazardous compounds of the European Union (EU) Water Framework Directive (WFD) [18,19]. Furthermore, European Directive 2003/53/EC [20] has also established restrictions on the use, production and marketing of these compounds. The total production of APEOs in the Western Europe has therefore considerably reduced in the past few years [21]. Despite this, ‘‘high’’ concentrations of EO compounds and derivatives are still found in environmental samples, especially in areas affected by wastewater discharges. Great public concern about the effects of APEOs and their metabolites on wildlife and human health, and about their environmental fate, has led to the development of different analytical methods for the determination of these substances in different matrices. Traditional methods of analysis are based on gas chromatography coupled to mass spectrometry (GC-MS), which is limited to NP, OP, and AP 1–3 EO, unless a derivatization step is included in the analytical procedure, which allows the determination of long-chain APEO compounds [22,23]. Nevertheless, the conversion of surfactants into volatile derivatives presents some disadvantages (e.g., more time for analysis) and, consequently, greater error [22]. To solve the problems presented by GC for the analysis of APEOs and their DPs, high-performance liquid chromatography (HPLC) methods have been employed with different detection systems, mainly spectrometric [24–27]. We review the last analytical methods for the analysis of APEOs and their DPs. We discuss and compare advanced extraction and clean-up techniques, and separation and quantification systems [e.g., LC with fluorescence detection (LC-FD), LC-MS and especially LC with tandem MS (LC-MS 2 )] for solid and liquid samples. 2. Sample-preparation methods The reliability of results obtained in the analysis of environmental samples is closely linked to sampling and storage procedures. In order to prevent the loss of analytes by abiotic reactions (e.g., hydrolysis) and biological degradation [28], several authors have used procedures to improve the stability of the analytes in complex environmental matrices and to ensure the integrity of the sample. Often, formaldehyde [29] and formalin (1% w/w of 37% solution of formaldehyde in water) [28] have been used to prevent biological degradation of APEOs, preserving the sample by freezing or by storage at 4C. For volatile organic compounds, the US Environmental Protection Agency (EPA) recommends acidification of the sample to pH <3, which is the most common procedure for conserving APEOs. To a lesser extent, chemical preservatives (e.g., mercury (II) chloride [30], sulfite and bisulfite [28]) have also been used, but have obtained worse results for long periods of storage. Some publications have reported on the stability of these compounds in solid-phase extraction (SPE) cartridges used to solve the problems of transporting, handling, and storing large volumes of sample after the extraction step. Petrovic and Barcelo ´[28] studied the stability of a mixture of NPEOs, with an average of 6 EO units, in C 18 SPE cartridges under different storage conditions and in different water matrices. For wastewater analysis, they obtained complete recovery at 20C for a period of 60 days. In the same way, Loyo-Rosales and co-workers [31] studied the stability of APs and APEOs with a range of 1–5 EO units in deionized water matrices. In this case, they employed hydroxylated poly(styrene-divinylbenzene) (PSDB) co-polymer SPE cartridges, finding recoveries of 63–116% for APEOs with less than 4 EO units after 1 year of storage at 20C. The results of both works [28,31] were in good agreement, and clearly demonstrated that the use of SPE cartridges is more effective method for stabilizing AP and APEOs than the simple storage using conventional procedures and under conventional conditions [31]. To preserve the target analytes in solid matrices, it is recommended to remove water from the solid matrix and to store the samples dry [32]. The more common and reliable procedure for dry solid samples is freeze drying [33–36]. However, in some studies, water is removed by air drying, despite the possible loss of more volatile compounds (e.g., APs). Subsequently, samples are homogenized, sieved and then stored at 4Cto20C or, less frequently, at room temperature while maintaining the sample in a desiccator [33]. When solid samples are collected as particulate matter, centrifugation is a common procedure before the drying step [11,37]. Nevertheless, some authors often choose to filter the samples using conventional glass-fiber filters (GF/Fs) and then stored them at 20C, extracting the compounds directly from the GF/Fs [38]. 2.1. Liquid matrices Since these organic compounds appear in the environment at trace and even ultra-trace levels, besides having systems of detection and quantification sufficiently sensitive and selective, it is necessary to use multi-step sample-pretreatment techniques with the major objective of eliminating potential matrix interferences to isolate the target compounds and finally to enrich the sample extracts. This type of procedure significantly improves the overall sensitivity of the analytical method. Trends in Analytical Chemistry, Vol. 28, No. 10, 2009 Trends http://www.elsevier.com/locate/trac 1187 24 Initially, the conventional analytical methods used to extract AP compounds from liquid samples were based on liquid-liquid extraction (LLE), followed by GC or LC and determination with different detection methods. Despite the LLE technique offering efficient and precise results, it is relatively time consuming, harmful, due to the use of large volumes of organic solvents that are frequently toxic, and very expensive. Although this technique is still in use in a few cases [39], there is an increasing tendency to replace LLE by SPE for liquid samples. Developed in the 1980s, SPE has emerged as a powerful tool for chemical isolation and purification. This method is an alternative to LLE because it reduces consumption of organic solvents and analysis time, and because it can be automated [40]. SPE has been developed in the off-line and on-line modes, although the on-line approach is preferred due to its advantages (e.g., greater sensitivity and fewer requirements for manipulation of samples). Several kinds of sorbents present in cartridges, columns and syringes have been employed successfully for the extraction or preconcentration of AP compounds. Basically, all the sorbents employed in SPE of APEOs can be conceived as modifications of three basic types of sorbents: silicabased, polymer-based (essentially PSDB) and carbonbased. Non-polar reversed-phase (RP) sorbents with a silica base were the first used in SPE of AP compounds from water samples. Among these sorbents (C 2 ,C 8 and C 18 ), C 18 was the most accepted. Retention of APEOs by C 18 cartridges is mainly due to non-polar Van der Waals interactions between the analytes and the sorbent. Despite evidence that C 18 sorbents present good recoveries for a wide range of APEO oligomers [41], they also show low breakthrough volumes for high oligomers, described as hydrophilic or ‘‘water-soluble’’ [42], and usually poor recoveries for NP and OP. Jonkers et al. [9] reported recoveries of 69% and 95% for NP and NP 10 EO respectively, but only by passing a sample volume of 100 ml through the cartridges. In the same way, Koh and coworkers [43] obtained similar recoveries (62–98% for APs, APEOs and APECs) for 250 mL of settled sewage samples. Subsequently, new highly cross-linked PSDB packing materials [e.g., Isolute ENV (International Sorbent Technology Ltd., Hengoed, UK) or LIChrolut EN (Merck, Darmstadt, Germany)] have obtained similar recoveries to those of C 18 silica-based materials [31,38]. Nevertheless, these hydrophobic polymeric sorbents are the most suitable, due to their broad range of physicochemical characteristics and their greater chemical stability [44]. The retention mechanism in these cases is based on not only non-polar Van der Waals interactions, but also p-pinteractions between the analytes and the aromatic rings that make up the sorbent structure. These hyper-cross-linked polymeric sorbents have a specific surface area up to 800 m 2 /g, which allows more p-pinteractions than traditional polymeric macroporous PS-DVB sorbents [44]. Overall, this means that the breakthrough volumes will be greater than those obtained when C 18 sorbents are used. Loyo-Rosales et al. developed an off-line SPE procedure to extract APs, short-chain APEOs (AP 1–5 EO) [31] and long-chain APEOs (AP 6–16 EO) [38] in water using Isolute ENV cartridges. Recoveries for the APs and shortchain APEOs were above 81% for all analytes, with variations in response in the range 1–14% (RSD). Although long-chain APEOs were extracted using the same protocol, the recoveries for this fraction were highly conditioned by the hydrophobic nature of the packing material. Thus, the greater the number of EO units, the smaller the recovery in the range 71–21% for NP 6 EO– NP 16 EO. Although recoveries obtained for long-chain APEOs were inefficient, Loyo-Rosales et al. achieved a rapid extraction of up to 4 L of sample, in contrast to the few hundred mL of sample that octadecyl-silica sorbents allowed to pass through the cartridges. To solve the lack of retention of the most polar compounds, other hydrophilic polymeric sorbents have already been studied in order to obtain better recoveries of the ‘‘water soluble’’ (EO > 5 units) fraction of APEO oligomers and acidic metabolites. Loos et al. [45] employed Oasis HLB cartridges (Waters, Milford, MA, USA) for the clean up and extraction of APs, their ethoxylates and their more polar carboxylates from textile-industry WWTP effluent and surface waters. This packing material is a hydrophilic macroporous poly(N-vinylpyrrolidone-divinylbenzene) (PVP-DVB) copolymer and has a specific surface area of 800 m 2 /g. Recoveries obtained in this study were in the range 50–90%. Results close to 50% corresponded to more hydrophobic compounds (NP and OP), while the better recoveries corresponded to high oligomers and carboxylated derivatives. Jahnke et al. [46] also employed Oasis HLB cartridges in the extraction of APs, AP 1–2 EO and more polar alkylphenoxy acetic-acid derivatives (AP 1 EC). They reported good recoveries for carboxylated compounds (110% and 99% for NP 1 EC and OP 1 EC, respectively), but the results shown for hydrophobic species are clearly lower, especially for NP and their ethoxylates (recoveries were <70%). Both OP and OP 1–2 EO presented higher recoveries (close to 80%) than their nonyl isomers, indicating the predominant influence of hydrophobic chain length in the extraction [7]. The fact that NP was not adequately recovered from Oasis HLB cartridges constituted a important disadvantage of these methods, due to the great relevance of NP for the control of potential endocrine disrupters (EDs), but the high recoveries that this sorbent shows for more polar analytes offer new perspectives [e.g., their use in sequential SPE (SSPE)] [47] to cover a broad range of EO compounds. SSPE has been employed successfully using Trends Trends in Analytical Chemistry, Vol. 28, No. 10, 2009 1188 http://www.elsevier.com/locate/trac 25 different sorbents (two cartridges of different SPE material coupled in series) [48] or selective elution employing solvents of different desorption potential and polarity [49,50]. Graphitized carbon black (GCB) is the third kind of sorbent usually employed in SPE of APEO species. Carbon is a non-porous sorbent, so cartridges filled with this material have a high resistance to the water flow, so the extraction is usually slower than that obtained using the sorbents described above. Another drawback of this kind of sorbent is that DVB polymers would allow a more selective extraction than GCB [38]. This means that the sample extract would contain more interfering compounds that would therefore affect the posterior quantification (e.g., matrix suppression effects in MS). Nevertheless, several procedures used for sample extraction of NPEOs employing GCB have reported [23,29,40] good recoveries, especially for high ethoxylated compounds and even passing volumes up to 1 L through the cartridges. Although most analytical approaches to the determination of APEOs and their DPs include SPE, conventional LLE and even steam distillation or solvent sublation [24], solid-phase microextraction (SPME) has gained importance in recent years, especially in extraction and enrichment of APs, short-chain APEOs (1–3 EO units) and their acidic metabolites followed by GC determination. It has been reported that SPME procedures without derivatization have been used to determine APs in water samples [51]. However, derivatization of APs (methylation, acetylation or silylation) is preferred due to some advantages (e.g., improving the quality and the sensitivity of GC analysis) [52]. Several kinds of fiber have therefore been tested for extracting these substances. Among commerciallyavailable SPME fibers, polyacrylate (PA) and polydimethylsiloxane-divinylbenzene (PDMS-DVB) have usually been recommended for the extraction of polar compounds, while PDMS fibers were recommended for enrichment of non-polar compounds [53,54]. For the relatively large partition coefficients of NP and NP 1–3 EO in the octanol/water system (log K ow values of 4.48 and 3.90, respectively) [55], the maximum extraction yields would be expected to be obtainable using PDMS fibers. Braun et al. [56] examined these three kinds of fiber for the determination of technical NP (t-NP), obtaining the best sensitivity with the 100-lm PDMS fiber. However, APs also exhibit good enrichment behavior on PA and PDMS-DVB fibers [56]. For the simultaneous determination of NP, NP 1–2 EO and their brominated derivatives, Dı´az et al. [57] developed and applied a SPME-GC-MS procedure using DVBCarboxen-PDMS (DVB-CAR-PDMS) fiber. By comparing the results with those obtained by SPE (C 18 cartridge), they reported that both results were in good agreement, but the SPME procedure showed some advantages [e.g., lower limits of detection (LODs), shorter analysis time and avoiding use of organic solvents]. Several procedures based on SPME-GC-MS for the determination of these ED substances have been published. Nevertheless, there are only a few relating to SPME of non-ionic surfactants by HPLC determination. Mitani et al. [58] developed an in-tube SPME-HPLC-UV method for determination of EDs (including NP and OP) in liquid medicines and intravenous solutions. Recoveries of these compounds spiked to the intravenous injection solutions were over 80%. Boyd-Boland and Pawliszyn reported another SPME-HPLC-UV method for the determination of NPEOs using a Carbowax-template resin [59]. Among all the methods reviewed based on SPME-HPLC, we have not found any work that employs MS detection, despite the clear advantages that this separation technique shows (described below). 2.2. Solid matrices As discussed above, APEO-breakdown processes in WWTPs or in the environment leads to the formation of more persistent, more toxic and more estrogenic metabolites [essentially APs and shorter-chain oligomers (Fig. 1)]. These (bio)DPs have high log K ow values, in the range 3.90–4.48 [7,55], with NP having the highest log K ow . They also show low solubilities in water, due to the small number of polar groups forming the hydrophilic part of the molecules [42]. Organic carbon-sorption constants (K oc ) have high values for these metabolites. Ferguson et al. [60] reported K oc for NP of 245 L/kg, while NP 1–4 EOs have K oc in the range 74–288 L/kg. K oc for OP was 151 L/kg, indicating the influence of the hydrophobic chain length (alkyl group) on the constant. These physico-chemical profiles suggest that these DPs have a strong affinity to aquatic particles and organic matter, due to their highly lipophilic nature and lower water solubilities. They therefore tend to bind tightly to sediments [42] and bioaccumulate in the aquatic organism [14]. These factors contribute to the persistence of these compounds in the environment, and, most importantly, allow the entry of these xenobiotics into foods of animal origin, which are thought to represent one of the major sources of human exposure to many organic pollutants. Classical approaches to the extraction of AP compounds in solid matrices are mainly based on Soxhlet extraction and steam distillation, which were employed almost exclusively in the 1980s and 1990s. Methanol, acetonitrile, dichloromethane, n-hexane or mixtures of hexane/acetone, dichloromethane/hexane, or hexane/ i-propanol were typical solvents in Soxhlet extraction [32]. Despite this technique offering high percentages of recovery for a broad range of oligomers (depending on appropriate selection of the solvent) [35], Soxhlet Trends in Analytical Chemistry, Vol. 28, No. 10, 2009 Trends http://www.elsevier.com/locate/trac 1189 26 Figure 1. Breakdown processes of long-chain alkylphenol polyethoxylates (APEOs) under aerobic and anaerobic conditions, and the formation of their halogenated derivatives. Trends Trends in Analytical Chemistry, Vol. 28, No. 10, 2009 1190 http://www.elsevier.com/locate/trac 27 extraction makes the analysis procedure excessively time consuming (up to 48 h) and, moreover, requires large amounts of hazardous organic solvents (50–300 mL) [32]. However, Soxhlet is still used to a lesser extent fundamentally for the extraction of APs and APEOs adsorbed into particulate matter (separated from the water matrix by simple filtration techniques) [38] and for comparing the results obtained by more versatile extraction systems [38,61]. During the past decade, a lot of extraction techniques have been developed to isolate these analytes in solid samples, to reduce the organic solvent consumption and to increase the speed of the process. Ultrasonic extraction has been presented by the scientific community as an attractive alternative to the conventional extraction systems. Although sonication is faster than Soxhlet extraction, it also requires relative large volumes of toxic and expensive organic solvents (30–60 mL). The solvents used most for the extraction of APEOs employing ultrasonic irradiation are acetone, hexane, dichloromethane and mixtures of them in different proportions [32]. Aparicio et al. [62] recently employed this approach to extract NP and NP 1–2 EO oligomers in sludge from WWTP samples. They used hexane as extraction solvent, with a global extraction time of 70 min for each sample. Fountoulakis et al. [63] showed much lower recoveries for the same analytes using a mixture of dichloromethane/methanol (30:70) as solvent. However, they took only 20 min for sonication and no further procedures to enhance the extraction efficiencies. Some modifications of this extraction technique have been reported. Ferguson et al. [41] employed a novel continuousflow, high-temperature sonication system to isolate short-chain-APEO metabolites from sediment samples. Similarly, Nu´nez et al. [64] developed a sonicationassisted extraction in small columns (SAESC) procedure to isolate NP and NPEOs in environmental solid samples. They used a mixture of water/methanol (70:30) in order to obtain better recoveries for more hydrophilic metabolites. They reported excellent recoveries for all compounds, in the range of 91% for long-chain NPEOs to 108% for NP. Pressurized liquid extraction (PLE), also known as pressurized fluid extraction (PFE) or accelerated solvent extraction (ASE), is, by far, the main extraction technique for these compounds in solid matrices. PLE offers a great reduction in solvent consumption (15–30 mL), and provides faster sample processing and a high level of automation [35,37]. This technique also offers the advantage that only two variables need to be optimized – extraction time and temperature – since the solvents chosen for PLE can be the same as those used in Soxhlet extraction [37] or sonication extraction [32]. High temperature and high pressure help to increase the diffusion rates, solubility and mass transfer and to maintain the solvent in the liquid state. Moreover, PLE provides cleaner extracts than Soxhlet and ultrasonic extraction, reducing the background noise in the subsequent determination, which is especially important in LC-MS analysis due to ion-suppression effects. PLE therefore provides rapid, efficient extraction for a wide range of APEO oligomers, even in complex matrices {e.g., sludge, soil [34,65,66] and biological tissues [67] (see Table 2)}. The high initial cost is the main drawback of these extraction systems; however, the large savings in solvent consumption and extraction time could depreciate that cost rapidly. In the past decade, microwave energy has been investigated and widely applied in analytical chemistry to accelerate sample digestion, and to extract analytes from different matrices and in chemical reactions. Microwave-assisted extraction (MAE) is an efficient extraction technique for solid samples. MAE is applicable to thermally stable compounds. Since its development, MAE has became a viable alternative to conventional methods due to it having had many substantial improvements over other sample-preparation techniques (e.g., shorter extraction time, smaller amounts of solvent and multiple samples analyzed at the same time) [68]. Temperature, extraction time and power, solvent volume and concentration of different solvent mixtures are the most common parameters to optimize. The number of papers reporting the use of MAE has therefore increased considerably [68]. Croce et al. [69] compared MAE versus PLE for the isolation of NP and NPEOs from river sediments. They concluded that MAE has an important disadvantage compared to PLE extraction – the need for sample centrifugation and filtration – which can have critical effects on analytical accuracy. However, this drawback can be overcome by using proper accessories for automatic sample handling. Moreover, MAE also offers the ability to extract several samples simultaneously, while, in PLE, samples are always run one at a time. MAE has therefore become the most suitable extraction system for monitoring programs due to its capability of handling a large number of samples in a short period of time [68]. To a lesser extent, supercritical-fluid extraction (SFE) has been reported for extraction of AP compounds in solid samples. SFE with solid trapping has proved better than conventional liquid-solvent-extraction methods, with several advantages (e.g., rapid extraction, low solvent requirement, low cost and higher efficiencies). Among all the solvents used in SFE, pure CO 2 is the most popular, due to its low critical properties, chemical inertness, low toxicity and cost, and its ability to dissolve a wide range of organic compounds, including those having high molecular mass. Nevertheless, pure CO 2 leads to low recoveries for polar compounds (e.g., longchain APEOs). Trends in Analytical Chemistry, Vol. 28, No. 10, 2009 Trends http://www.elsevier.com/locate/trac 1191 28 The lack of extraction efficiency for these polar compounds can be overcome by adding modifiers or co-solvents to the pure CO 2 , with water and methanol being the most common solvent modifiers used. Lee et al. [70] were among the first to set up an SFE method for the extraction of NPEOs and carboxylated derivatives from dried sewage-treatment-plant sludge. By using water as the CO 2 -solvent modifier, they obtained recoveries in the range 86–105% for NP 1–17 EOs oligomers, with NP 17 EO showing the worst result. With the same objectives, Minamiyama et al. [71] recently studied the effect of adding methanol in the extraction of NP and NPEOs using SFE for sewage-sludge samples. They reported that adding methanol to the pure CO 2 increased the quantities of NP and NP n EO extracted by 1.7–5 times. Earlier in this decade, some attempts were made to use environmentally-friendly approaches employing non-ionic surfactants for the extraction of lipophilic AP compounds from sediment and biological matrices [61,69]. The use of surfactant solutions offers several advantages over organic solvent-like extractants (e.g., reduction in the amounts of solvents used, low cost, easy handling and non-toxic procedures). In this way, Patrolecco et al. [61] developed a readily applicable method using aqueous non-ionic surfactant solutions (Tween 80) for the extraction of EDs, including NP, NP 1 EO and NP 2 EO as target compounds. They reported recoveries of 89–95% after 3 h of extraction. This extraction time is relatively long compared to those for conventional techniques, and poses the biggest disadvantage of the method. Employing the same extraction protocol, Croce et al. [69] reported recoveries of 84–95% for the same analytes. They also compared this ‘‘new trend’’ with ASE and MAE, concluding that the recovery and the reproducibility of these different extraction methods are comparable and all methods give reliable results for the extraction of short-chain AP compounds. Due to the complexity of many solid matrices, especially biological tissues and sludge samples, purification of the extracts by different clean-up procedures after extraction has become crucial in order to obtain the maximum sensitivity in the subsequent detection of analytes. Among all methods employed for this purpose, the conventional approaches based on use of offline SPE cartridges or solid-liquid adsorption chromatography remain the most important clean-up and fractionation methods to eliminate interfering compounds. In order to reduce the sample-pretreatment time and to improve the isolation of analytes, advanced methods {e.g., coupling in series two HPLC columns or integrating LC-sample preparation and analysis on a dual-column system (column switching) [65]} have been developed for purification of APEOs from solid matrices. 3. Analysis 3.1. Liquid chromatography Chromatographic separation of APEOs and their metabolites in a single run presents several difficulties that make it complicated to choose an appropriate analytical column and a mode of separation. The great complexity of commercially-available technical products (mixtures of EO homologues and alkyl isomers), besides the broad range of polarities that APEOs exhibit from the more lipophilic APs to the more hydrophilic long-chain oligomers, is the main drawback to obtaining a good chromatographic performance needed to conduct proper quantitative analysis. Reversed-phase liquid chromatography (RPLC) has been used extensively for LC-MS analysis of APEOs and their DPs. RP columns (silica-based C 18 and C 8 , aluminabased C 18 or polyethylene-coated alumina) separate according to the nature of the hydrophobic moieties, so mixtures of surfactants containing several hydrophobic moieties (e.g., nonyl, octyl and heptyl) can be determined correctly since the quantification is simplified [47]. However, RPLC separation on C 18 columns is not affected by the length of EO units, and only the APs and AP 1–2 EO can be successfully separated from other EO oligomers, which often co-elute in a single peak. Each peak therefore represents a single alkyl chain length and contains the whole range of EO chain lengths. Despite many authors describing this aspect as an advantage, claiming that co-elution enhances signal-peak response and hence sensitivity, quantification is severely compromised because the response factors of each homologue vary significantly, with poor sensitivity for the monoethoxylate compounds [72]. Moreover, this coelution also leads to competitive ionization during the electrospray processes [40,41] and to isobaric interferences between singlyand doubly-charged adducts [34] when MS systems are employed (as discussed below). Broadly, this approach is not suitable for the determination of the full range of APEOs [39]. In this case, the distinction between the long-chain oligomers is made only by MS detection [see mass spectrum (Fig. 2)], and other detection techniques [e.g., FD or ultraviolet-diode array (UV-DAD)] are not particularly recommended for the determination of every single APEO oligomer. Given the difficulties from co-elution of alkyl homologues, normal-phase liquid chromatography (NPLC) has been tested for LC analysis of APEOs. NPLC allows the separation of APEO oligomers according to the increasing number of EO units, while the alkyl chain has virtually no effects on the chromatographic separation [64]. Thus, oligomers with the same number of EO units, but different alkyl chains, co-elute in a single peak (e.g., NP 4 EO and OP 4 EO). Trends Trends in Analytical Chemistry, Vol. 28, No. 10, 2009 1192 http://www.elsevier.com/locate/trac 29 Figure 2. Mass spectra of oligomeric mixture of nonylphenol ethoxylates (NPEOs) with an average of 4 EO units using an electrospray ionization (ESI) interface [47]. Figure 3. Chromatographic separations of oligomeric mixture of nonylphenol ethoxylates (NPEOs) and nonylphenol (NP) using different highperformance liquid chromatography (HPLC) columns. 1) reversed-phase LC (RPLC) with C 18 column: a) co-elution of several EO oligomers (NPEO x ) in a single peak. 2) normal-phase LC (NPLC) with silica column: b) co-elution of short-chain NPEOs (NP 1–2 EO) and NP; c) separation of alkylphenol-polyethoxylate (APEO) oligomers according to the increasing number of ethylene oxide units. 3) RPLC with C 8 column: d) complete separation of NPEO oligomeric mixture with the typical dome-shaped base line [64]. Trends in Analytical Chemistry, Vol. 28, No. 10, 2009 Trends http://www.elsevier.com/locate/trac 1193 30 The major drawback of NPLC approaches is that the separation of short-chain APEOs, especially mono-ethoxylated and di-ethoxylated compounds, becomes very difficult under certain conditions, and the co-elution of AP 1 EO, AP 2 EO, and even APs, is almost unavoidable (Fig. 3). Nevertheless, working correctly on the gradientelution profiles, especially in the first minutes of the chromatograms, this drawback can be partly solved [64]. Other disadvantages of NPLC are that it requires longer equilibration times than RPLC [29,35] and a polar modifier may have to be added post-column to support ionization of target analytes in MS detection [35]. However, complete separations of APs and a wide range of their EO homologues have been reported using NP columns [29,35]. Another approach to the separation of these compounds is also possible. RPLC using C 8 columns provides intermediate separation between RPLC and NPLC. In this case, separation is also done according to the character of the hydrophobic moieties. However, the number of EO units exerts a greater influence on the separation than those observed on C 18 columns, allowing a good performance of the highly ethoxylated compounds and, most importantly, clear separation between the shortchain compounds. Nevertheless, the hydrophobic behavior of this kind of material compromised the baseline resolution of long-chain APEOs, which are usually dome-shaped, hampering their proper quantification (Fig. 3). In this way, some authors have successfully employed C 8 columns for the complete separation of mixtures of AP compounds of up to 17 EO units in a single chromatographic run [9,23,36,41,46]. Important advances in APEO separation have been reported using so-called mixed-mode chromatography [72]. This mode of HPLC combines two mechanisms of separation: size exclusion and RP, so compounds are separated according to their molecular weight, allowing the separation of compounds based on their number of EO units, and according to the character of the hydrophobic moieties, allowing the separation of the different alkyl groups (Fig. 4). Ferguson and co-workers [39] were pioneers in using these columns for the chromatographic separation of this family of compounds. Subsequently, Loyo-Rosales et al. employed them to perform strongly in separating short-chain compounds [31] as well as highmolecular-weight oligomers [38] for octyl and nonyl polyethoxylated compounds into the same chromatogram. Liu and Pohl [73] tested a new mixed-mode silicabased stationary phase for the separation of non-ionic EO surfactants. They prepared a new stationary phase that combined both hydrophilic interaction chromatography (HILIC) and RP characteristics, providing two modes of operation that allowed a great performance in separation based on the nature of both alkyl chains and EO units. Surveying the current literature on LC analysis of target AP compounds, we can observe that applications based on mixed-mode separation technology are represented to Figure 4. Separation of nonylphenol (NP) and nonylphenol-ethoxylate (NPEO) oligomeric mixture using a mixed-mode high-performance liquid chromatography (HPLC) column: a) complete separation of alkylphenol-polyethoxylate (APEO) oligomers according to the increasing number of ethylene oxide units; b) separation between the target compound and its corresponding internal standard (A and B) [39]. Trends Trends in Analytical Chemistry, Vol. 28, No. 10, 2009 1194 http://www.elsevier.com/locate/trac 31 Capítulo)I:)Introducción) ! 38! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Capítulo)I:)Introducción) ! I.2.2.)Hormonas)esteroideas!39! ) I.2.2.!Hormonas!esteroideas.!! !Las! hormonas! esteroideas! engloban! a! un! grupo! de! compuestos! químicos,!! biológicamente! activos,! que! actúan! como! hormonas! en! el! sistema! endocrino! de! los! seres!vivos,!pudiendo!ser!tanto!de!origen!natural!como!antropogénico!(xenobióticos).! Este!tipo!de!esteroides!son!sintetizados!a!partir!del!colesterol,!teniendo!todos!ellos!en! común! un! anillo! de! ciclopentanoDoDperhidroxifenantreno! en! su! estructura! química! (Figura!I.3.)! ·∙)Hormonas)esteroideas)de)origen)natural) !La!hormonas!esteroideas!de!origen!natural!se!sintetizan!mayoritariamente!en!el!tejido! suprarrenal,!ovarios,!testículos!y!placenta.!La!clasificación!de!estas!sustancias!se!realiza! en!función!a!los!receptores!estrogénicos!a!los!que!se!unen,!existiendo!5!grupos!bien! diferenciados:! (a)! estrógenos,! (b)! andrógenos! (c)! mineralcorticoides,! (d)! glucocorticoides!y!(e)!progestágenos![50].!! !Los! estrógenos! naturales! (estradiol,! estrona! y! estriol)! son! predominantemente! hormonas! femeninas,! cumpliendo! con! un! rol! determinante! en! el! mantenimiento! de! diferentes! procesos! reproductivos! y! de! desarrollo.! Los! andrógenos! (testosterona,! dehidroepiandrosterona! and! androstenediona)! regulan! diversos! procesos! implicados! en!la!regeneración!de!tejidos!musculares,!óseos!y!en!la!piel.!! !Los!glucocorticoides! (cortisol)! se! generan!en!las! glándulas! adrenales! en!respuesta!a! diferentes!situaciones!de!estrés,!como!por!ejemplo,!durante!la!realización!de!ejercicio! físico,!trastornos!emocionales,!enfermedad,!inanición!e!incluso!durante!intervenciones! Capítulo)I:)Introducción) ! 40!I.2.2.)Hormonas)esteroideas! ! quirúrgicas.! Los! progestágenos! participan! en! el! balance! hormonal,! controlando! los! niveles!de!estrógenos!y!andrógenos!en!el!organismo![51].!! ·∙)Hormonas)esteroideas)de)origen)antropogénico) !Los!esteroides!sintéticos!han!sido!empleados!desde!hace!décadas!en!el!campo!de!la! medicina!humana!y!en!la!ganadería!con!diferentes!propósitos,!como!por!ejemplo,!en! distintos!métodos!anticonceptivos,!como!promotores!del!crecimiento,!o!en!diferentes! terapias! clínicas! [52D54].! Hoy! en! día! se! considera! que! son! los! medicamentos! más! prescritos!a!nivel!mundial.! !Dentro!de!este!grupo!de!compuestos!artificiales,!las!hormonas!esteroideas!!de!mayor! relevancia! medioambiental! son! las! de! naturaleza! estrogénica,! como! por! ejemplo! ! el! 17αDetinilestradiol,! el! mestranol,! la! noretindrona! o! el! dietilestilbestrol5,! compuestos! ampliamente!utilizados!como!anticonceptivos,!en!el!tratamiento!de!la!menopausia!y!el! síndrome!postDmenopáusico,!en!terapias!de!reemplazo!fisiológico!y!en!el!tratamiento! de!cáncer!de!mama!y!de!próstata![54].!! !El! norgestrel! es! otro! esteroide! sintético! (progestina)! que! también! se! emplea!en! grandes!cantidades!como!principio!activo!en!algunos!métodos!anticonceptivos,!como! por!ejemplo!los!implantes!subcutáneos!o!los!sistemas!anticonceptivos!de!emergencia! (píldora! del! día! después! o! dispositivos! intrauterinos)! [55].! Esta! sustancia! tiene! la! peculiaridad! de! presentarse! como! una! mezcla! de! dos! estereoisómeros,! dextroD norgestrel!y!levoDnorgestrel,!aunque!sólo!el!levoDnorgestrel!es!biológicamente!activo! [56].! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 5!El!uso!de!dietilbestrol!(DES)!ha!sido!fuertemente!restringido!desde!la!década!de!los!80!como! consecuencia!del!“caso!Sindell”.!En!1997!se!detuvo!definitivamente!la!fabricación!de!DES!en!USA![55].!! Capítulo)I:)Introducción) ! I.2.2.)Hormonas)esteroideas!41! ) !Al! igual! que! las! hormonas! naturales,! estas! sustancias! ejercen! su! función! pasando! a! través!de!la!membrana!plasmática!para!unirse!posteriormente!al!receptor!intracelular! correspondiente.!Los!mecanismos!de!acción!de!estas!sustancias!han!sido!ampliamente! discutidos!en!varias!publicaciones![57D59].! ! Figura!I.3.!Estructuras!químicas!de!las!hormonas!esteroideas!analizadas!en!esta!Tesis!Doctoral.!a)! hormonas!esteroideas!sintéticas.!b)!Hormonas!esteroideas!naturales.! Capítulo)I:)Introducción) ! 42!I.2.2.)Hormonas)esteroideas! ! ·∙)Principales)fuentes)de)emisión:) !Todas! las! hormonas! esteroideas,! bien! sean! endógenas! o! exógenas,! naturales! o! sintéticas,! están! sujetas! a! una! variedad! de! reacciones! metabólicas! en! el! interior! de! cualquier!organismo!vivo:!oxidación,!hidroxilación,!metilación,!o!la!conjugación!con!el! ácido! glucorónico! o! sulfúrico.! No! obstante,! una! cantidad! significativa! del! esteroide! original!sale!del!organismo!a!través!de!las!excreciones!(bilis,!orina!y!heces!fecales)![60].! !A!modo!de!ejemplo,!el!17αDetinilestradiol!se!excreta!tanto!en!su!forma!libre!como!en! sus!formas!conjugadas!de!glucuronida!y!sulfatos.!Por!su!parte,!el!estradiol!endógeno! (17βDestradiol)! esta! sujeto! a! procesos! oxidativos! en! los! cuales! se! bioDtransforma! en! estrona,! la! cual! a! su! vez! se! puede! oxidar! para! dar! lugar! al! estriol.! En! un! artículo! publicado!por!Johnson!et!al.![61]!se!realiza!una!estimación!de!las!excreciones!diarias! de!diferentes!hormonas!esteroideas!en!diferente!grupos!poblacionales,!obteniendo!los! resultados!que!se!muestran!en!la!siguiente!tabla:! ! Grupo&poblacional& 17β3! estradiol& Estrona! Estriol! 17α3 etinilestradiol! Hombre! 1.6! 3.9! 1.5! D! Mujeres!menstruantes! 3.5! 8! 4.8! D! Mujeres!menopáusicas! 2.3! 4! 1! D! Mujeres!embarazadas! 259! 600! 6000! D! Mujeres!(anticonceptivos)a! D! D! D! 35! ! Tabla!I.3.!Excreciones!diarias!(μg)!de!esteroides!estrogénicos!en!humanos.!a!Calculo!realizado!teniendo! en!cuenta!la!cantidad!17αDetinilestradiol!presente!en!las!píldoras!anticonceptivas.! !Este! tipo! de! excreciones! suponen! la! mayor! fuente! de! entrada! de! hormonas! esteroideas!al!medioambiente,!la!cuales,!en!su!mayor!parte,!se!canalizan!a!través!de! las!estaciones!depuradoras!de!aguas!residuales.!Así,!son!numerosos!los!artículos!que! coinciden! en! calificar! a! las! EDARs! como! las! principales! fuentes! de! emisión! de! estos! Capítulo)I:)Introducción) ! I.2.2.)Hormonas)esteroideas!43! ) contaminantes!al!medioambiente![62D64].!También!se!han!descrito!otro!tipo!vertidos! de!menor!relevancia!medioambiental!asociados!a!residuos!ganaderos,!de!acuicultura!y! a!otros!vertidos!incontrolados![51,!62].! !Los! principales! problemas! asociados! a! las! EDARs! con! respecto! a! las! hormonas! esteroideas!se!pueden!resumir!de!la!siguiente!manera:!a)!La!mayor!parte!de!este!tipo! de! instalaciones! carecen! de! los! sistemas! apropiados! para! eliminar! por! completo! a! estas!sustancias,!habiéndose!estimado!porcentajes!de!degradación!que!oscilan!entre!el! 60! y! el! 90! %! en! las! EDARs! convencionales! [65D69].! b)! Aunque! la! mayoría! de! los! estrógenos! se! excretan! como! subproductos! menos! activos! que! los! esteroides! de! partida,! en! las! EDARs! se! puede! producir! una! “deconjugación”,! de! forma! que! en! los! efluentes!volvemos!a!encontrar!a!los!productos!originales![67].!c)!Los!tratamientos!de! cloración! convencionales! también! pueden! producir! subproductos! clorados! que! presentan!actividad!estrogénica,!siendo!ésta,!por!norma!general,!ligeramente!superior! que!la!de!los!compuestos!originales![68].! ·∙)Propiedades)físico=químicas) !Algunas! de! las! propiedades! físicoDquímicas! de! las! hormonas! esteroideas! más! importantes!a!nivel!medioambiental,!han!sido!mostradas!previamente!en!la!Tabla!I.1.! Tal!y!como!se!observa,!los!estrógenos!naturales!muestran!una!solubilidad!en!torno!a! los!13!mg!·∙!LD1,!mientras!que!las!solubilidades!descritas!para!los!estrógenos!sintéticos! son!significativamente!inferiores:!4.8!mg!·∙!LD1!para!el!etinilestradiol,!3.32!mg!·∙!LD1!para!el! dietilestilbestrol!y!0.3!mg!·∙!LD1!para!el!mestranol![51].!! !Por!su!parte,!los!coeficientes!de!partición!octanolDagua!(log!Kow)!para!las! hormonas! naturales! están! comprendidos! entre! 2.81! (estriol)! y! 3.94! (estradiol).! Los! estrógenos! Capítulo)I:)Introducción) ! 44!I.2.2.)Hormonas)esteroideas! ! sintéticos! muestran! valores! superiores:! 4.15! para! el! etinilestradiol,! 4.67! para! el! mestranol,!y!5.64!para!el!dietilestilbestrol![54].!Tanto!las!hormonas!naturales!!como!las! sintéticas!muestran!presiones!de!vapor!muy!bajas!(2.3!·∙!10D10!D!6.7!·∙!10D10!mm!Hg),!lo! cual!indica!que!se!trata!de!compuestos!muy!poco!volátiles.! !Con!estos!datos!como!base,!se!puede!afirmar!que!este!tipo!de!hormonas!esteroideas! son,! en! conjunto,! compuestos! orgánicos! que! presentan! una! hidrofobicidad! relativamente! alta! y! una! baja! volatilidad,! por! lo! que! es! de! esperar! se! produzca! adsorción! importante! de! estas! sustancias! a! los! sedimentos! y! a! los! sólidos! en! suspensión! presentes! en! las! muestras! medioambientales,! reduciendo! así! su! concentración!en!la!fase!disuelta.! ·∙)Mecanismos)de)transporte)y)destino)medioambiental) !La!partición,!el!trasporte!y!el!destino!final!de!estos!compuestos!en!el!medioambiente! están! estrechamente! ligados! con! las! propiedades! físicoDquímicas! de! los! mismos.! Muchos!de!estos!aspectos!ya!fueron!descritos!en!las!sección!I.1!de!esta!introducción.! !En! líneas! generales,! los! niveles! de! solubilidad! presentados! tanto! por! los! esteroides! sintéticos!como!por!los!naturales,!sugieren!que!estas!sustancias!podrían!encontrarse! en!la!fase!disuelta!en!diferentes!tipos!de!muestras,!aunque!no!deberían!permanecer! durante! demasiado! tiempo! en! la! misma.! Esto! ha! quedado! demostrado! en! la! bibliografía!científica!dado!que!este!tipo!de!EDCs!ha!sido!determinado!a!niveles!traza!y! ultra!traza!en!diferentes! tipos! de! matrices! acuosas:! ! aguas!residuales![69D71],!aguas! superficiales![72D74],!e!incluso!en!aguas!subterráneas![75].! Capítulo)I:)Introducción) ! I.2.2.)Hormonas)esteroideas!45! ) !Los! valores! relativamente! altos! de! las! constantes! de! partición! (log! Kow!y! log! Koc)! sugieren!que!la!fracción!sólida!(biota,!sedimentos!y!material!particulado)!puede!actuar! como! un! sumidero! para! estos! compuestos,! especialmente! para! los! esteroides! sintéticos,! reduciendo! así! la! concentración! en! la! fase! acuosa! e! incrementando! la! biodisponibilidad.!La!bioacumulación!de!estas!sustancias!también!ha!sido!demostrada! en!varios!artículos,!tal!y!como!se!explicó!en!apartados!anteriores![42].!! !Finalmente,!varios!estudios!afirman!que!estas!sustancias!presentan!tiempos!de!vida! medio!(t1/2)!relativamente!cortos!en!ambiente!aeróbicos!(entre!4!y!20!días),!tanto!en! aguas!como!en!sedimentos![48,!49],!pero!una!degradación!casi!nula!bajo!condiciones! de!anoxia,!lo!cual!aumenta!sus!tiempos!de!residencia!en!el!medio.! ·∙)Estrogenicidad) !Con!frecuencia,!los!estudios!de!evaluación!y!monitorización!de!hormonas!esteroideas! se! centran! casi! exclusivamente! en! la! estimación! de! los! niveles! de! concentración! de! estas! sustancias! en! el! medio.! No! obstante,! estos! resultados! ofrecen! poca! o! nula! información! acerca! del! efecto! estrogénico! de! las! muestras! analizadas.! En! otras! palabras,! no! se! asocian! los! niveles! de! concentración! con! la! actividad! disruptora! endocrina!a!la!que!se!ven!sometidos!los!organismos!expuestos.!De!tal!forma,!el!análisis! químico! instrumental! resulta! ineficaz! en! este! sentido,! requiriéndose! ensayos! biológicos!(BBAs)!para!conocer!la!estrogenicidad!de!los!EDCs.!! !Los! BBAs! se! fundamentan! en! la! medición! de! una! determinada! señal! biológica! (anomalías!fisiológicas,!proliferación!celular,!bioluminiscencia,!etc.),!como!respuesta!a! la!exposición! de! una! concentración! conocida! de! un! determinado! EDCs! o! mezcla! de! ellos.! Este! tipo! de! ensayos! se! pueden! dividir! en! tres! grandes! grupos:! a)! Ensayos) en) Capítulo)I:)Introducción) ! 46!I.2.2.)Hormonas)esteroideas! ! organismos)vivos,!como!por!ejemplo!peces,!anfibios!o!insectos;!b)!ensayos)celulares)in) vitro,!empleando!por!ejemplo!levaduras!(p.e.!“Yeast!estrogen!assays!(YES)”)!o!células! humanas! (p.e.! EDSCREEN,! dónde! se! mide! la! proliferación! de! células! mamarias! cancerígenas);!y!c)!ensayos)no)celulares,!empleando!análisis!inmunoenzimáticos!(p.e.! “enzyme)linked)inmunosorbent)assays”!(ELISA))![37].! !Este! tipo! de! estudio! han! permitido! conocer! la! respuesta! estrogénica! que! genera! cualquier! sustancia! química! sospechosa! de! ejercer! disrupción! endocrina,! y! normalizarla! con! respecto! a! la! hormona! natural,! el! 17βDestradiol,! cuyo! valor! normalmente!se!establece!en!1.!Los!valores!resultantes!de!la!normalización!se!conocen! como! factores! de! equivalencia! de! estradiol! (EEF! según! sus! siglas! en! la! literatura! anglosajona).! !En!la!siguiente!tabla!(tabla!I.4)6!se!muestran!algunos!valores!de!EEFs!para!diferentes! hormonas!esteroideas,!lo!cuál!nos!permite!hacernos!una!idea!del!potencial!disruptor! endocrino!de!cada!uno!de!ellos.! ! Tabla!I.4.!Valores!de!factores!de!equivalencia!de!estradiol!estimados!para!el!17βDestradiol,!estrona,! estriol,!17α–etinilestradiol,!and!dietilestilbestrol.!a!Factor!de!equivalencia!de!estradiol.! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 6!Datos!obtenidos!de!C.G.)Campbell)et)al.)[37].! ! 17β3estradiol& Estrona& Estriol& 17α3etinilestradiol& Dietilestilbestrol& EEF&a& 1! 0.38!! 2.4!x!10D3!! 1.19!! 2.6!! & 1! 0.05!! 0.34!! 1.62!! D! & 1!! 0.02!! 0.01!! 1.20!! D! & 1! 0.14!! 0.08!! 0.8!! D! & 1! 0.096!! D! 1.9! D! & 1!! 0.1!! D! 0.8!! D! & 1!! 0.01!! D! D! D! Average&& 1! 0,11! 0,11! 1,25! 2.6! Capítulo)I:)Introducción) ! I.2.2.)Hormonas)esteroideas!47! ) !De! estos! valores! se! puede! sacar! en! claro! que! los! esteroides! sintéticos! 17αD etinilestradiol! y! ! dietilestilbestrol! producen! una! mayor! respuesta! estrogénica! en! los! organismos! que! la! propia! hormona! natural! (17βDestradiol),! lo! cual! indica! la! elevada! capacidad! de! ambas! sustancias! para! ejercer! como! disruptores! endocrinos.! Por! el! contrario,! los! metabolitos! de! la! hormona! natural! (estrona! y! estriol)! suponen! una! menor!“amenaza)estrogénica”!dado!que!su!capacidad!para!activar!una!respuesta!en! los!receptores!estrogénicos!es!10!veces!inferior!a!la!observada!en!el!estradiol![37].!! !Con! respecto! al! levoDnorgestrel,! se! ha! observado! que! sus! afinidades! de! unión!! relativas,!obtenidas!mediante!ensayos!in!vitro!en!los!receptores!humanos!específicos! de! hormonas! esteroideas,! son:! 323%! que! el! de! la! progesterona! en! el! receptor! de! progesterona,!58%!que!el!de!la!testosterona!en!el!receptor!androgénico!(AR),!17%!que! el!de!la!aldosterona!en!el!receptor!mineralocorticoide,!7.5%!que!el!del!cortisol!en!el! receptor!glucocorticoide,!y!<0,02%!que!el!del!estradiol!en!el!receptor!estrogénico.!! !A!modo!de!ejemplo,!se!ha!demostrado!que!concentraciones!tan!bajas!como!1!ng!·∙!LD1! de!17βDestradiol!(o!concentraciones!relativas!equivalentes)!inducen!la!producción!de! vitelogenina! en! ejemplares! macho! de! trucha! [76],! lo! cual! nos! indica! los! niveles! de! concentración!a!los!cuales!las!hormonas!esteroideas!podrían!ejercer!como!disruptores! endocrinos.!! ·∙)Concentraciones)en)el)medioambiente) !En! la! Tabla! I.2! del! apartado! anterior! se! muestran! los! niveles! de! concentración! de! varios!esteroides!estrogénicos!(naturales!y!sintéticos)!en!diferentes!tipos!de!muestras! sólidas! y! líquidas.! ! Como! se! puede! observar,! los! niveles! de! estas! sustancias! varían! Capítulo)I:)Introducción) ! 54!I.2.2.2.)Técnicas)de)separación)y)sistemas)de)detección! ! ! ! ! Tabla!I.5.!Diferentes!metodologías!empleadas!para!la!determinación!cuantitativa!de!hormonas! esteroideas!naturales!y!sintéticas![105].! Compuestos! Matriz& Extracción& Determinación& Límite!de! detección! (ng·∙L31;!ng·∙g31)& ! E2,E3,!E1,!EE,!PROG,! DES,!LEV,!NOR! ! Agua! OnDLine!SPE! (HysphereDResinDGP)! column! LCDESIDMS! <1! ! E2,E3,!E1! & Agua!de!río! SPE!(SDBDSD!disk)! LCDESI(NI)DMS! 1D50! ! E2,!E1! & Efluente!EDAR! SPE(LiChrolutEN+C18)! +inmunoaffinity! extraction! LCDESI(NI)DMS! 0.07D0.18! ! E2,EE! & Agua!de!acuario! SPE!(SepDPak!C18)! LCDAPCI(PI)D MS! 0.6D1! ! E2,E3,!E1,!EE,!DES! ! Agua!de!río! SPE!(C18!column)! LCDIS(NI)DMS! 3.2D10.6! ! E2,E3,!E1,!EE! & Agua!de!río,! efluente!y! entrada!de!EDAR! SPE!(CarbographD4! column)! LCDESI(NI)D MS/MS! 0.08D0.6! ! E2,E3,!E1,EE,!E3D3G,! E2D3G,!E1D3G,!E3D 16G,!E2D17G,!E3D3S,! E2D3S,!E1D3S! & Agua!residual!y! agua!de!río! SPE!(CarbographD4)! LCDESI(NI)D MS/MS! 0.03D15! ! E2,E3,!E1,!EE,!DES! & Agua!de!río,! efluente!de!EDAR! SPE!(C18!column)! LCDESI(NI)D MS/MS! 5! ! E2,E3,!E1,!EE! ! Efluente!y! entrada!de!EDAR! SPE!(ENVIDCARB! column)! LCDAPCI(PI)D MS! 0.5D1! ! E2,E3,!E1,!EE,!MES,! equilin!testosterone,! dehydrotestosterone ,!cyproterone! ! Agua!de!río! SPE!(C18!column)! LCDAPCI(PI)D MS! 1D10! ! E2,E3,!E1,!EE,!PROG,! DES,!LEV,!NOR! ! Sedimentos!de!río! PLE!! (acetona:metanol!1:1)! +!SPE!(Lichrospher!ADS! C4)! LCDESI(NI)DMS! (SIM)! 0.5D5! ! E2,E3,!E1,!EE,!PROG,! DES,!LEV,!NOR! & Sedimentos!de!río! Ultrasonicación! (acetona:metanol!1:1)! +!SPE!(C18!column)! LCDESI(NI)DMS! (SIM)! 0.04D1! Capítulo)I:)Introducción) ! I.2.3.)Bisphenol9a!55! ) ! I.2.3.!Bisphenol/a.! El!bisfenol,a!(2,2,bis(4,hidroxifenil)propano)!(Figura!I.5),!también!conocido!como!BPA! según!sus!siglas!en!la!literatura!anglosajona,!es!un!compuesto!químico!extensamente! utilizado! en! la! industria! como! monómero! en! la! producción! de! resinas! epoxi! y! de! policarbonatos![106,108].!Este!tipo!de!sustancias!se!emplean!a!su!vez!en!la!fabricación! de! innumerables! productos,! los! cuales! incluyen! envases! plásticos,! materiales! adhesivos,! CDs,! DVDs,! lentes! ópticas,! pinturas,! materiales! de! construcción,! o! en! las! cubiertas!de!ordenadores!y!electrodomésticos,!por!citar!algunos!ejemplos.! ! Figura!I.5.!Estructura!molecular!del!bisfenol,a.! !El!consumo!de!bisfenol,a!para!la!fabricación!de!policarbonato!constituye!casi!el!70%! de!la!producción!total!de!esta!sustancia.!Por!otra!parte,!las!resinas!epoxi!constituyen! casi! el! 30%.! Este! tipo! de! resinas! se! seleccionan! con! frecuencia! debido! a! sus! propiedades! protectoras! contra! la! corrosión,! su! estabilidad! térmica! y! resistencia! mecánica,! empleándose! fundamentalmente! como! recubrimientos! para! un! gran! número!de!aplicaciones!industriales!de!consumo,!tales!como!envases!de!alimentos!y! bebidas!y!recubrimientos!protectores.! ·∙)Síntesis)del)bisfenol9a) Capítulo)I:)Introducción) ! 56!I.2.3.)Bisphenol9a! ! !El! BPA! se! sintetiza! fundamentalmente! mediante! dos! métodos! bastante! similares.! El! primero!de!ellos!se!basa!en!la!condensación!de!fenol!con!acetona!(de!ahí!el!sufijo!–a)!! en!un!ambiente!de!pH!bajo!y!temperaturas!altas!(>!150°!C)!,!realizándose!todo!ello!en! presencia!de!un!catalizador!(normalmente!ácido!clorhídrico!(HCl)).!A!continuación,!el! bisfenol!“crudo”!se!destila,!mediante!distintas!tecnologías,!para!purificarlo.!El!producto! final!se!filtra!y!se!seca.!! !En!la!segunda!metodología!de!síntesis,!las!únicas!diferencias!residen!en!el!uso!de!un! diferente! catalizador! (poliestireno! sulfonado)! y! de! técnicas! de! purificación! más! eficientes.! El! bisfenol,a! seco,! obtenido! por! cualquiera! de! las! dos! técnicas,! se! comercializa!en!forma!de!copos,!escamas!y!cristales.! ·∙)Propiedades)físico9químicas) !Las! propiedades! físico,químicas! del! bisfenol,a! se! detallan! en! la! Tabla! I.67.! Como! se! observa,!el!BPA!se!muestra!como!un!sólido!a!temperatura!ambiente,!presentado!un! punto!de!fusión!comprendido!entre!los!150!y!los!157°!C.! !Diferentes! valores! de! solubilidad! ha! sido! descritos! para! esta! sustancia,! habiéndose! publicado! valores! comprendidos! entre! los! 120,300! mg/L! [109,112].! Un! trabajo! publicado!por!la!USEPA!afirma!que!el!BPA!presenta!una!mayor!solubilidad!a!pH!básicos! como!consecuencia!directa!de!sus!constantes!de!disociación!o!pKa!(9.6,10.2)![109].! !Los!coeficientes!de!partición!octanol,agua!(log!Kow)!se!muestran!en!la!Tabla!I.78.!Los! valores!de!este!parámetro!se!encuentran!entre!2.20!y!3.40,!indicando!que!el!BPA!va!a! sentir! una! ligera! atracción! por! asociarse! al! material! particulado! y! a! los! sedimentos.! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 7!Datos!tomados!de!C.A.!Staples!et!al.![106].! Capítulo)I:)Introducción) ! I.2.3.)Bisphenol9a!57! ) Esta!afirmación!se!halla!en!concordancia!con!los!valores!de!las!constantes!de!adsorción! calculados!por!Howard!y!colaboradores!para!suelos!y!sedimentos![112].!!! !Los!valores!de!las!presiones!de!vapor!mostrados!en!la!Tabla!I.6!indican!que!el!BPA!es! un! compuesto! poco! volátil! en! el! medioambiente! [112],! dado! que! normalmente! se! considera! como! poco! volátiles! a! aquellos! compuestos! que! presenten! valores! de! constante!de!Henry!inferiores!a!1.0!·∙!10,7!atm!·∙!m3!/!mol.!! Parámetro) Valor) Nº!CAS! 80,05,7! Peso!molecular! 228!g!·∙!mol,1! Fórmula!química! C15H16O2! Densidada! 1060!g!·∙!cm,3! 1195!g!·∙!cm,3! ! Punto!de!ebullición! 220°!C!(a!4!mm!de!Hg)! 398°!C!(a!760!mm!de!Hg)! ! Punto!de!fusión! 150,155°!C! 157°!C! ! pKa! 9,59,10.2! 11.3! ! Solubilidad!en!aguaa! 120!mg!·∙!L,1! 300!mg!·∙!L,1! ! Presión!de!vapora! 4.0!·∙!10,8!mm!de!Hg! 3.96!·∙!10,7!mm!de!Hg! 8.70!·∙!10,10!mm!de!Hg! 3.96!·∙!10,9!mm!de!Hg! ! ! Tabla!I.6.!Propiedades!físico,químicas!del!bisfenol,a.!! a!Valores!medidos!a!temperaturas!entre!20,25°!C.! ·∙)Fuentes)de)contaminación) En!el!mundo!se!produce!más!de!un!millón!de!toneladas!al!año!de!bisfenol,a![113],! existiendo!varias!rutas!potenciales!de!entrada!de!esta!sustancia!al!medioambiente.!La! migración!de!BPA!desde!los!recipientes!o!recubrimientos!de!plástico!empleados!en!el! almacenamiento! de! alimentos! (envases,! botellas,! biberones,! etc.)! hacia! los! propios! Capítulo)I:)Introducción) ! 58!I.2.3.)Bisphenol9a! ! alimentos! es! un! hecho! que! se! ha! demostrado! en! varias! ocasiones! en! la! bibliografía! [114,!115],!y!es!la!vía!que!supone!un!mayor!riesgo!para!la!población!general.!! El!uso!de!BPA!como!antioxidante!en!plásticos!del!tipo!PVC,!empleados!ampliamente! en!envases!de!alimentos,!tuberías!y!tanques!de!almacenamiento,!también!supone!una! fuente!de!entrada!de!este!compuesto!a!los!diferentes!compartimentos,!especialmente! a!los!torrentes!de!agua!que!terminan!por!llegar!a!las!EDARs![114,116].!! El!extensivo!uso!que!se!hace!en!actividades!industriales!de!algunos!productos!como! las!pinturas! o! los! materiales!adhesivos,! o! de! su!presencia! en! diversos! materiales!de! construcción,!también!facilita!su!entrada!al!medio.!! Finalmente,!también!se!ha!demostrado!la!liberación!de!pequeñas!cantidades!de!BPA! a!partir!de!las!instalaciones!de!manufacturado!y!procesamiento,!las!cuales!vierten!esta! sustancia!tanto!a!la!atmósfera!como!a!las!aguas!residuales![106].!!! ·∙)Estrogenicidad)y)toxicidad)) !La!estrogenicidad!del!bisphenol,a!ha!sido!demostrada!desde!hace!décadas.!Krishnan! et!al.![117]!demostraron!a!principios!de!los!90!mediante!ensayos!in!vitro!que!el!BPA! mostraba! estrogenicidad! a! concentraciones! comprendidas! entre! 2! y! 5! ng! ·∙! mL,1,! compitiendo!con!la!hormona!endógena!por!unirse!a!los!receptores!estrogénicos!en!el! útero!de!ratas.!! !Von! Saal! et! al! [118]! encontraron! que! durante! la! administración! oral! de! BPA! en! ratones,! una! dosis! de! 2! ng! ·∙! g,1! era! suficiente! para! incrementar! el! tamaño! de! los! órganos!reproductivos!de!crías!machos!durante!el!embarazo,!y!que!una!dosis!de!20!ng! Capítulo)I:)Introducción) ! I.2.3.)Bisphenol9a!59! ) ·∙!g,1!reducía!significativamente!la!producción!de!esperma,!concretamente!un!20%!con! respecto!al!control.! !Hoy!en!día!existen!varios!ensayos!in)vitro!comercialmente!disponibles!para!medir!la! actividad!estrogénica!del!bisphenol,a!(p.e.!“E,SCREEN”,!“YES”,!etc.),!los!cuales!han!sido! recientemente! analizados! en! una! extensa! revisión! publicada! por! A.M.! Soto! y! colaboradores! [119].! De! tal! forma,! se! han! estimado! factores! de! equivalencia! de! estradiol! (EEFs)! comprendidos! entre! 1.1! ·∙! 10,4!y! 3.6!·∙! 10,5! [120],! lo! cual! denota! una! estrogenicidad!relativamente!débil.! !Diferentes!estudios!toxicológicos!han!determinado!que!la!dosis!máxima!tolerada!en! seres!humanos!es!de!1000!mg!por!kg!de!masa!corporal!(MC)!y!por!día!(mg!·∙!kg,1!·∙!d,1).! Por!su!parte,!la!USEPA!ha!calculado!una!dosis!de!referencia!de!50!mg!·∙!kg,1!·∙!d,1!usando! un!factor!de!seguridad!de!1000![113].!Este!factor!se!suele!aplicar!para!considerar!el! riesgo! humano! a! partir! de! estudios! con! animales,! considerando! la! variabilidad! poblacional!y!extrapolando!los!niveles!de!exposición!crónicos!y!subcrónicos![14].!Por! su! parte,! la! Autoridad! Europea! de! Seguridad! Alimentaria! (EFSA)! establece! que! la! ingesta!diaria!tolerable!por!el!cuerpo!humano!es!de!0.05mg!por!kg!de!masa!corporal.! ·∙)Mecanismos)de)transporte)y)destino)medioambiental) !Al!igual!que!ocurre!con!el!resto!de!los!EDCs,!la!mecanismos!de!transporte!que!actúan! sobre! el! bisfenol,a! se! hallan! regulados! principalmente! por! sus! propiedades! físico, químicas,!especialmente!por!su!solubilidad!(Tabla!I.6)!y!sus!coeficientes!de!partición! (Tabla!I.7).! ! Capítulo)I:)Introducción) ! 60!I.2.3.)Bisphenol9a! ! Parámetro) Valor) Comentario) Log!Kow! 2.20! 3.32! 3.40! 3.82! ! LC!en!fase!reversa! pH!3! Log!Koc!para!suelos!y! sedimentos! 314! 1524! ! a!partir!de!la!solubilidad! a!partir!del!Kow! ! Bioconcentración! ! 5.1,13.8! ! 150!μg!·∙!L,1! ! Factor!(BFC)! <20,68! 42! 196! ! ! 15!μg!·∙!L,1! a!partir!de!la!solubilidad! a!partir!de!Kow!=!3.32! Constante!de!Henry! 1!·∙10,10!atm!·∙!m3!·∙!mol,1! a!partir!de!la!presión!de! vapor!y!la!solubilidad! ! Tabla!I.7.!Parámetros!que!caracterizan!la!partición!del!bisphenol,a.! !Tal!y!como!se!comentó!anteriormente,!tanto!los!valores!de!Kow!como!los!de!Koc!para! suelos!y!sedimentos!indican!una!ligera!tendencia!a!asociarse!con!los!sedimentos!y!el! material!particulado.!Por!su!parte,!los!valores!de!solubilidad!indican!que!el!BPA!podría! permanecer!disuelto!durante!largo!periodos.! !En!el!trabajo!de!C.E.!Staples!et!al.![106]!se!aplica!el!modelo!matemático!Mackay!para! analizar!la!distribución!del!BPA!en!el!medioambiente,!basándose!en!estimaciones!de!la! fugacidad! del! bisfenol,a! en! diferentes! compartimentos! (atmósfera,! suelos,! sedimentos,! biota,! aguas! superficiales! y! sólidos! en! suspensión).! En! esta! caso,! la! fugacidad! se! puede! definir! como! la! tendencia! del! bisfenol,a! a! escapar! de! una! determinada!fase.!! !Este!parámetro!se!mide!en!unidades!de!presión!(p.e.!Pascales),!y!esta!relacionada!con! una! concentración! determinada! mediante! un! valor! de! fugacidad! constante! que! se! Capítulo)I:)Introducción) ! I.2.3.)Bisphenol9a!61! ) aplica!a!cada!una!de!las!fases.!Los!valores!que!obtuvieron!se!presentan!en!la!siguiente! tabla!(Tabla!I.8)8:! Compartimento) Volumen) (m3)) Densidad)media) (kg)·∙)m:3)) M) (moles)) Porcentaje) (%)) Aire) 6!·∙!109! 1.19! 0.0002! <<1! Suelos) 4.5!·∙!104! 2400! 24.63! 25! Agua) 7!·∙!106! 1000! 52.36! 52! Biota) 7! 1000! 0.0036! <1! Sólidos)en)suspensión) 35! 1500! 0.0239! <1! Sedimentos) 2.4!·∙!104! 2400! 22.98! 23! ! Table!I.8.!Nivel!de!distribución!medioambiental!haciendo!uso!de!modelo!Mackay!level!I.! !Los!resultados!obtenidos!mostraron!que!más!del!50%!del!BPA!presente!en!el!medio! permanece! en! la! fase! disuelta,! mientras! que! el! resto! se! reparte! mayoritariamente! entre! las! fracciones! sólidas,! especialmente! los! suelos! y! los! sedimentos! (48%).! Estos! resultados! se! correlacionan! bastante! bien! con! los! deducidos! a! partir! de! sus! coeficientes!de!partición.! ·∙)Concentraciones)en)el)medioambiente) !!Aún! conociendo! la! posible! distribución! del! BPA,! se! puede! afirmar! que! las! concentraciones! de! esta! sustancia! en! el! medioambiente! están! mayormente! supeditadas! a! los! diferentes! procesos! de! degradación! que! sufre! este! compuesto! cuando!se!encuentra!inmerso!en!los!distintos!compartimentos.! !De!tal!forma,!varias!publicaciones!coinciden!en!que!el!BPA!no!es!una!sustancia!que! persista! durante! largos! períodos! en! el! medioambiente! [121,123].! Se! ha! demostrado! que!el!BPA!es!degradado!fácilmente!tanto!en!aguas!residuales!y!sus!efluentes,!como! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 8!Datos!tomados!de!C.A.!Staples!et!al.![106].! Capítulo)I:)Introducción) ! 62!I.2.3.)Bisphenol9a! ! en! diferentes! tipos! de! aguas! superficiales,! donde! se! estima! que! sus! tiempo! de! vida! medio!(t1/2)!se!encuentran!comprendidos!entre!los!2.5!y!los!4!días![106].!! !A!esta!biodegradación!se!le!suma!la!rápida!descomposición!que!sufre!el!BPA!debido!a! los! procesos! de! fotólisis! y! foto,oxidación.! Howard! et! al.! [112]! sugieren! que! la! foto, oxidación!del!BPA!en!agua,!la!cual!ocurre!tanto!por!fotolisis!como!por!la!propia!foto, oxidación,! podría! materializarse! en! t1/2! que! oscilan! entre! las! 66! h! y! los! 160! d,! dependiendo!del!tipo!de!agua.!Mientras!que!los!t1/2!estimados!para!la!foto,oxidación! de!este!compuesto!en!la!atmósfera!oscilan!entre!las!0.74!y!7.4!h.!! Por! otra! parte,! también! se! ha! descrito! que! el! BPA! no! tiene! una! gran! tendencia! a! asociarse! con! el! tejido! graso! de! los! seres! vivos,! habiéndose! descrito! factores! de! bioconcentración! que! varían! entre! <20! y! 196! [124,126],! lo! cuál! denota! un! bajo! potencial!para!bioacumularse.! !Todos!estos!datos!hacen!presagiar!que!el!BPA!debería!ser!una!sustancia!difícilmente! detectable! en! el! medioambiente,! no! obstante,! son! mucho! los! artículos! que! han! proporcionado! concentraciones! de! esta! sustancia! en! diferentes! tipo! de! muestras! medioambientales.! En! la! siguiente! tabla! (Tabla! I.9)! se! hace! acopio! de! algunos! resultados!obtenidos!por!la!comunidad!científica:!! !Las! técnicas! de! análisis! más! habituales! para! determinar! esta! sustancia! en! muestras! medioambientales! se! presentan! a! continuación,! incluyendo! los! sistemas! de! preparación! de! muestras! y! las! técnicas! instrumentales! empleadas! para! la! posterior! identificación!y!cuantificación.! ! Capítulo)I:)Introducción) ! I.2.3.1.)Técnicas)de)extracción!63! ) Aguas! superficiales! (ng/l)! Sedimentos! (μg/g)! Muestras! biológicas! (ng/g)! Aguas! residuales! (ng/l)! Lodos!de! depuradoras! (μg/g)! Aire!(ng/m3)! ! <1,145![127]! ! 470![128]! ! 95,855![135]! ! ! ! ! <2,118! [127]! ! 5,9![134]! ! 11![127]! ! 52,220! [133]! ! 2,13![135]! ! ! ! 560![128]! ! 60,300![130]! ! 407![131]! ! 250,1550! [132]! ! <!LODa![131]! ! 2.89![136]! ! 0.125,0.780! [137]! ! ! ! 5!ng!·∙!m,3! [129]! ! Tabla!I.9.!Concentraciones!de!BPA!halladas!en!diferentes!matrices!medioambientales.! ! I.2.3.1.!Técnicas!de!extracción.!!!!!!! !!En! los! siguiente! apartados! se! discute! acerca! de! las! técnicas! de! extracción! y! pre, concentración!más!empleadas!en!el!análisis!de!BPA,!abarcando!tanto!las!empleadas!en! muestras!líquidas!como!en!muestras!sólidas.! I.2.3.1.1.!Muestras!líquidas.!!!!!!! !Tal!y!como!se!describió!en!apartados!anteriores,!es!de!esperar!que!más!del!50%!del! BPA! presente! en! el! medioambiente! se! encuentre! en! la! fase! disuelta,! lo! cual! ha! propiciado! que! la! mayoría! de! los! trabajos! publicados! hasta! la! fecha! se! centren! en! matrices!líquidas.! !Durante!las!últimas!dos!décadas!el!número!de!técnicas!empleadas!para!extraer!el!BPA! en!este!tipo!de!muestras!ha!sido!muy!amplio,!abarcando!tanto!técnicas!convencionales! como! avanzadas.! No! obstante,! las! técnicas! clásicas! como! por! ejemplo! la! extracción! líquido,líquido![138]!o!algunas!técnicas!de!destilación![139],!han!sido!paulatinamente! sustituidas!por!algunas!técnicas!más!eficientes!y!versátiles!como!la!extracción!en!fase! sólida!(SPE)!o!la!microextraccción!en!fase!sólida!(SPME).!! Capítulo)I:)Introducción) ! 70!I.3.1.)Análisis)estadístico! ! ! I.3.1.!Análisis!estadístico.!!!!!!! !Todas! las! metodologías! analíticas! presentadas! en! esta! Tesis! han! sido! evaluadas! en! términos! de! precisión,! linealidad,! límites! de! detección,! límites! de! cuantificación! y! recuperación.! ·∙)Precisión! !La!precisión!fue!evaluada!mediante!el!cálculo!de!las!desviaciones!estándares!relativas! (RSD).!Los!resultados,!expresados!como!porcentajes,!fueron!estimados!a!partir!de!un! tamaño!de!muestra!de!N!=!6.!! !La!RSD!se!define!según!la!siguiente!expresión:! 𝑅𝑆𝐷 =! 𝜎 𝒳 !𝑥!100! siendo:! ! 𝒳=! ! 𝒳! ! !!!!!! ! ! (media)aritmética)! !!!!!!!!!y! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!𝜎=!! !!!(𝒳!−𝒳)! ! !!!!!!! !!(desviación)estándar)) ·∙)Linealidad) !Las!linealidades!de!cada!uno!de!los!métodos!desarrollados!fueron!estimadas!en!base!a! los!coeficientes!de!correlación!de!Pearson!(r!ó!R),!calculados!a!partir!de!sus!curvas!de! calibrado.!Los!coeficientes!de!determinación!estimados!(r2)!fueron!superiores!a!0.991! en!todos!los!casos.! 𝑟 !,!=! 𝜎!" 𝜎!𝜎! =! 𝑛𝑥!𝑦!−𝑥!𝑦! 𝑛𝑥! !−(𝑥!)!𝑛𝑦! !−(𝑦!)! ! Capítulo)I:)Introducción) ! I.3.1.)Análisis)estadístico!71! ) ·∙)Límites)de)detección) !Los! límites! de! detección! (LODs)! de! las! diferentes! metodologías,! y! de! los! diferentes! analitos! analizados,! han! sido! calculados! en! base! a! la! siguiente! definición:!! “Concentración)de)analito)que)genera)una)relación)señal/ruido)(S/R))igual)a)tres”:! 𝐿𝑂𝐷 =! 𝑋!"#$%& 𝑋!"#$% =3! !Los! cálculos! de! dicho! valor! se! realizaron! por! triplicado! (n=3)! a! partir! del! nivel! de! concentración!más!bajo!estimado!en!las!curvas!de!calibrado.! ·∙)Límites)de)cuantificación) Por! su! parte,! los! límites! de! cuantificación! (LOQs)! se! determinaron! en! base! a! la! siguiente! definición:! ! “Concentración) de) analito) que) genera) una) relación) señal/ruido) (S/R))igual)a)diez”:! 𝐿𝑂𝐷 =! 𝑋!"#$%& 𝑋!"#$% =10! !Los!cálculos!de!dicho!valor!también!se!realizaron!por!triplicado!(n=3)!a!partir!del!nivel! de!concentración!más!bajo!estimado!en!las!curvas!de!calibrado.! ·∙)Recuperación) !Las!cantidades!de!analito!que!somos!capaces!de!extraer!mediante!el!uso!de!cada!una! de! las! metodologías! desarrolladas! han! sido! expresadas! en! porcentaje.! Por! norma! general,! los! valores! obtenidos! han! sido! calculados! por! triplicado,! expresándose! además!las!desviaciones!estándares!relativas!obtenidas!para!cada!compuesto.!! Capítulo)I:)Introducción) ! 72!I.3.1.)Análisis)estadístico! ! I.3.1.1.!Análisis!de!los!resultados!en!muestras!reales.!!!!!!! !Las! concentraciones! de! los! EDCs! en! las! muestras! reales! han! sido! determinadas! haciendo! uso! de! curvas! de! calibrado! externas! (apartados! III.1,! III.2,! III.3! y! III.4)! ó! aplicando!el!método!de!las!adiciones!estándar!(apartados!III.5!y!III.6).!!! !No! obstante,! en! cualquiera! de! los! dos! casos,! la! regresión! o! ajuste! lineal! se! realizó! empleando! seis! niveles! de! concentración! diferentes,! estimándose! cada! uno! de! ellos! por! triplicado.! Los! rangos! lineales! variaron! en! función! de! la! metodología! analítica! empleada!y!del!analito!a!determinar,!estando!estos!indicados!de!forma!específica!en! cada!uno!de!los!apartados!del!Capítulo!III.!! ·∙)Efecto)matriz) !El! efecto! de! supresión! iónica! (ó! realzamiento! en! algunas! ocasiones)! sobre! la! determinación! de! EDCs! en! muestras! medioambientales! complejas! es! un! hecho! ampliamente! demostrado,! especialmente! cuando! se! emplea! LCFESIFMS/MS! cómo! técnica! de! análisis! [84].! ! Los! cambios! en! la! intensidad! de! la! señal! generados! como! consecuencia!directa!del!efecto!matriz!pueden!conllevar!a!la!obtención!de!resultados! erróneos!o!poco!significativos![172F174].! !En!cada!uno!de!los!trabajos!presentados!en!esta!Tesis!hemos!tenido!en!cuenta!en!qué! grado! o! medida! estaba! afectando! la! naturaleza! de! las! muestras! a! los! resultados! obtenidos.! La! medida! del! efecto! matriz! ha! sido! estimada! mediante! el! uso! de! dos! modelos!matemáticos!diferentes.!! F Expresión!de!Vieno!et!al.![175]:! Capítulo)I:)Introducción) ! I.3.1.)Análisis)estadístico!73! ) 𝑀𝐸 =! 𝐴!!–(𝐴!" −!𝐴!"#) 𝐴! !𝑥!100! dónde:!As!se!corresponde!con!el!área!del!pico!registrada!en!una!disolución!pura!del! estándar,! Asp! se! corresponde! con! el! área! del! pico! registrada! en! una! matriz! contaminada! a! una! concentración! conocida! (“spiked”),! y! Ausp)se!corresponde! con! el! área!del!pico!en!la!matriz!sin!contaminar!(“nonNspiked”).!! F Expresión!de!Matuszewski!et!al.![176]:! 𝑀𝐸 =! 𝐴 𝐵!𝑥!100! dónde:!A!se!corresponde!con!el!área!del!pico!en!el!extracto!de!la!matriz!contaminado! (“spiked”),!y!B!se!corresponde!con!el!área!del!pico!registrada!en!una!disolución!pura! del!estándar.!! !En! ambos! modelos,! un! valor! del! 100%! indicaría! la! ausencia! de! efecto! matriz! en! el! análisis;!valores!inferiores!al!100%!indicarían!la!existencia!de!una!reducción!en!la!señal! obtenida! como! consecuencia! de! la! supresión! iónica! y;! valores! superiores! al! 100%! indicarían!la!existencia!de!un!incremento!en!la!señal!obtenida!como!consecuencia!de! un!realzamiento!iónico.!! I.3.1.2.!Diseño!factorial.!!!!!! !La!optimización!de!las!metodologías!de!extracción!empleadas!para!la!determinación! de!EDCs!en!lodos!de!depuradora!(apartados!III.4!y!III.6)!se!llevó!a!cabo!mediante!el!uso! de! diversos! diseños! factoriales,! empleándose! para! la! construcción! de! los! mismos! el! “software”!Statgraphics!Plus!versión!5.1!(Manugistic,!Rockville,!MD,!USA).! Capítulo)I:)Introducción) ! 74!I.3.1.)Análisis)estadístico! ! !Este! tipo! de! diseño! estadístico! ha! sido! previamente! aplicado! en! el! desarrollo! de! metodologías! de! extracción! en! diferentes! tipos! de! muestras! sólidas! [98,! 177,! 178].! Según! Miller! y! Miller! [179],! esta! herramienta! matemática! permite! reducir! significativamente! el! número! de! ensayos! requeridos! durante! el! proceso! de! optimización,! y! además,! permite! obtener! la! influencia! de! cada! variable! sobre! el! proceso!de!extracción!y!las!correlaciones!con!el!resto!de!variables.!! !En!nuestro!caso!particular,!la!primera!etapa!del!proceso!de!optimización!consistió!en! el!empleo!de!un!diseño!factorial!23!(tres!variables!a!dos!niveles!diferentes),!mediante! el!cual!se!determinaban!las!variables!e!interacciones!que!ejercían!un!mayor!peso!sobre! la!recuperación!de!los!diferentes!EDCs!en!estudio!(Tabla!I.11).! !En!la!segunda!etapa!del!proceso!se!empleó!un!diseño!factorial!32,!en!el!cual!las!dos! variables!que!mostraron!mayor!influencia!sobre!la!recuperación!de!los!analitos!en!la! etapa! anterior! fueron! estudiadas! a! tres! niveles! diferentes.! Este! diseño! se! dividió! en! tres!bloques!con!un!punto!central!para!cada!uno!(Tabla!I.12).!! !Con!motivo!de!evitar!la!influencia!de!otros!factores!no!controlados!que!pudieran!tener! efecto!sobre!los!resultados!finales,!tanto!el!diseño!23!como!el!32!fueron!construidos!de! forma! aleatoria.! Los! cálculos! de! las! correlaciones! parciales! y! bivariadas! entre! las! diferentes! variables! (dependientes! e! independientes)! se! realizaron! mediante! el! “software”!SPSS!versión!11.0!(SPSS!Inc.,!Illinois,!USA).! ! ! ! Capítulo)I:)Introducción) ! I.3.1.)Análisis)estadístico!75! ) ! ! ! Run) ! Volúmen! (mL)! Potencia! (W)! Tiempo! (min)! 1! 5! 100! 10! 2! 15! 100! 10! 3! 5! 300! 10! 4! 5! 300! 5! 5! 5! 100! 5! 6! 15! 100! 5! 7! 15! 300! 5! 8! 15! 300! 10! ! Tabla!I.11.!Diseño!factorial!23!empleado!en!la!optimización!de!la!metodología!de!MAE!presentada!en!el! apartado!III.4!de!esta!Tesis!Doctoral.! ! ! ! Run! ! Block! Power! (W)! Time! (min)! 1! 1! 200! 5! 2! 1! 300! 10! 3! 1! 100! 2! 4! 1! 200! 2! 5! 2! 100! 10! 6! 2! 200! 5! 7! 2! 200! 5! 8! 2! 300! 2! 9! 3! 200! 5! 10! 3! 200! 10! 11! 3! 100! 5! 12! 3! 300! 5! ! Tabla!I.12.!Diseño!factorial!32!empleado!en!la!optimización!de!la!metodología!de!MAE!presentada!en!el! apartado!III.6!de!esta!Tesis!Doctoral. Capítulo)I:)Introducción) ! 76!I.3.2.)Evaluación)de)la)actividad)disruptora)endocrina! ! I.3.2.!Evaluación!de!la!actividad!disruptora!endocrina.!!!!!!! !La!estrogenicidad!de!las!muestras!analizadas,!incluyendo!cada!una!de!la!fracciones!en! estudio!(material!particulado,!lodos!y!aguas!residuales),!ha!sido!evaluada!en!términos! de!concentración!equivalente!de!estradiol!(EEQs)![127,!180,!181].!!Este!parámetro!se! puede!definir!de!la!siguiente!manera:!! 𝐸𝐸𝑄!=𝐶!!𝑥!𝐸𝐸𝐹 ! (1)! dónde:! Ci! es! la! concentración! del! compuesto! i) en! la! muestra,! y! EEFi! el! factor! de! equivalencia!del!compuesto!i!con!respecto!al!estradiol.! ·∙)Factores)de)equivalencia)de)estradiol)! !El!factor!de!equivalencia!de!estradiol!(EEF)!se!define!en!la!siguiente!expresión:! 𝐸𝐸𝐹 !=!"!"!! !"!"! (2) dónde:!EC50E2!es!la!concentración!que!genera!la!mitad!de!la!respuesta!máxima!para!el! estradiol,! ! y! EC50i! es! la! concentración! que! genera! la! mitad! de! la! respuesta! máxima! para!el!compuesto!i.! !Las! respuestas! máximas! indicadas! en! dicha! expresión! se! obtienen! generalmente! de! diferentes! tipos! de! ensayos! biológicos! (BBAs),! aunque! la! mayoría! de! las! veces! se! emplean!ensayos!in)vitro)[14,!27,!81,!180F182].!! !Hoy! en! día! existe! una! gran! cantidad! de! ensayos! in! vitro,! muchos! de! ellos! comercialmente!disponibles,!capaces!de!determinar!de!forma!rápida!y!sensible!tanto! la!presencia!(concentración)!de!casi!cualquier!compuesto!químico,!como!su!actividad! estrogénica!asociada.!Por!ende,!también!permiten!normalizar!los!resultados!obtenidos! Capítulo)I:)Introducción) ! I.3.2.)Evaluación)de)la)actividad)disruptora)endocrina!77! ) para! un! compuesto! i! con! respecto! al! observado! para! el! estrógeno! natural! (17βF estradiol),!lo!cual!posibilita!la!estimación!de!los!EEFi.! !Los! test! o! ensayos! in) vitro! más! empleados! en! la! bibliografía! científica! para! la! estimación!de!los!potenciales!estrogénicos!se!detallan!a!continuación:! ·∙! “Receptor) binding) assays”:! Miden! la! afinidad! de! una! sustancia! para! unirse! con! un! receptor! hormonal! específico,! como! por! ejemplo! los! receptores! estrogénicos! (ERα)! (p.e.!ERFCALUX).! ·∙! “Cell) proliferation) assays”:! Miden! la! capacidad! de! una! sustancia! para! estimular! un! crecimiento! celular! originado! como! consecuencia! de! una! respuesta! hormonal! específica,! como! por! ejemplo,! la! proliferación! de! células! mamarias! cancerígenas! (EF SCREEN).! ·∙! “Reporter) gene) assays”:! Miden! la! capacidad! de! una! sustancia! para! activar! algún! proceso! específico! de! transcripción! genética! en! las! células! (p.e.! “yeast! estrogen! screen”!(YES)).! !Debido!a!la!alta!variabilidad!observada!en!la!bibliografía!con!respecto!a!los!valores!de! EEFs! para! los! EDCs,! en! el! análisis! de! la! estrogenicidad! de! nuestras! muestras! hemos! optado!por!compilar!diversos!valores!de!factores!de!equivalencia!y!promediarlos.!Este! procedimiento!ya!ha!sido!anteriormente!empleado,!encontrándose!que!dichos!valores! promedios! aplicados! en! la! ecuación! (1),! generan! valores! de! EEQs! mucho! más! significativos!que!si!nos!basásemos!en!un!solo!BBA![127,!180,!183].!En!la!Tabla!I.4!se! mostraron!los!valores!de!EEFi!estimados!para!algunos!EDCs!estudiados!en!esta!Tesis! Doctoral.! Capítulo)I:)Introducción) ! 78!I.3.2.)Evaluación)de)la)actividad)disruptora)endocrina! ! ·∙)Cálculo)de)los)potenciales)estrogénicos:! !El! efecto! aditivo! de! los! EDCs! a! la! actividad! estrogénica! es! un! hecho! que! se! ha! demostrado!científicamente![120,!184].!De!tal!forma,!la!actividad!estrogénica!total!de! una! muestra! puede! ser! definida! como! la! suma! de! cada! una! de! las! concentraciones! equivalentes!de!estradiol!(EEQi)!registradas!para!cada!EDC!de!manera!individual:! 𝐸𝐸𝑄!=!𝐸𝐸𝑄!! (3)!! 𝐸𝐸𝑄!=𝐶!+!𝐸𝐸𝐹 !+!𝐶!+!𝐸𝐸𝐹 !+!𝐶!+!𝐸𝐸𝐹 !…!!(4) !Este!tipo!de!análisis!nos!ofrece!resultados!muy!significativos!desde!el!punto!de!vista! medioambiental.!Por!una!parte,!mediante!el!cálculo!de!los!EEQt!podemos!estimar!el! peligro! potencial! de! cualquier! tipo! de! muestra! como! generador! de! disrupción! endocrina,!y!por!otra,!mediante!la!estimación!de!los!EEQi!podemos!identificar!cuál,!o! cuales! de! los! EDCs! en! estudio,! son! los! que! ejercen! mayor! influencia! sobre! la! estrogenicidad!!total!observada,!y!por!lo!tanto,!los!que!mayor!peligro!representan!para! los!organismos!expuestos.! !En! los! trabajos! presentados! en! el! Capítulo! III! 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Objetivos! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Capítulo!II:!Objetivos! ! 108!II.!Objetivos! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Capítulo!II:!Objetivos! ! II.!Objetivos!109! ! ! !La!presencia!de!EDCs!en!el!medioambiente,!así!como!sus!efectos!negativos!sobre!la! salud! de! las! poblaciones! animales,! son! hechos! ampliamente! reconocidos! a! escala! global,!y!representan!un!problema!de!interés!creciente!en!la!comunidad!científica!y!en! la!sociedad.!No!obstante,!hoy!en!día!no!existe!una!normativa!o!legislación!específica! que!regule!los!niveles!de!emisión!de!este!tipo!de!contaminantes!al!medioambiente.!! !Este! vacío! legal,! existente! para! la! mayoría! de! compuestos! catalogados! cómo! EDCs,! exime!de!responsabilidades!jurídicas!a!las!estaciones!depuradoras!de!aguas!residuales! (EDARs),! las! cuales,! como! se! comentó! en! el! capítulo! anterior,! no! son! capaces! de! eliminar! completamente! a! estas! sustancias! durante! el! tratamiento,! y! suponen,! además,!la!mayor!fuente!de!entrada!de!estos!contaminantes!al!medio.! !Durante!los!últimos!cinco!años,!las!principales!instituciones!de!la!Unión!Europea!y!de! Norteamérica,!como!por!ejemplo!la!“Water!Framework!Directive!(WFD)”!o!la!agencia! de! protección! medioambiental! de! los! Estados! Unidos! (USEPA),! han! comenzado! a! tomar!medidas!con!respecto!a!la!regulación!de!estos!contaminantes,!incluyendo!en!las! listas!de!contaminantes!prioritarios!a!varios!EDCs!(p.e.!el!nonilfenol),!y!comenzando!a! establecer! los! niveles! de! concentración! mínimos! para! garantizar! unos! estándares! apropiados!de!calidad!medioambiental.! !Teniendo!en!cuenta!todos!estos!factores,!e!incluyendo!una!posible!regularización!de! los! niveles! de! EDCs! en! un! futuro! cercano,! es! de! interés! tanto! a! nivel! internacional! como!local,!conocer!y!referenciar!por!primera!vez!la!presencia!de!estos!contaminantes! en! los! efluentes! de! EDARs! en! la! isla! de! Gran! Canaria! (España).! De! tal! forma,! los! objetivos!que!se!presentan!en!la!presente!Tesis!Doctoral!son!los!siguientes:! Capítulo!II:!Objetivos! ! 110!II.!Objetivos! ! a) !Optimizar! y! desarrollar! metodologías! de! extracción! de! los! compuestos! de! interés!en!muestras!de!aguas!residuales,!mediante!el!uso!de!la!extracción!en! fase! sólida! (SPE).! Dichas! metodologías! deben! permitirnos,! de! forma! rápida! y! sencilla,!extraer!los!compuestos!de!interés!para!su!posterior!detección!a!niveles! de!trazas.! b) !Optimizar!y!desarrollar!una!metodología!de!extracción!de!los!compuestos!de! interés!en!muestras!del!material!particulado!(<!0.45!μm)!asociado!a!las!aguas! residuales! analizadas,! mediante! la! extracción! asistida! por! ultrasonidos.! La! metodología! empleada! debe! ser! rápida,! emplear! bajos! volúmenes! de! disolventes! orgánicos,! y! permitir! la! extracción! de! varias! muestras! de! manera! simultánea.! c) !Optimizar!y!desarrollar!metodologías!de!extracción!de!los!diferentes!EDCs!en! estudio!en!muestras!de!lodos!de!depuradora,!mediante!el!uso!de!la!extracción! asistida! por! microondas! y! un! posterior! paso! de! limpieza! y! preconcentración! basado!en!las!técnicas!de!SPE!desarrolladas!previamente.!Dichas!metodologías! han! de! presentar,! con! respecto! a! las! técnicas! convencionales,! una! reducción! significativa! en! el! tiempo! de! extracción! y! en! el! consumo! de! disolventes! orgánicos,! permitiendo! además! la! determinación! de! los! compuestos! seleccionados!a!niveles!traza!(ng!·∙!g_1).! d) Establecer! las! condiciones! cromatográficas! de! separación! y! detección! de! los! compuestos!bajo!estudio!mediante!la!cromatografía!líquida!de!alta!resolución! (LC)! y! la! cromatografía! líquida! de! ultra! resolución!(UHPLC),! acopladas! a! detectores! de! fluorescencia! (FD)! y! de! espectrometría! de! masas! de! triple! cuadrupolo!(MS/MS).! ! ! Capítulo!II:!Objetivos! ! II.!Objetivos!111! ! e) Realizar!un!muestreo!temporal!de!muestras!líquidas!y!sólidas!procedentes!de! diversas!EDARs!de!la!isla!de!Gran!Canaria,!con!diferentes!tipos!de!instalaciones,! y!diferentes!etapas!de!tratamiento.!! f) Aplicar! las! metodologías! optimizadas! al! análisis! de! las! muestras! recolectadas! (aguas!residuales,!material!particulado,!y!lodos!activos).! g) Evaluar! las! tasas! de! eliminación! de! los! compuestos! de! interés! en! las! EDARs! estudiadas.! h) Realizar!un!análisis!estadístico!e!interpretar!los!resultados!obtenidos!para!los! niveles!de!EDCs!encontrados.! i) Evaluar! los! potenciales! estrogénicos! y! la! actividad! disruptora! endocrina! asociada!a!las!diferentes!muestras!analizadas.! ! 702 T. Vega-Morales et al. / Journal of Hazardous Materials 183 (2010) 701–711 Table 1 Physiochemical properties of the compounds studied [4,6]. Chemical name Molecular weight Water solubility (mgL−1at 20◦C) logKow Bisphenol-A (BPA) 228.0 120 3.32 17␤-Estradiol (E2) 272.4 13 3.94 Estrone (E1) 270.4 13 3.43 Estriol (E3) 288.4 13 2.81 17␣-Ethynylestradiol (EE) 296.4 4.8 4.15 4-Octylphenol (OP) 206.0 12.6 4.12 Octylphenol monoethoxylate (OP1EO) 250.0 8.0 4.10 Octylphenol diethoxylate (OP2EO) 294.0 13.2 4.00 Octylphenol triethoxylate (OP3EO) 338.0 18.4 3.90 Octylphenol tetraethoxylate (OP4EO) 384.0 24.5 3.90 4-Nonylphenol (NP) 220.0 1.57 4.48 Nonylphenol monoethoxylate (NP1EO) 264.0 3.02 4.17 Nonylphenol diethoxylate (NP2EO) 308.0 3.38 4.21 Nonylphenol triethoxylate (NP3EO) 352.0 5.88 4.20 Nonylphenol tetraethoxylate (NP4EO) 396.0 7.65 4.30 quantitative information, facilitating the correct and unambiguous identification of each analyte and an increase in sensitivity. The major weakness of “traditional” methodologies for EDCs analysis can be solved by coupling LC to MS, particularly to a tandem mass spectrometer (LC/MS/MS) [13]. The objective of this study was to develop a simple and rapid analytical procedure for the simultaneous extraction and determination of nonylphenol, octylphenol and corresponding ethoxylates (1–12), 17␣-ethynylestradiol, 17␤-estradiol and its two metabolites estrone (E1) and estriol (E3), and bisphenol-A (BPA) in sewage samples and to apply the optimised method to the quantification of EDCs in wastewater samples obtained from a wastewater treatment plant (WWTP) in Las Palmas of Gran Canaria (Spain). Compounds were determined in both the dissolved and particulate phases at different stages of the WWTP process, including its final effluent located on the city’s coast. 2. Experimental 2.1. Chemicals and reagents Methanol and water used to dissolve standards or Igepal mixturesandto preparemobile phaseswere LC–MSgrade andobtained fromPanreacQuímica(Barcelona,Spain).Methanolandwaterwere filtered through a 0.22␮m acetate membrane filter. Glacial acetic acid used to adjust the pH of the mobile phase was high performance liquid chromatography (LC) grade and was purchased from Scharlau Chemie S.A. (Barcelona, Spain). Ultra-high-quality water obtained by a Milli-Q (Millipore, Bedford, MA, USA) water purification system was used in solid phase extraction. All standards and Igepal technical mixtures were purchased from Sigma Aldrich (Madrid, Spain). Short ethoxylated chains APnEOs (n≤2), 17␤-estradiol, estrone, estriol, 17␣- ethynylestradiol and bisphenol-A were greater than 98% pure and were used as standards. Stock solutions (1000␮gmL−1) of alkylphenols, steroidal hormones, and bisphenol-A were prepared by dissolving the standards into methanol. Stock solutions were stored in glass-stoppered bottles at −20◦C prior to use. 10␮gmL−1of nonylphenol monoethoxylate, nonylphenol diethoxylate, octylphenol monoethoxylate and octylphenol diethoxylate were obtained as stock solutions (1mL) in acetone and were stored at −20◦C. Long-chain APnEOs (n≥3) were only available in technical mixtures. Igepal CO210, CO520 and CO720 contained a range of NPnEO oligomers with 3–12 ethoxy units (EO) while Igepal CA210, CA520 and CA720 contained the same EO range of OPnEO oligomers. Stock solutions (1000␮gmL−1) of long-chain alkylphenolic ethoxylated surfactants were also prepared by dissolving appropriate amounts of each mixture into methanol and were stored in glass-stoppered bottles at 20◦C. The cartridges (6mL) employed in this study included Sep-Pak Vac C18 (500mg) and Oasis HLB (200 mg) from Waters (Madrid, Spain), Bond Elut-ENV (500mg) and Bond Elut Plexa (500 mg) from Varian (Madrid, Spain), and LiChrolut EN (500mg) from Merck (Darmstadt, Germany). A Varian Vac Elut 20 SPE Manifold coupled to a Sartorius vacuum pump was used for extractions. 2.2. Sample collection To test the applicability of the method, three samples from primary, secondary and final effluent were collected from one of the major wastewater treatment plants in Las Palmas de Gran Canaria (Spain). Treatment in the WWTP relies on settling and flotation (primarytreatment)and biologicaltreatmentwithactivatedsludge (secondary treatment). A tertiary treatment was not conducted in the WWTP process. Samples were collected in July 2009 and acidified to a pH< 3 to prevent the loss of analytes by abiotic reactions (such as hydrolysis) and by biological degradation [21]. The samples were stored at 4◦C in 2.5 L glass bottles and extracted within 48h. Primary and secondary treatment samples were taken from the effluent of each stage. Prior to extraction, wastewater samples were filtered through 0.65␮m filter paper and 0.45 ␮m membrane filters (Millipore, Bedford, MA, USA). A pore size of 0.45␮m was used to separate dissolved and particulate phases [22]. 2.3. Extraction Compounds were isolated from the dissolved phase using solid phase extraction (SPE). The cartridge was conditioned with 3× 5mL of methanol and 3×5 mL of Milli-Q water at a flow-rate of 5mLmin−1beforeeach extraction.The sample(250 mL)percolated through the cartridge at a flow-rate of 10mL min−1. A wash step was conducted using 2×5mL of Milli-Q water to remove impurities retained in the cartridge. Subsequently, the cartridge was dried under vacuum for 10minutes, and the retained analytes were eluted at a low flow-rate (approximately 1mL min−1) with 2mL of methanol. Blanks were run to confirm the absence of carryover. 0.45␮m membrane filters with retained particulate matter (between 0.1 and 0.2g for all samples) were immersed in an ultrasonicbath with10mL ofmethanol for10min. Themethanolextract was collected in a flask, evaporated to dryness under a gentle stream of nitrogen and reconstituted in 100␮L of methanol. The final extracts were analysed separately and concentrations of dissolved and particulate phases are reported separately for each sample. 118 T. Vega-Morales et al. / Journal of Hazardous Materials 183 (2010) 701–711 703 Table 2 Characteristic of ESI/MS/MS parameters for each compound studied. Compound m/zPrecursor [M+NH4]+m/zPrecursor [M−H]−Cone (V) Fragment ions (collision potential) Ion mode NP1EO 282.3 – 30 265.3 (6)a, 127.1 (8) ESI+ NP2EO 326.3 – 30 183.1 (9)a, 121 (20) ESI+ NP3EO 370.3 – 32 353.3 (8)a, 227.1 (11) ESI+ NP4EO 414.5 – 32 397.4 (8)a, 271.2 (13.5) ESI+ NP5EO 458.6 – 48 441.5 (12)a, 315.2 (15.5) ESI+ NP6EO 502.6 – 52 485.5 (13.5)a, 359.3 (17) ESI+ NP7EO 546.7 – 56 529.6 (14.5)a, 403 (18) ESI+ NP8EO 590.8 – 64 573.6 (15.5)a, 447.5 (20) ESI+ NP9EO 634.8 – 64 617.8 (16)a, 335 (21) ESI+ NP10EO 678.9 – 68 661.8 (16.5)a, 132.7 (24.5) ESI+ NP11EO 722.9 – 72 704.9 (17.5)a, 291 (26.5) ESI+ NP12EO 766.9 – 88 749.9 (18)a, 291 (28.5) ESI+ NP13EO 811.1 – 80 794 (18)a, 291.1 (29) ESI+ NP14EO 855.2 – 88 838 (18)a, 291 (32.5) ESI+ OP1EO 268.1 – 30 251.1 (6)a, 113 (7.5) ESI+ OP2EO 312.3 – 30 183.0 (9.5)a, 121 (19.5) ESI+ OP3EO 356.4 – 32 339.4 (8)a, 227.1 (14) ESI+ OP4EO 400.4 – 32 383.4 (10)a, 271.2 (14) ESI+ OP5EO 444.5 – 48 427.5 (12)a, 315.2 (15.5) ESI+ OP6EO 488.5 – 52 471.5 (13.5)a, 359.3 (17) ESI+ OP7EO 532.8 – 52 516.6 (15)a, 403.3 (18) ESI+ OP8EO 576.7 – 60 559.7 (15.5)a, 277.2 (24) ESI+ OP9EO 620.8 – 64 603.7 (16.5)a, 277.2 (25.5) ESI+ OP10EO 664.9 – 68 647.8 (17.5)a, 277.2 (27.5) ESI+ OP11EO 708.9 – 68 691.8 (18)a, 277.2 (29) ESI+ OP12EO 752.9 – 68 735.9 (19)a, 277.2 (29.5) ESI+ OP13EO 797.1 – 72 780 (18.5)a, 277.2 (30.5) ESI+ OP14EO 841.2 – 84 823.2 (20)a, 132.6 (27) ESI+ NP – 218.7 −64 105.7 (20.5)aESI− OP – 204.7 −72 134 (16.5)a, 106 (19.5) ESI− BPA – 226.7 −60 211.7 (17.5)aESI− E2 – 271.1 −60 183.5 (14.5)a, 145.2 (20.5) ESI− E1 – 269.3 −55 145.4 (16.5)a, 143.2 (19.5) ESI− E3 – 287.2 −67 171.0 (16.5)a, 145.2 (19.5) ESI− EE – 295.3 −71 159.5 (17.5)a, 145.2 (21.5) ESI− aFragment ion used for quantitation (MRM). 2.4. Instrumentation and chromatographic conditions 2.4.1. LC/MS/MS analysis Analysis was performed by reversed phase liquid chromatography coupled to a triple quadrupole (TQ) mass spectrometer equippedwithan electrosprayinterface (LC/ESI/MS/MS). Theapparatus was composed of a Varian 320-MS TQ Mass Spectrometer (Varian Inc., CA, USA) equipped with a Varian LC system consisting of a binary pump, autosampler and temperature controlled column compartment. Chromatographic separation was performed on a Pursuit XRs Ultra-C18 reversed phase column (2.8␮m particle size, 50mm ×2mm i.d.) from Varian Inc. (CA, USA). The mobile phase consisted of water (solvent A) and methanol (solvent B) with 0.1% (v/v) glacial acetic acid and 15mM ammonium acetate. Gradient elution consisted of a solution of 30:70 (v/v) methanol: water for 9min,followed byanincreaseinmethanolto100%over10 min.The injection volume was 10␮L and the flow-rate was 200␮Lmin−1 for 10min. The temperature in the column compartment was set to 40◦C. Multiple reaction monitoring (MRM) parameters were optimisedforsubsequentquantitativeanalysis.Precursor ionsincluded [M+NH4]+for APnEOs in positive ion mode and [M−H]−for APs, steroidal hormones, and BFA in negative ion mode. This procedure was conducted using a 1mL syringe pump (Hamilton Company, Reno, NV, USA), employing a continuous flow-rate of 20␮Lmin−1. Each standard or mixture was prepared as 10mg L−1in methanol. 0.1mL of each solution was taken up by the Hamilton syringe and the remaining 0.9mL of syringe volume were filled with mobile phase. The composition of the mobile phase depended on the ionisation of each analyte in ESI. Ionisation in the ESI source was achieved using nitrogen as a nebuliser and drying gas. Housing and desolvation temperature were set to 60◦C and 250 ◦C, respectively, for the optimisation of syringepumpinjectionsforMS/MS.However,toobtainastrongsignal for each analyte, the desolvation temperature was set to 200◦C during the first 4min and was increased linearly to 350◦C until the end of the chromatographic run. The drying and nebulising gas pressures were fixed at 30psi and 65 psi, respectively. The capillary voltage was set to 4.5kV in positive mode (ESI+) and −3 kV in negative mode (ESI−). The shield voltage was maintained at −600/600V (ESI+/ESI−) and the cone voltage was optimised for each individual compound (Table 2). Collision induced dissociation (CID) was conducted with argon as the collision gas at a fixed pressure of 2mTorr. The fragment ions obtained for each compound and the collision potential are displayed in Table 2. 3. Results and discussion 3.1. Optimisation of MS/MS conditions The optimisation of MS/MS conditions for each compound was performed by direct infusion of pure standards or Igepal mixtures. Under positive ion mode (ESI+), APnEOs possess a high affinity for alkali metal ions in unmodified mobile phases and often lead to the formation of sodium adducts [M−Na]+rather than protonated molecules [M−H]+[23]. These adducts are reluctant to fragment in the collision cell and cannot be used in MRM detection [19]. For this reason, an aqueous mobile phase of 0.1% (v/v) glacial acetic acid and 20mM ammonium formiate was used to force the formation of ammonium adducts [M+NH4]+, which have a relevant and reproducible fragmentation in the collision cell [19,20]. 119 704 T. Vega-Morales et al. / Journal of Hazardous Materials 183 (2010) 701–711 Fig. 1. Recoveries obtained by different types of SPE cartridges. (a) Recoveries obtained for NPEOs oligomers; (b) recoveries obtained for OPEOs oligomers; (c) recoveries obtained for BPA and steroidal hormones. In negative ion mode (ESI−), transitions from the [M−H]−ion for APs, steroidal hormones and bisphenol-A were monitored. For these compounds, mobile phases with different percentages of methanol were used for direct flow injections. The optimal response for these transitions was observed with a 70% (v/v) methanol, 30% (v/v) aqueous solution, which is consistent with previous studies [24]. 3.2. Optimisation of SPE process The optimisation of SPE for wastewater samples included the evaluation of several experimental variables to achieve maximum extraction efficiency for each compound. Hence, cartridge type, pH, ionic strength, sample volume, wash step and desorption volume were optimised. A sample volume of 100mL of Milli-Q water containing 500ng L−1of each analyte and a desorption volume of 2mL of methanol were used initially. Samples were passed through cartridges under the conditions described in Section 2.3. 3.2.1. Type of cartridge Five solid phase materials with different characteristics were tested to obtain an optimal extraction for the analytes, including a Sep-Pak Vac C18 (500mg), Oasis HLB (200 mg), Bond Elut-ENV (500mg),BondElutPlexa(500mg)andLiChrolutEN(500 mg).Fig.1 shows the results obtained with the aforementioned cartridges. Among the cartridges tested, Sep-Pak Vac C18 showed superior performance under the initial conditions, even for long-chained APnEO oligomers (recoveries >90%). Despite this result, it was expected that an increase in sample volume passed through the cartridges would decrease the recoveries of polar compounds considerably [25]. However, due to the nature of wastewater samples, especially those taken from primary treatment, a few hundred millilitres (∼300mL) of sample were enough to saturate the cartridges [19], even when the samples had been properly filtered. The Oasis HLB cartridge showed poor recoveries for APs and short-chain alkylphenolic compounds (from 1 to 5 ethoxylate units). However, due to its hydrophilic nature [26,27], a satisfactory extraction was obtained for “water soluble” APnEO (n> 5 units) oligomers (recoveries between 78% and 92%). The Bond Elut Plexa cartridge showed the best recoveries for polar compounds (n>9), but the results obtained for hydrophobic species were clearly inferior to those achieved with the C18 cartridge, especially for nonylphenol (NP) and its ethoxylates. Due to the highly cross-linked materials of LiChrolut EN and Bond Elut120 T. Vega-Morales et al. / Journal of Hazardous Materials 183 (2010) 701–711 705 Fig. 2. Relative extraction efficiencies at several pH (a) and ionic strength (b) values for estriol, 17␤-estradiol, bisphenol-A, nonylphenol, NP1EO, NP5EO, NP12EO, octylphenol, OP1EO, OP5EO and OP12EO. Fig. 3. (a) Normalized peaks areas obtained for different volumes of Milli-Q water samples. A volume of 2mL of methanol was employed for desorption of analytes. (b) Normalized peak areas obtained for extraction of 250mL of Milli-Q water samples spiked with 500ngL−1of each analyte using different desorption volumes. 121 706 T. Vega-Morales et al. / Journal of Hazardous Materials 183 (2010) 701–711 ENV, recoveries obtained under the initial conditions were inferior for all analytes studied. Typically, large volumes of organic solvents such as dichloromethane, trichloromethane, hexane, ethyl acetate oracetonearerequiredfortheelutionofadsorbedcompoundsfrom thiskindofadsorbents[28].Theuseoflargevolumesofthesehighly toxic organic solvents generally leads to the inclusion of an evaporation step to allow the final extract to be compatible with the mobile phase and analytical instrument (e.g., LC/MSninterfaces) or to reduce the injection volume and increase the preconcentration factor [28]. This additional step lengthens analysis time, increases the final cost and leads to the loss of volatile analytes, which affects the quality of the results. On the basis of the results presented in this section, the SepPak Vac C18 (500mg) cartridge was selected for the analysis of the selected compounds. 3.2.2. pH and ionic strength Fig.2 showsthe effectsof pH(a) andionic strength(b) onextraction recoveries of EDCs using Sep-Pak Vac C18 (500mg) cartridges. The results revealed that a pH between 3 and 9 did not significantly affectextractionrecovery[29,30]. Thus,a pHbetween6 and7.5 was selected as the sample pH for SPE. The effect of ionic strength on the recovery of EDCs was performed by the addition of 0% to 30% (w/v) sodium chloride to the aqueous medium. The results show that an increase in ionic strength did not produce an increase in the signals of EDCs. Thus, a sample solution was used without salt additions for subsequent experiments. 3.2.3. Sample volume The effect of sample volume between 100 and 1000mL on signal intensity was evaluated. To compare the signals with the same concentration (500ng L−1) of analyte, the data was normalised and preconcentration was applied in each case. Two millilitres of methanol was employed for the desorption of analytes. Fig. 3a showsthenormalised signalsfor selectedvolumes ofaqueoussamples. Similar responses between 100 and 250mL were observed for all analytes. However, a significant decrease in the APnEO water soluble fraction (n>5) was obtained with 500 mL of sample. For the other compound, equal signal intensities were recorded for sample volumes ranging from 100 to 1000mL. Even though samples had been thoroughly filtered (0.45␮m), cartridges were completely saturated when 300mL of sample was used in identical experiments on wastewater matrices (primary and secondary treatment). Based on these results, a sample volume of 250mL was used to obtain the best analytical results. 3.2.4. Wash step and desorption volume Theeffect ofthe washsolution compositiononextractionrecovery was also evaluated. The proper elution of analytes is significant becauseaselective extractionand thoroughsample cleaningallows for minimal ion suppression effects in the ESI interface [31]. The percentage of methanol was varied (0% (v/v), 1% (v/v), 5% (v/v), 10% (v/v), and 20% (v/v)) in 5mL of Milli-Q water and the resultant solutions were used to elute the analyte. Equal signal intensities were obtained with the use of 0% (v/v) to 5% (v/v) methanol for all compounds; however, a decrease in signal intensity of the most polar compounds was observed with 10% (v/v) methanol and was most significant with estriol and the water soluble fraction of APnEOs (n>5). Thus, 5 mL of a Milli-Q water/methanol (5% (v/v)) solution was selected for the wash step. Desorption volume was optimised to ensure the complete extraction of analytes. Volumes of methanol between 1 and 7mL were tested. Fig. 3b shows normalised peaks obtained for EDCs with different volumes of wash solution. Similar responses were Table 3 Evaluation of the analyte signal suppression in the primary and secondary treatments and final effluent. Compound Primary treatmenta(%) Secondary treatmenta(%) Final effluenta(%) NP1EO 14.3 15.2 10.6 NP2EO 12.1 14.5 10.8 NP3EO 14.3 14.9 11.8 NP4EO 14.9 15.2 10.8 NP5EO 16.6 15.5 10.5 NP6EO 16.6 16.7 10.4 NP7EO 17.6 15.9 10.8 NP8EO 17.8 15.2 11.5 NP9EO 18.4 14.2 10.5 NP10EO 18.0 14.9 11.8 NP11EO 21.0 14.5 12.3 NP12EO 19.6 15.2 11.8 OP1EO 14.3 12.7 9.1 OP2EO 13.9 12.4 10.3 OP3EO 17.6 13.3 10.6 OP4EO 17.0 12.5 10.5 OP5EO 16.8 12.3 11.0 OP6EO 17.8 12.7 11.4 OP7EO 20.8 12.7 11.6 OP8EO 18.6 13.6 11.1 OP9EO 18.0 14.0 11.9 OP10EO 19.4 12.5 12.6 OP11EO 19.4 12.8 11.4 OP12EO 20.4 13.6 11.4 NP 23.8 18.6 13.4 OP 19.7 18.1 12.9 BPA 19.0 14.9 10.3 E1 17.8 13.7 10.1 E2 16.4 14.5 11.4 E3 18.7 13.6 11.0 EE 18.4 13.9 9.9 aMean of three determinations. observed for desorption volumes of 2–7mL; however, 1 mL of methanol was clearly insufficient for proper elution of the analytes. Thus,adesorptionvolumeof2 mLofmethanolwaschosentoobtain the highest preconcentration possible. In summary, the optimal conditions for the extraction of the target compounds included a Sep-Pak Vac C18 (500mg) cartridge with a sample volume of 250mL at a pH between 6 and 7.5, a wash step with 5mL of Milli-Q water/methanol (5%, v/v) and a desorption volume of 2mL of methanol. Under these conditions, a preconcentration factor of 125 was obtained. 3.3. Matrix effects Despitethe highsensitivity andlowchemical noiseinLC/MS/MS systems, the sample composition has a great influence on the analyte signal [31]. Thus, a low analyte signal may be the result of co-eluting compounds that impair ionisation [13]. To evaluate the relative signal suppression in the dissolved phase, the algorithm published by Vieno et al. [32] was used and is shown in Eq. (1). The results are expressed as a percentage and presented in Table 3. As−(Asp −Ausp) As×100 (1) where Ascorresponds to the peak area of the analyte in pure standard solution, Asp corresponds to the peak area in the spiked matrix extract, and Ausp corresponds to the matrix extract. This procedure was applied to primary and secondary wastewaters, as well as to final effluent matrices. Greater matrix effects were observed during the analysis of primary treatment samples, resultinginsignalsuppressions from14%to 24%.Moreseveresignal suppression was observed for hydrophobic compounds, especially for NP, OP, AP1,2EO, and EE. Ion effect suppressions were signifi122 T. Vega-Morales et al. / Journal of Hazardous Materials 183 (2010) 701–711 707 cantly lower for secondary treatment samples (between 12% and 18%) and final effluent samples (between 9% and 3%). The results obtained are in agreement with those reported in similar studies [33]. 3.4. Quantification and quality control The developed method was evaluated for recovery, precision and detection limit. Calibration curves were evaluated by analysing external standard solutions in triplicate at six different concentration levels. Linearity was established from 50ng L−1to 3000ng L−1. Satisfactory linearity was obtained with correlation coefficients over 0.991 for all analytes studied. The instrumental detection limits (IDLs) were defined and determined to equal the concentration of analyte that yielded a signal to noise ratio of 3. The limit of detection for the entire method (MDLs) in different matrices was calculated with the following equation: MDL =IDL ×100 R(%) ×c(2) where IDL is the instrumental detection limit, R(%) is the recovery of each analyte in the corresponding matrix and cis the preconcentration factor (125 for all matrices in the dissolved phase and 10 in the particulate phase). The results obtained are shown in Table 4. Recovery and reproducibility were evaluated from replicate analysisinMilli-Q water,wastewaters fromprimary and secondary treatment, and particulate matter (n=3). Replicate samples were spiked with 500ng L−1of the standard mixture in each matrix (dissolved phases) or 10␮gg−1in approximately 0.2g of particulate dry weight (particulate phase). The replicate samples were extracted and analysed according to the respective methodology. Reproducibility was expressed as the relative standard deviation (% Table 4 Method detection limits obtained in the different matrices: MDLwin Milli-Q water, MDLpt in primary treatment, MDLst in secondary treatment, and MDLpm in particulate matter (primary treatment). Compound MDLw(ngL−1)MDL pt (ngL−1)MDL st (ngL−1)MDL pm (ngg−1) NP1EO 1.2 3.5 3.2 6.2 NP2EO 0.5 1.5 1.4 3.1 NP3EO 0.6 1.6 1.6 2.8 NP4EO 0.7 2.0 1.9 3.4 NP5EO 1.8 5.4 4.7 8.2 NP6EO 0.3 0.9 0.7 1.4 NP7EO 1.1 3.2 2.8 4.6 NP8EO 1.1 3.0 3.0 4.2 NP9EO 0.9 2.7 2.6 3.7 NP10EO 1.6 4.3 4.4 6.0 NP11EO 0.5 1.3 1.4 1.5 NP12EO 0.5 1.3 1.3 1.7 OP1EO 2.1 7.2 6.0 12.7 OP2EO 1.2 3.7 3.5 6.6 OP3EO 0.9 2.6 2.4 3.8 OP4EO 1.3 3.9 3.7 5.8 OP5EO 1.6 4.5 4.7 6.8 OP6EO 1.3 3.6 3.7 5.1 OP7EO 0.8 2.2 2.3 2.6 OP8EO 0.7 2.0 1.9 2.7 OP9EO 1.1 2.9 2.8 3.8 OP10EO 1.2 2.9 3.4 3.8 OP11EO 1.4 3.8 3.9 5.0 OP12EO 1.0 2.7 2.7 3.4 NP 1.3 2.7 2.7 5.1 OP 1.8 5.7 5.5 9.4 BPA 1.9 5.7 5.2 9.8 E1 1.3 4.0 3.9 5.7 E2 1.2 3.3 2.8 5.1 E3 1.3 3.7 3.3 6.9 EE 0.9 2.8 2.6 5.0 Table 5 Recovery percentages and RSD (%) (n=3) for different matrices spiked with 500ngmL−1(dissolved phase) or 10␮gg−1(particulate phase from primary treatment samples) of each analyte. Compound Milli-Q waterRecovery (%) Primary treatmentRecovery (%) Secondary treatmentRecovery (%) Particulate matterRecovery (%) NP1EO 97 ±10 85 ±593±771±9 NP2EO 96 ±11 86 ±289±560±9 NP3EO 96 ±12 94 ±691±671±8 NP4EO 98 ±886±11 93 ±674±7 NP5EO 102 ±884±10 95 ±782±6 NP6EO 101 ±783±6 102 ±582±7 NP7EO 98 ±986±197±687±9 NP8EO 97 ±692±293±10 88 ±5 NP9EO 101 ±684±10 87 ±891±6 NP10EO 95 ±794±791±389±8 NP11EO 96 ±898±889±7 104 ±9 NP12EO 97 ±794±993±797±6 OP1EO 102 ±13 73 ±288±871±10 OP2EO 108 ±11 82 ±586±11 69 ±12 OP3EO 99 ±12 85 ±692±987±9 OP4EO 92 ±984±687±584±5 OP5EO 93 ±988±785±883±7 OP6EO 98 ±891±788±688±6 OP7EO 100 ±793±788±9 103 ±14 OP8EO 104 ±889±894±392±6 OP9EO 100 ±795±11 97 ±495±8 OP10EO 99 ±6 101 ±587±496±9 OP11EO 94 ±791±789±596±8 OP12EO 98 ±891±894±11 101 ±8 NP 94 ±12 82 ±14 84 ±12 67 ±7 OP 97 ±979±12 82 ±13 76 ±7 BPA 93 ±13 82 ±10 91 ±773±7 E1 91 ±781±784±988±6 E2 91 ±891±689±581±8 E3 87 ±788±683±767±9 EE 96 ±11 79 ±785±771±8 123 708 T. Vega-Morales et al. / Journal of Hazardous Materials 183 (2010) 701–711 Table 6 Concentrations of each analyte in sewage from WWTP: Cpt in primary treatment, Cst in secondary treatment, Cfe in final effluent, and Cpm in particulate matter (primary treatment). Compound Cpta(ngL−1)Csta(ng L−1)Cfea(ng L−1)Cpma(␮gg−1) NP1EO 170.9 278.8 274.3 2.6 NP2EO 188.0 154.4 161.7 2.9 NP3EO 305.2 140.9 135.2 2.8 NP4EO 413.1 51.8 42.8 0.9 NP5EO 485.2 42.6 41.3 1.1 NP6EO 615.4 47.8 41.3 0.3 NP7EO 927.2 34.0 29.8 0.5 NP8EO 1097 33.0 27.3 0.5 NP9EO 981.1 25.7 19.4 0.5 NP10EO 890.0 27.0 21.5 0.4 NP11EO 759.2 27.5 20.5 0.4 NP12EO 589.0 24.7 16.1 0.3 OP1EO 41.92 58.6 51.8 0.2 OP2EO 205.7 10.3 9.2 0.3 OP3EO 216.9 9.2 9.1 0.4 OP4EO 113.8 10.9 9.9 0.2 OP5EO 68.3 8.6 8.5 32.8c OP6EO 39.2 8.0 7.0 18.8c OP7EO 28.0 7.2 6.3 12.3c OP8EO 29.7 7.6 7.2 10.7c OP9EO 33.8 7.0 7.0 16.2c OP10EO 23.6 5.1 5.0 11.3c OP11EO 17.0 4.9 4.9 8.1c OP12EO 9.8 4.3 4.2 4.7c NP 9.7 18.7 17.5 0.8 OP 6.1 9.3 8.6 0.2 BPA 13.4 7.8 6.4 0.4 E2 16.3 5.0 3.5 0.4 E1 15.5 13.4 11.6 0.5 E3 22.2 18.7 16.1 0.4 EE 9.3 n.d.bn.d.b0.1 aMean of three determinations. bNot detected. c×10−3. RSD) and RSDs lower than 14% were achieved for all EDCs. Recoveries into different matrices were tested for each analyte and are displayed in Table 5. 3.5. Determination of EDCs in sewage samples 3.5.1. Dissolved phase concentrations To demonstrate that the proposed method can be used to adequately quantify EDCs in actual environmental samples, the methodology was applied to sewage samples from a wastewater treatment plant of Las Palmas of Gran Canaria (Spain). Dissolved phase concentrations are presented in Fig. 4.Fig. 4a shows the results of NP and 1–12 ethoxylate concentrations in different treatment stages. The total dissolved concentration of NP and NP1–12EO was reduced from 7.60␮gL−1in the primary treatment to 0.84␮gL−1 in the final effluent (Table 6 shows the concentration of single compounds). Thus, an 89% decrease in total NP was observed. However, the relative composition of the homologous mixture was enriched in short-chained NP1–2EO and NP as the treatment progressed, whichisinagreement withtheresults ofother publications[29,33]. This phenomenon is a direct consequence of APnEOs breakdown in WWTPs, especially during biological treatments. These compounds progressively lose ethoxylate units, which results in the formation of APs (a raw material for microorganisms), short-chain APEOs, and other biotransformation products such as carboxylated and halogenated derivatives [19]. The total dissolved OP and OP1–12EO concentration showed a similar biotransformation pattern to nonylphenol ethoxylates (NPEOs) family. A reduction of approximately 84% (from 0.83␮gL−1to 0.13␮gL−1) was observed in the waste water treatment process (Fig. 4b). Nonetheless, only OP and OP1EO showed a slightincreaseinconcentrationasthetreatmentprogressed.Differences between NPnEO and OPnEO concentrations can be attributed to the global production of non-ionic surfactants, as nonylphenolic compounds represent approximately 80% of APnEOs worldwide production [3]. The absence of a mass balance between AP0–12EO concentrations in the primary treatment and the biotransformation products in the final effluent could be explained by two distinct processes. Logarithmic values of octanol/water partition coefficients (logKow) for AP1–4EO metabolites are between 3.90 and 4.48, suggesting that these substances might become associated with organic matter in particulate matter and sediment [4]. Additionally, APnEOs under aerobic conditions are susceptible to oxidation to more polar short-chain and long-chain AP ethoxycarboxylate (APEC) and carboxylated AP ether carboxylate (CAPEC) derivatives [19].Ithas been reported that short-chain AP1–2ECs account for the majority of APnEO-related compounds, especially in secondary and final effluents, where 80% of APnEOs exist as AP1–2ECs in the dissolved phase [26,34]. Fig. 4c shows the concentrations of BPA and steroidal hormones during the treatment process. Bisphenol-A concentration was reduced by approximately 47%, from 13.4 ngL−1in the primary treatment to 6.36ng L−1in the final effluent. Throughout all stages of treatment, steroidal hormones were present at low ngL−1levels. 17␤-estradiol (E2) showed an efficient biodegradation during the activated sludge treatment, reflected in the rapid decline in concentration from the primary treatment (16.3ng L−1) to the final effluent (3.45ng L−1). This result is in agreement with those of previous publications [35,36]. Concentrations of E1 (from 15.54ng L−1in the primary treatment to 11.57ng L−1in the final effluent) and E3 (from 22.25ng L−1to 16.12ng L−1) remained relatively constant throughout the treatment, which can be explained by the continuous enrichment of these metabolites by E2 biotransformation. A small decline in the concentration of E2 and E3 may in part be due to the affinity of these metabolites to organic matter present in sediments and particulate matter (E3 has a logKow of 2.81 and E1 has a logKow of 3.43).This affinityis particularlyplausible for17␣-ethynylestradiol (EE), which was found in primary treatment samples (9.34ng L−1) but was not observed in the secondary treatment and final effluent. However, EE concentrations were determined throughout all stages of treatment for particulate phase samples. 3.5.2. Particulate phase concentrations Concentrations of EDCs in the particulate phase (Table 6) demonstrated the tendency of hydrophobic compounds to bind tightly to particulate matter and sediments. In Fig. 5, particulate phase concentrations in different stages of wastewater treatment are highlighted. The concentration profiles of APnEOs homologues in the particulate phase were similar to the profiles of dissolved compounds,except thatshort-chain APnEOswere presentin higher proportions (Fig. 5a and b). In primary treatment samples, more than 60% of short-chain APnEOs (n<3) were associated with particulate matter. Moreover, more than 80% of total NP and more than 60% of total OP were found in the particulate phase. The partitioning of target compounds between the dissolved and particulate fraction of the sample was similar for each treatment stage and only the relative concentrations of the analytes varied between stages. BPA and steroidal hormones were also found in the particulate phase (Fig. 5c). The partitioning of these compounds between the particulate and dissolved phase was significant, especially for hydrophobic compounds. Up to 60% of total BPA and steroidal hormones was present in the particulate phase. 124 T. Vega-Morales et al. / Journal of Hazardous Materials 183 (2010) 701–711 709 Fig. 4. Dissolved phase concentrations of NPEOs (a), OPEOs (b), BPA, and steroidal hormones (c) in primary treatment, secondary treatment, and final effluent with the change in concentrations during the process highlighted. 125 710 T. Vega-Morales et al. / Journal of Hazardous Materials 183 (2010) 701–711 Fig. 5. Particulate phase concentrations of NPEOs (a), OPEOs (b), BPA, and steroidal hormones (c) in primary treatment, secondary treatment, and final effluent with the change in concentrations during the process highlighted. 4. Conclusions In this work, an analytical method for the simultaneous extraction, identification, and quantification of nonylphenol, octylphenol and corresponding ethoxylates (1–12), bisphenol-A, 17␣-ethynylestradiol, and 17␤-estradiol and two of its metabolites (estrioland estrone)fromwastewatermatriceshas beendeveloped and applied to sewage samples. An SPE method (dissolved phase) coupled with LC/MS/MS allows for rapid extraction and analysis, offeringthelowdetectionlimits(from 0.5ng L−1to6ngL−1inMRM mode) and high selectivity required to detect these analytes in complex environmental matrices. Ultrasonic extraction of the particulate phase with methanol has several advantages including low desorption volumes and fast extraction times. Moreover, recoveries between 60% and 104% with a RSD lower than 14% and detection limits from 1.4ng g−1to 12.7ng g−1were obtained. The application of the methodology to samples from a WWTP revealed that almost all of the target analytes were present in every stage of wastewater treatment, with concentrations on the order of ngL−1or ␮gg−1in the dissolved and particulate phase, respectively. Moreover, the results show that a complex mixture of endocrine-disrupting compounds occurs in the effluent of the waste water treatment plant under study, and that this mixture will eventually enter the environment where aquatic organisms are exposed to these pollutants. Acknowledgement This work was supported by funds provided by the Spanish Ministry of Education of Science. Research Project No. CTM2006/06503. References [1] R.P. Schwarzenbach, B.I. Escher, K. Fenner, T.B. Hofstetter, C.A. Johnson, U. von Gunten, B. Wehrli, The challenge of micropollutants in aquatic systems, Science 313 (2006) 1072–1077. [2] C. Sonnenschein, A.M. Soto, An updated review of environmental estrogen and androgen mimics and antagonists, J. 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[31] S. González, M. Petrovic, D. Barceló, Advanced liquid chromatography–mass spectrometry (LC–MS) methods applied to wastewater removal and the fate of surfactants in the environment, Trends Anal. Chem. 26 (2007) 116–124. [32] N.M. Vieno, T. Tuhkanen, L. Kronberg, Analysis of neutral and basic pharmaceuticals in sewage treatment plants and in recipient rivers using solid phase extraction and liquid chromatography–tandem mass spectrometry detection, J. Chromatogr. A 1134 (2006) 101–111. [33] K.K. Koh, T.Y. Chiu, A.R. Boobis, E. Cartmell, S.J.T. Pollard, M.D. Scrimshaw, J.N. Lester, A sensitive and robust method for the determination of alkylphenol polyethoxylates and their carboxylic acids and their transformation in a trickling filter wastewater treatment plant, Chemosphere 73 (2008) 551–556. [34] J.E. Loyo-Rosales, C.P. Rice, A. Torrents, Octyl and nonylphenol ethoxylates and carboxylates in wastewater and sediments by liquid chromatography/tandem mass spectrometry, Chemosphere 68 (2007) 2118–2127. [35] H.B. Lee, D. Liu, Degradation of 17ß-estradiol and its metabolites by sewage bacteria, Water Air Soil Pollut. 134 (2002) 353–368. [36] T.A. Ternes, P. Kreckel, J. Mueller, Behavior and occurrence of estrogens in municipal sewage treatment plants. II. Aerobic batch experiments with activated sludge, Total Environ. 225 (1999) 91–99. 127 Capítulo)III:)Parte)Experimental)y)Resultados) ! 134!III.3.)T.)Vega<Morales)et)al./)Journal)of)AOAC)International)(en)prensa)! ! la! extracción! en! fase! sólida! (SPE)! y! la! extracción! asistida! por! ultrasonidos! (UAE),! respectivamente,!obteniéndose!recuperaciones!comprendidas!entre!el!69%!y!el!102%.!! !Las!combinaciones!de!ambas!metodologías!de!extracción!con!el!sistema!de!HPLCRFD! demostraron! ser! los! suficientemente! sensibles! para! la! determinación! de! los! compuestos! estudiados! en! muestras! reales,! alcanzándose! límites! de! detección! y! de! cuantificación! comparables! a! los! obtenidos! en! otros! trabajos! que!emplearon! la! espectrometría!de!masas!(MS)!como!sistema!de!detección.!! !Los!métodos!optimizados!fueron!aplicados!satisfactoriamente!a!la!determinación!de! los!EDCs!seleccionados!en!muestras!de!aguas!residuales,! las!cuales!fueron! recogidas! trimestralmente! desde! Julio! de! 2009! hasta! Julio! de! 2010! en! la! mayor! EDAR! de! Las! Palmas! de! Gran! Canaria! (España).! En! este! caso,! también! se! evaluaron! diferentes! etapas! del! tratamiento! (entrada,! salida! de! tratamiento! secundario! y! efluente! final),! obteniéndose!concentraciones!entre!<!10!ng!·∙!LR1!hasta!aproximadamente!1300!ng!·∙!LR1! en!la!fase!disuelta,!y!entre!0.1!µg!·∙!g!R1!y!7.7!µg!·∙!g!R1!en!la!fase!particulada.!!!!!! !Por! último,! también! se! expone! un! análisis! detallado! de! los! cálculos! de! las! concentraciones!de!estradiol!equivalentes!(EEQs)!correspondientes!a!cada!una!de!las! muestras! y! fracciones! analizadas,! lo! cuál! nos! permitió! obtener! conclusiones! significativas! acerca! de! la! actividad! disruptora! endocrina! durante! cada! etapa! del! tratamiento!y!acerca!de!los!compuestos!que!ejercían!una!mayor!influencia!sobre!dicha! actividad.!! !! ! Capitulo)III:)Parte)Experimental)y)Resultados) ! III.3.)T.)Vega<Morales)et)al./)Journal)of)AOAC)International)(en)prensa)!135! ) ! Dear!Prof.!Santana:! ! It!is!a!pleasure!to!accept!your!manuscript!entitled!"Determination!and!assessment!of! estradiolRmimicking!compounds!in!the!dissolved!and!particulate!phases!of!wastewater! treatment! plant! samples"! in! its! current! form! for! publication! in! the! Journal! of! AOAC! INTERNATIONAL.! ! Thank!you!for!your!fine!contribution.!!On!behalf!of!the!Editors!of!the!Journal!of!AOAC! INTERNATIONAL,!we!look!forward!to!your!continued!contributions!to!the!Journal.! ! Sincerely,! Mr.!Alexander!Krynitsky! Section!Editor,!Journal!of!AOAC!INTERNATIONAL! [email protected]! ! ! ! ! Capítulo)III:)Parte)Experimental)y)Resultados) ! 136!III.3.)T.)Vega<Morales)et)al./)Journal)of)AOAC)International)(en)prensa)! ! ! III.3.$T.Vega$Morales$et$al./$Journal$of$AOAC$International$(en$prensa)$ 137$ Determination$and$assessment$of$estradiol2mimicking$ compounds$in$the$dissolved$and$particulate$phases$of$ wastewater$treatment$plant$samples$$ $ T.$Vega<Morales,$R.Guedes<Alonso,$Z.$Sosa<Ferrera$and$J.J.$Santana<Rodríguez$ Departamento$de$Química,$Universidad$de$Las$Palmas$de$Gran$Canaria,$$ 35017$Las$Palmas$de$Gran$Canaria,$Spain.$ Telephone:$+34$928$452$915;$Fax:$+34$928$452$922;$Email:[email protected]$ $ Abstract( (During$the$last$two$decades,$a$large$number$of$publications$have$clearly$shown$that$anthropogenic$compounds$ that$disrupt$the$endocrine$system$of$wildlife$species$are$a$major$cause$for$concern,$and$this$concern$has$led$to$a$ demand$ for$ new$ screening$ methods.$ In$ this$ work,$we$ have$ optimised$ and$ applied$a$ new$ method$ to$ identify$ endocrine$disrupting$chemicals$(EDCs)$such$as$nonylphenol,$octylphenol$and$their$corresponding$ethoxylates$(122),$ 17α2ethynylestradiol,$ bisphenol2A,$ 17β2estradiol$ and$ estriol,$in$ sewage$ samples.$ For$ the$ extraction$ and$ preconcentration$ of$ all$ the$ analytes$ from$ the$ dissolved$ and$ particulate$ phases,$we$ have$ employed$ solid$ phase$ extraction$ (SPE)$ and$ ultrasonic$ assisted$ extraction$ (UAE)$ respectively.$ Identification$ and$ quantification$ were$ achieved$ by$ high$ performance$ liquid$ chromatography$ with$ fluorescence$ detection.$ Satisfactory$ detection$ limits$ (between$ 0.5$–$7.6$ng$·∙$L21$in$ the$ dissolved$phase$and$ 12.3$ 2$21.4$ng$·∙$g$ 21$in$the$ particulate$phase)$and$ analyte$ recoveries$(between$67$2$102%)$were$achieved$for$target$compounds.$The$optimised$method$was$applied$to$the$ determination$ of$ EDCs$ in$ liquid$ sewage$ samples$ collected$during$ a$ year$ (from$ July$ 2009$ to$ July$ 2010)$from$ a$ wastewater$treatment$plant$(WWTP)$in$Las$Palmas$de$G.C.$(Spain).$Concentrations$of$EDCs$ranged$from$<$10$ng$·∙$L21$ to$nearly$1300$ng$·∙$L21$in$the$dissolved$phase,$and$from$0.1$µg$·∙$g$21$to$7.7$µg$·∙$g$21$in$the$suspended$particulate$ matter.$ ( Keywords:$Alkylphenol$ethoxylates,$sex$hormones,$sewage$samples,$liquid$chromatography,$fluorescence$detection.$ ( ( $ 138$ 1. Introduction( ( $Presently,$it$is$known$that$a$significant$exposure$to$ anthropogenic$ and$ natural$ chemicals$ is$ able$ to$ disrupt$the$endocrine$system$(1,$2).$Although$these$ endocrine2disrupting$ chemicals$ (EDCs)$ can$ alter$ the$ endocrine$ system$ in$ various$ ways,$ one$ of$ the$ most$ widely$demonstrated$modes$of$hormonal$disruption$ corresponds$to$the$mimicry$of$endogenous$estradiol$ by$binding$to$and$activating$oestrogen$receptors$(ER$ agonist)$(3).$ $Among$ all$ of$ the$ EDCs$ which$ behave$ this$ way,$ alkylphenolic$ ethoxylated$ surfactants$ (APEOs),$ and$ more$ specifically,$ their$ main$ biodegradation$ metabolites,$nonylphenol$(NP)$and$octylphenol$(OP),$ have$ aroused$ enormous$ interest$ in$ the$ scientific$ community$ due$ to$ their$ considerable$ worldwide$ production$ levels$(4),$ their$ extensive$ use$ in$ several$ industrial,$ agricultural$ and$ household$ applications$ (5),$and$their$chronic$toxicity$(4,$6).$Bisphenol2A$(2,22 bis(42hydroxyphenyl)propane)$ (BPA),$ a$ chemical$ substance$ widely$ employed$ in$ industry$ as$ an$ antioxidant$in$PVC$plastics$and$as$a$monomer$in$the$ production$ of$ epoxy$ resins$ and$ polycarbonates$ (7),$ also$ has$ the$ capacity$ to$ mimic$ the$ endogenous$ estradiol.$ $More$ recently,$ it$ has$ been$ reported$ that$ natural$ steroid$ hormones,$ such$ as$ 17β2estradiol$ and$ its$ metabolites,$ oestrone,$ estriol$ and$ 16α2 hydroxyestrone,$ and$ synthetic$ steroid$ hormones,$ such$ as$ 17α2ethynylestradiol,$ are$ released$ into$ the$ aquatic$ environment$ through$ discharges$ from$ WWTP$and$may$act$as$EDCs$(8,$9).$These$oestrogens$ have$ ER$ potencies$ of$ at$ least$ three$ orders$ of$ magnitude$ higher$ than$ that$ of$ the$ alkylphenolic$ compounds$(10)$and$are$suspected$to$be$the$major$ contributors$of$oestrogenic$activities$in$sewage,$even$ when$their$concentrations$are$usually$in$the$range$of$ parts$per$trillion$(ppt)$(11).$ $Moreover,$ the$ complete$ degradation$ of$ EDCs$ in$ conventional$ wastewater$ treatment$ plants$ is$ not$ achieved;$ therefore,$ a$ complex$ mixture$ of$ xenobiotics$could$eventually$enter$the$environment$ where$ aquatic$ organisms$ will$ be$ exposed$ to$ them$ (12).$ In$ many$ cases,$ the$ biodegradation$ process$ leads$to$the$formation$of$sub2products$that$are$even$ more$ toxic,$ more$ lipophilic,$ more$ oestrogenic$ and$ more$ persistent$ than$ the$ parent$ substances,$ as$ occurs$with$polyethoxylated$alkylphenols$(13).$$ $Based$ on$ their$ physico2chemical$ properties$ (Table$ 1),$ these$ compounds$ tend$ to$ bind$ tightly$ to$ particulate$ matter$ and$ sediments$ and$ bio2 accumulate$ in$ aquatic$ organisms$ (13,$14),$ which$ contributes$ to$ the$ persistence$ of$ these$ compounds$ in$the$environment.$Therefore,$to$know$the$real$fate$ of$ these$ emerging$ pollutants,$ it$ is$ necessary$ to$ develop$ complementary$ analytical$ methodologies$ for$both$the$dissolved$and$solid$phases.$$ $The$ main$ objective$ of$ the$ present$ work$ is$ the$ development$ of$a$ simple,$ cheap$and$ rapid$ method$ suitable$ for$ the$ identification$ and$ quantification$ of$ nonylphenol,$ octylphenol$ and$ the$ corresponding$ shorted2chained$ethoxylates$(122),$bisphenol2A,$17α2 ethynylestradiol,$ and$ 17β2estradiol$ and$ one$ of$ its$ III.3.$T.Vega$Morales$et$al./$Journal$of$AOAC$International$(en$prensa)$ 139$ $ $ $ Table$1.$Physiochemical$properties$of$the$compounds$investigated.$$ $ $ $ 140$ metabolites,$estriol,$in$both$dissolved$and$particulate$ phases$in$sewage$samples.$$ $For$ extraction$ in$ the$ dissolved$ phase,$ we$ have$ employed$ a$ solid$ phase$ extraction$ (SPE)$ methodology,$which$is$a$powerful$tool$for$chemical$ isolation$ and$ purification.$ For$ the$ extraction$ of$ the$ analytes$ in$ the$ particulate$ phase,$ we$ have$ used$ ultrasonic$ assisted$ extraction$ (UAE),$ which$ offers$ relatively$high$recovery$percentages,$low$volumes$of$ organic$solvents$and$short$extraction$times$(15,$16).$ $Finally,$ the$ extracts$ were$ determined$ by$ high$ performance$ liquid$ chromatography$ with$ fluorescence$detection.$We$have$tested$the$capacity$ of$ fluorescence$ detection$ (FD)$ as$ a$ cheaper$ alternative$ to$ mass$ spectrometry$ (MS)$ detection.$ Despite$ the$ undeniable$ superiority$ of$ MS$ detectors$ in$terms$of$identification$power,$it$has$been$reported$ on$ several$ occasions$ that$ the$ sensitivity$ to$ compounds$ that$ fluoresce$ significantly$ does$ not$ differ$ much$ between$ the$ two$ instrumental$ techniques$(17,$18).$Therefore,$the$proper$extraction$ and$ isolation$ of$ each$ analyte$ from$ the$ matrix$ play$ fundamental$ roles$ in$ performing$ a$ suitable$ and$ competitive$ analytical$ methodology$ for$ the$ determination$of$these$emerging$pollutants.$ $The$ proposed$ method$ was$ applied$ to$ the$ determination$ of$ target$ compounds$ in$ sewage$ samples$collected$over$one$year$from$a$wastewater$ treatment$ plant$ (WWTP)$ located$ in$ Las$ Palmas$ de$ Gran$Canaria$(Spain).$ ( 2. Experimental( 2.1.$Chemicals$and$reagents( $High$ performance$ liquid$ chromatography$ (HPLC)2 grade$methanol,$acetonitrile$and$water$were$used$to$ dissolve$ the$ standards$ and$ to$ prepare$ the$ mobile$ phases$ and$ were$ obtained$ from$ Panreac$ Química$ (Barcelona,$Spain).$They$were$filtered$through$a$0.22$ μm$ acetate$ membrane$ filter.$ HPLC2grade$ glacial$ acetic$acid$was$used$to$adjust$the$pH$of$the$mobile$ phase$and$samples$and$was$purchased$from$Scharlau$ Chemie$ S.A.$ (Barcelona,$ Spain).$ Ultra2high2quality$ water,$obtained$by$a$Milli2Q$(Millipore,$Bedford,$MA,$ USA)$water$purification$system,$was$used$in$the$solid$ phase$extraction$(SPE)$protocol.$ $All$ of$ the$ standards$ were$ purchased$ from$ Sigma$ Aldrich$(Madrid,$Spain).$Individual$compounds$were$ used$ as$ standards$ (≥$ 98%$ of$ purity)$ for$ short$ ethoxylated$ chains$ APnEOs$ (n$ ≤$ 2),$ 17β2estradiol,$ oestrone,$ estriol,$ 17α2ethynylestradiol$ and$ bisphenol2A.$The$stock$solutions$(1000$µg$·∙$mL21)$of$ alkylphenols,$ steroidal$ hormones,$ and$ bisphenol2A$ were$prepared$by$dissolving$appropriate$amounts$of$ the$ commercial$ products$ in$ methanol$ and$ then$ storing$ in$ glass2stoppered$ bottles$ at$ 220ºC$ prior$ to$ use.$ Nonylphenol$ monoethoxylate,$ nonylphenol$ diethoxylate,$ octylphenol$ monoethoxylate$ and$ octylphenol$ diethoxylate$ were$ prepared$ in$ stock$ solutions$(1$ml)$at$10$µg$·∙$mL21$in$acetone$and$were$ also$stored$at$220ºC.$ $The$ cartridges$ (6$ mL)$ employed$ in$ this$ study$ were$ Sep2Pak$ Vac$ C18$ (500$ mg)$ from$ Waters$ (Madrid,$ Spain).$A$Varian$Vac$Elut$20$SPE$Manifold$(Varian$Inc,$ III.3.$T.Vega$Morales$et$al./$Journal$of$AOAC$International$(en$prensa)$ 141$ CA,$ USA)$ coupled$ to$ a$ Sartorius$ vacuum$ pump$ was$ used$for$the$extractions.$ $ 2.2.$Sample$collection$ $ $To$ test$ the$ applicability$ of$ the$ method,$ primary,$ secondary$and$final$effluent$samples$were$collected$ from$one$of$the$major$wastewater$treatment$plants$ in$Las$Palmas$de$Gran$Canaria$(Spain).$Treatment$at$ the$monitored$WWTP$relies$on$settling$and$flotation$ (primary$ treatment)$ and$ biological$ treatment$ with$ activated$ sludge$ (secondary$ treatment).$ Samples$ were$collected$quarterly$from$July$2009$to$July$2010,$ acidified$at$pH$<$3$to$prevent$the$loss$of$analytes$by$ abiotic$ reactions,$ such$ as$ hydrolysis,$ and$ by$ biological$ degradation$ (19),$ stored$ at$ 4ºC$ in$ glass$ bottles$and$extracted$within$24$hours.$ $Prior$ to$ the$ extraction,$ wastewater$ samples$ were$ filtered$through$0.65$ and$ 0.45$μm$glass$fibre$filters$ (GF/F)$ (Millipore,$ Bedford,$ MA,$ USA),$ respectively.$ The$ 0.452μm2pore$ size$ was$ used$ to$ separate$ the$ dissolved$and$particulate$phases$(20).$Therefore,$the$ particulate$phase$analyzed$only$contains$particles$in$ the$ range$ 0.4520.65$ µm$ since,$ above$ 0.65$ µm$ the$ particles$may$tend$to$settle$quickly,$becoming$part$of$ the$solid$fraction$(sludges$and$sediments)$and$below$ 0.45$µm,$it$is$considered$that$the$particulate$matter$ becomes$part$of$the$dissolved$phase$of$the$samples$ (20).$ $ 2.3.$Extraction$ $ $The$ EDCs$ were$ isolated$ from$ the$ dissolved$ phase$ using$solid$phase$extraction$(SPE).$The$ Sep2Pak$ Vac$ C18$cartridge$was$conditioned$with$5$mL$of$methanol$ and$ 5$ mL$ of$ Milli2Q$ water$ at$ a$ flow2rate$ of$ 5$ mL$ ·∙$ min21$ before$each$ run.$A$ sample$ volume$ of$ 250$mL$ was$passed$ through$ the$ C18$sorbents$ regardless$ of$ the$type$of$sample$under$study.$The$flow$rate$for$the$ sample$extraction$was$kept$constant$between$5$and$ 10$mL$·∙$min21$using$a$vacuum$manifold.$The$cartridge$ was$ washed$ twice$ with$ 5$ mL$ of$ a$ Milli2Q$ water/methanol$ (5%$ (v/v))$ solution$ to$ remove$ any$ remaining$ impurities.$ Subsequently,$ the$ cartridge$ was$ dried$ under$ vacuum$ for$ 10$ minutes$ and$ the$ retained$analytes$were$eluted$at$a$low$flow$rate$(~$1$ mL$⋅$min21)$ with$ two$ portions$ of$ 1$ mL$ of$ methanol$ prior$ to$ analysis$ with$ LC2FD.$ Blanks$ were$ run$ to$ confirm$ the$ carryover$ absence.$ The$ optimisation$ of$ the$ whole$ SPE$ protocol$ has$been$ previously$ described$by$Vega2Morales$et$al$(12).$$ $The$ retained$ particulate$ from$ the$ 0.45$ μm$ glass$ fibre$ filters,$ with$ amounts$ varying$ between$ 0.103$ and$ 0.286$ g,$was$ immersed$ in$ an$ ultrasonic$ bath$ with$ 10$ mL$ of$ methanol$ for$ 10$ minutes.$ $ Then$ the$ particulate$ was$ collected$ in$ a$ flask,$ evaporated$ to$ dryness$ under$ a$ gentle$ stream$ of$ nitrogen$ and$ reconstituted$ with$ 250$ µL$ of$ methanol.$ Then,$ to$ purify$the$sample,$the$extract$was$diluted$with$10$mL$ of$ Milli2Q$ water$ (1/40$ methanol/water,$ v/v)$ and$ passed$ through$ a$ Sep2Pak$ Vac$ C18$ cartridge$ using$ the$ aforementioned$ protocol$ before$ the$ quantification$by$LC2FD.$ $ $ 142$ $Based$ on$ the$ volume$ of$ the$ filtered$ samples,$ the$ concentrations$(i.e.,$µg$·∙$L21)$were$calculated$for$the$ particulate$phase.$Before$the$extraction,$the$weights$ of$ the$ particles$ on$ the$ filters$ were$ measured;$ therefore,$ the$ particulate$ phase$ concentrations$ are$ also$ reported$ on$ a$ weight$ basis$ (i.e.,$ ng$ ·∙$ g21).$ This$ extract$ was$ injected$ separately,$ and$ the$ concentrations$ of$ the$ dissolved$ and$ particulate$ phases$are$reported$separately$for$each$sample.$$ $$ 2.4.$LC<$FD$analysis$ $ 2.4.1.$Instrumentation$and$chromatographic$ conditions$ $ $The$ chromatography$ system$ consists$ of$ a$ Varian$ pump$ fitted$ with$ a$ Varian$ Autosampler$ 410$ with$ a$ volume$selector$and$a$Column$Valve$Module$with$an$ internal$ oven.$ Detection$ of$ the$ samples$ was$ performed$ on$ a$ Varian$ Fluorescence$ detector.$ The$ system$ and$ data$ management$ were$ controlled$ by$ Star$software$from$Varian$(Varian$Inc.,$CA,$USA).$ $The$column$was$a$Phenomenex$Luna$C8$(5μm,$250$ mm$ ×$ 4.6$ mm)$ preceded$ by$ a$ Phenomenex$ guard$ column$ (4$ mm$ length$ ×$ 3$ mm$ i.d.)$ (Phenomenex,$ Torrance,$CA,$USA)$of$the$same$packing$material.$For$ separation$and$quantification$of$the$EDCs,$a$gradient$ elution$ profile$ with$ a$ mobile$ phase$ consisting$ of$ methanol$ and$ water$ was$ employed.$ The$ gradient$ profile$consisted$of$a$linear$increase$from$55:45$(v/v)$ methanol:water$to$75:25$(v/v)$methanol:water$for$5$ min,$ followed$ by$ an$ increase$ in$ methanol$ to$ 90%$ over$ 25$ min.$ The$ column$ oven$ temperature$ was$ 30ºC,$ the$ flow$ rate$ was$ 1$ mL$ ·∙$ min21,$ and$ the$ injection$ volume$ was$ 30$ μL.$ $The$ fluorescence$ detector$ operated$ at$ an$ excitation$ wavelength$ of$ 228$ nm$ and$ an$ emission$ wavelength$ of$ 315$ nm.$$ Linear$ range$ was$ set$ from$ 0.1$ to$ 500$ μg$ ·∙$ L21,$ obtaining$correlation$coefficients$over$0.991$for$all$of$ the$analytes$under$study.$ $ 3. Results(and(discussion( $ 3.1.$Optimisation$of$ultrasonic$assisted$extraction$ (UAE)$processes$ $ $ $The$ optimisation$ of$ UAE$ for$ particulate$ phase$ samples$ included$ the$ evaluation$ of$ several$ experimental$ variables$ to$ achieve$ the$ maximum$ extraction$efficiency$for$each$compound.$Hence,$the$ type$ of$ extraction$ solvent,$ the$ extraction$ time,$ the$ solvent$ volume,$ the$ sample$ dilution$ and$ the$ final$ clean$up$were$optimised.$To$perform$this$procedure,$ spiked$samples$(10$μg$⋅$g21$in$approximately$0.2$g$of$ dry$ particulate$ weight)$ were$ employed.$ An$ extraction$volume$of$10$mL$of$organic$solvent$and$an$ extraction$time$of$15$minutes$were$used$initially.$ $ 3.1.1.$Choice$of$extraction$solvent$ $ $Two$ polar$ solvents,$ methanol$ (ε’$ =$ 33)$ and$ acetonitrile$ (ε’$ =$ 37),$ and$ a$ mixture$ of$ 1/1$ (v/v)$ methanol/water$were$used.$Similar$peak$areas$were$ III.3.$T.Vega$Morales$et$al./$Journal$of$AOAC$International$(en$prensa)$ 143$ observed$ for$ all$ compounds$ after$ using$ either$ methanol$ or$ acetonitrile,$ obtaining$ slightly$ higher$ results$ for$ more$ polar$ compounds$ using$ methanol$ and$slightly$higher$results$for$less$polar$compounds$ using$ acetonitrile.$ Thus,$ given$ that$ both$ solvents$ showed$ similar$ properties$ and$ considering$ that$ methanol$ was$ employed$ for$ the$ solid$ phase$ extraction$ protocol,$ we$ opted$ to$ carry$ out$ this$ process$ with$ methanol.$ Moreover,$ methanol$ has$ been$ employed$ for$ the$ extraction$ of$ the$ same$ compounds$ in$ particulates$ in$ other$ works$ (21),$ offering$ better$ extraction$ recoveries$ than$ acetonitrile$ or$ even$ a$ mixture$ of$ hexane/acetone$ (1/1,$v/v)$for$the$NP$and$OP.$$The$addition$of$water$ to$ the$ extraction$ solvent$ was$ completely$ rejected$ since$ the$ peak$ areas$ obtained$ for$ this$ experiment$ were$ significantly$ lower$ than$ those$ obtained$ with$ the$pure$organic$solvent.$$ $ 3.1.2.$Extraction$time$ $ $With$ the$ motive$ of$ obtaining$ the$ optimal$ time$ of$ extraction,$ four$ different$ values$ were$ estimated,$ covering$an$initial$range$from$1$min$to$15$min$(2,$5,$ 10$ and$ 15$ min.).$ Figure$ 1$ shows$ the$ peak$ areas$ obtained$ for$ each$ value$ of$ time$ estimated$ in$ this$ study.$$ $As$the$extraction$time$increased$from$2$to$10$min,$ the$peak$area$of$each$compound$also$increased.$This$ effect$ was$ less$ pronounced$ for$ more$ polar$ compounds,$ which$ showed$ better$ extraction$ recoveries$ at$ faster$ rates$ than$ the$ less$ polar$ compounds.$ From$ 10$ to$ 15$ minutes$ there$ were$ no$ significant$increases$in$the$peak$area$values$for$any$ compound$in$this$study.$For$this$reason,$we$selected$ 10$min$as$the$optimum$extraction$time.$ $ 3.1.3.$Solvent$volume$ $ $Four$different$methanol$ volumes$ (5,$10,$15$and$20$ mL)$were$tested$to$obtain$the$optimal$value.$$From$5$ to$ 10$ mL,$ a$ general$ increase$ in$ the$ value$ of$ the$ normalised$ peak$ area$ was$ observed$ for$ all$ compounds.$ From$ 10$ to$ 20$ mL,$ the$ values$ of$ normalised$ areas$ remained$ constant$ or$ slightly$ decreased.$As$a$result,$and$to$minimise$the$amount$ of$ organic$ solvent$ used,$ due$ to$ its$ high$ cost$ and$ toxicity,$we$chose$10$mL$as$the$optimum$volume$for$ the$extraction.$ $ 3.1.4.$Dilution$of$the$extract$and$clean$up$$ $ $For$the$cleanup$and$preconcentration$of$the$extract,$ we$ selected$ the$ previously$ optimised$ SPE$ process$ described$ by$Vega2Morales$et$ al$(12),$ which$ was$ employed$ in$ wastewater$ samples$ for$ the$ same$ analytes.$ However,$ it$ is$ necessary$ to$ know$ how$ affects$ the$ dilution$ of$ the$ extract$ in$ different$ volumes$of$water$on$the$retention$of$the$analytes$in$ the$ SPE$ cartridges.$ To$ achieve$ this$ goal,$ various$ spiked$samples$with$the$same$concentration$of$EDC$ (1$ µg$ of$ each$ analyte)$ were$ prepared$ in$ various$ volumes$of$Milli2Q$water$(2.5,$5,$10,$25,$100$mL),$to$ which$ was$ added$0.250$ mL$ of$ methanol$ to$ obtain Capítulo)IV:)Conclusiones) ! 246!IV.)Conclusiones! ! a.4. La! automatización! de! la! SPE! mediante! un! sistema! “On>Line”! incrementó! notablemente!los!niveles!de!preconcentración,!redujo!el!tiempo!necesario!para! realizar!el!procedimiento!extractivo!y!de!purificación,!y!mejoró!notablemente!la! repetitividad!(RSDs)!de!los!análisis!gracias!a!la!minimización!de!las!operaciones! manuales!durante!el!procedimiento.! ! ! a.5. La! técnica! de! extracción! asistida! por! ultrasonidos! (UAE),! empleada! para! “lixiviar”!a!los!analitos!del!material!particulado!retenido!en!los!filtros!de!0.45! μm,! se! reveló! como! un! procedimiento! rápido,! de! bajo! coste,! y! altamente! efectivo!para!extraer!a!estos!contaminantes!de!dicha!fracción.! ! a.6. En! comparación! con! otras! técnicas! convencionales,! las! metodologías! optimizadas!para!la!extracción!de!EDCs!mediante!MAE\SPE!y!MAE\On\Line\SPE! aportaron! una! mayor! rapidez! a! los! análisis! y! unos! límites! de! detección! que! resultaron!adecuados!para!la!determinación!de!dichos!contaminantes!en!lodos! de!EDARs.! ! ! a.7. Los! parámetros! analíticos! obtenidos! en! cada! una! de! las! metodologías! desarrolladas!en!la!presente!Tesis! resultaron!satisfactorios!para!su!utilización! en! la! determinación! de! muestras! reales,! obteniéndose! límites! de! detección! comprendidos!entre!0.3\7.6!ng!·∙!L\1,!1.4\21.4!ng!·∙!g\1,!y!0.1\3.5!ng!·∙!g\1,!en!la!fase! disuelta,! material! particulado! y! lodos! activos,! respectivamente.! Las! desviaciones! estándares! relativas! obtenidas,! incluyendo! todos! los! EDCs! y! los! diferentes!tipos!de!muestras!en!estudio,!fueron!inferiores!al!14.7!%.!A!su!vez,! Capítulo)IV:)Conclusiones) ! IV.)Conclusiones!247! ) las! recuperaciones! obtenidas! mediante! la! optimización! de! los! distintos! métodos!se!hallaron!entre!el!60%!y!el!110%.! !De! acuerdo! a! lo! expuesto! anteriormente,! podemos! concluir! que! Las! metodologías! desarrolladas! para! la! determinación! de! EDCs! en! muestras! asociadas! a! EDARs! se! mostraron! como! procedimientos! de! fácil! aplicación,! rápidos,!y!lo!suficientemente!sensibles!como!para!determinar!la!presencia!de! estos! contaminantes! en! la! fase! disuelta,! material! particulado! y! lodos! activos! procedentes!de!tres!estaciones!depuradoras!localizadas!en!la!región!noreste!de! la!isla!de!Gran!Canaria!(España).!!! ! b))De)la)aplicación)de)las)metodologías)a)muestras)reales:) b.1. Los! niveles! encontrados! para! cada! familia! de! compuestos! y! en! cada! tipo! de! matriz!en!estudio!se!hallaron!entre!los!siguientes!intervalos:!! \ En) la) fase) disuelta:! Alquilfenoles! (3.1! \! 110.0! ng! ·∙! L\1),! alquilfenoles! polietoxilados!(56.6!\!958.9!ng!·∙!L\1),!hormonas!esteroideas!(1.9!\!65.4!!!ng!·∙! L\1),!y!bisfenol\a!(3.9!\!39.5!ng!·∙!L\1).! \ En)el)material)particulado:!Alquilfenoles!(5.0!\!1693.3!ng!·∙!g\1),!alquilfenoles! polietoxilados! (16.6! \! 618.3! ng! ·∙! g\1),! hormonas! esteroideas,! y! bisfenol\a! (3.7!\!174.9!ng!·∙!g\1).! \ En) los) lodos) activos:! Alquilfenoles! (5.8! \! 383.1! ng! ·∙! g\1),! alquilfenoles! polietoxilados!(19.5!\!942.7!ng!·∙!g\1),!hormonas!esteroideas!(1.3!\!127.5!ng!·∙! g\1),!y!bisfenol\a!(0.6!\!19.9!ng!·∙!g\1).! ! Capítulo)IV:)Conclusiones) ! 248!IV.)Conclusiones! ! b.2. La!monitorización!de!surfactantes!alquilfenólicos!polietoxilados!(APnEOs)!en!la! fase!disuelta!de!las!diferentes!etapas!de!tratamientos!de!las!EDARs!reveló!una! degradación! de! la! concentración! total! de! estas! sustancias! mayor! al! 90%.! No! obstante,!la!pérdida!progresiva!de!unidades!etoxiladas!a!medida!que!el!proceso! de! tratamiento! avanzaba,! terminaba! por! liberar! a! los! alquilfenoles! usados! como! base! (nonilfenol! y! octilfenol),! los! cuáles! son! más! tóxicos,! más! estrogénicos,! y! mas! persistentes! que! las! cadenas! largas.! De! tal! forma,! se! observaron!mayores!concentraciones!de!ambos!alquilfenoles!en!los!efluentes! de!las!EDARs!que!en!las!aguas!“brutas”!de!entrada.! ! b.3. Las! hormonas! esteroideas! (tanto! sintéticas! cómo! naturales),! así! como! el! bisfenol\a,! estuvieron! presentes! en! la! fase! disuelta! de! la! mayoría! de! las! muestras!analizadas.!En!la!mayoría!de!los!casos!se!observaron!degradaciones! de! estas! sustancias! entre! el! 70%! y! el! 90%,! lo! cual! denota,! por! un! lado,! la! incapacidad!de!las!EDARs!en!estudio!para!degradar!a!dichos!compuestos,!y!por! otro,! la! necesidad! de! tratamientos! terciarios! específicos! para! alcanzar! una! eliminación!completa.! ! ! b.4. La!naturaleza!altamente!lipofílica!de!la!mayoría!de!los!EDCs!estudiados!quedó! patente!en!la!determinación!de!la!presencia!de!estos!compuestos!en!la!mayoría! de! las! muestras! sólidas! analizadas.! Las! concentraciones! observadas! en! el! material! particulado! y! en! los! lodos! activos! fueron! bastante! similares,! hallándose!en!el!rango!comprendido!entre!unidades!de!ng!·∙!g\1!y!unidades!de! μg!·∙!g\1.! Capítulo)IV:)Conclusiones) ! IV.)Conclusiones!249! ) ! b.5. Se! observó! que! más! de! el! 60! %! de! la! cantidad! total! de! los! compuestos! más! estrogénicos!(p.e.! 17α\etinilestradiol,! dietilestilbestrol,!nonilfenol! y! octilfenol)! se! hallaba! asociada! al! material! particulado! presente! a! las! muestras,! lo! cuál! indica! la! importancia! medioambiental! del! análisis! de! EDCs! en! las! fracciones! sólidas.! ! b.6. En!cuanto!a!la!evolución!temporal!registrada!durante!los!más!de!dos!años!de! muestreo! se! puede! decir! que,! en! la! mayoría! de! los! casos,! no! fue! posible! determinar! fluctuaciones! evidentes! que! se! relacionaran! con! los! parámetros! físicos! asociados! a! variaciones! estacionales,! ó! con! los! volúmenes! y! características!de!las!aguas!de!entrada!y!tratamientos!biológicos!empleados!en! las! diferentes! EDARs,! observándose! una! gran! aleatoriedad! en! los! resultados! obtenidos.!! ) c))De)la)estimación)de)la)actividad)estrogénica:) c.1. A! pesar! de! que! la! aplicación! de! las! diferentes! metodologías! analíticas! mostraron!concentraciones!realmente!bajas!de!los!EDCs!analizados!(del!orden! de!unidades!de!ng!·∙!L\1!y!ng·∙!g\1!en!la!mayoría!de!los!casos),!el!estudios!de!la! actividad! disruptora! endocrina,! realizado! mediante! el! cálculo! de! las! concentraciones!de!estradiol!equivalentes!(EEQs),!reveló!que!la! gran!mayoría! de!las!muestras!analizadas!poseían!la!capacidad!suficiente!para!ejercer!efectos! negativos!sobre!la!salud!de!los!organismos!expuestos.! Capítulo)IV:)Conclusiones) ! 250!IV.)Conclusiones! ! ! c.2. Este! tipo! de! análisis! también! demostró! que! los! compuestos! disruptores! endocrinos!que!ejercían!una!mayor!influencia!sobre!los!potenciales!endocrinos! eran!la!hormona!natural!17β\estradiol!en!la!fracción!disuelta,!y!las!hormonas! sintéticas!17α\etinilestradiol!y!dietilestilbestrol!en!las!fracciones!sólidas.!! ! ! c.3. Se!observó! que! las! fracciones! sólidas!representan!un! riesgo! potencial! mucho! más!elevado!que!el!estimado!para!las!fases!disueltas,!registrándose!valores!de! EEQs! para! las! primeras! entre! 10! y! 50! veces! superiores! a! los! obtenidos! en! muestras!líquidas.! ! c.4. Aunque! dicho! valores! de! EEQs! sobrepasan! notablemente! el! nivel! de! concentración! mínimo! establecido! para! catalogar! una! muestra! como! potencialmente! estrogénica! (1! ng! E2! ·∙! L\1),! los! fenómenos! físicos! (dilución,! fotooxidación,! adsorción! etc.),! químicos! (reacciones! con! halógenos,! transformaciones,! oxidación,! metilación,! etc.)! y! biológicos! (degradación! bacteriana)! reducen! significativamente! la! actividad! disruptora! endocrina! a! la! cual! están! realmente! expuestas! las! poblaciones! y! sub\poblaciones! animales,! incluyendo!los!seres!humanos.!!! ! ! Anexos' ! Anexos!251! ' ! ! ! ! ! ! ! ! ! ! ! ! ! Anexos' ! ! ! ! ! ! ! ! ! ! Anexos' ! 252!Anexos! ! ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' Anexos' ! I.'Acrónimos!253! ' ' I. Acrónimos' ' ACN:'Acetonitrile.' APCI:'Atmospheric'pressure'chemical'ionization'interface.' APnECs:'Alkylphenol'carboxylated'derivatives.' APnEOs:'Alkylphenol'polyethoxylates.'' APs:'Alkylphenols.' AR:'Androgenic'receptor.' ASE:'Accelerated'solvent'extraction.' AST:'Activated'sludge'treatment.' BBAs:'Biological'based'assays.! BPA:'Bisphenol>a.' CAPEC:'Carboxylated'alkylphenol'ether'carboxylate.' CAR:'Carboxen.' CAS:'Conventional'activated'sludge.' DAD:'Diode>array'detector.' DCM:'Dichloromethane.' DDT:'Dichlorodiphenyltrichloroethane.' DES:'Diethylstilbestrol.' DPs:'Degradation'products.' E1:'Estrone.' E2:'17β>estradiol.' E3:'Estriol.' EAU:'Extracción'asistida'por'ultrasonidos.' EC:'Electrochemical'detector.' EDARs:!Estaciones'depuradoras'de'aguas'residuales.' Anexos' ! 254!I.'Acrónimos! ! EDCs:'Endocrine'disrupting'compounds.' EE:'17α>ethynylestradiol.' EEFs:'Estradiol'equivalency'factors.' EEQs:'Estradiol'equivalent'concentrations.' EFSA:'European'food'safety'agency.' EI:'Electronic'impact.' ELISA:'Enzyme'linked'inmunosorbent'assays.' ELSD:'Evaporative'line'scattering'detector.' EO:'Ethoxy'unit.' ERα:!estrogen'receptor.' ESI:'Electrospray'interface.' FD:'Fluorescence'detector.' GC:'Gas'chromatography.' hERα:!Human'estrogen'receptor.! HPLC:'High'performance'liquid'chromatography.' IPA:'Isopropanol.' Koc:'Coeficiente'de'partición.' Kow:'Coeficiente'de'partición'octanol>agua.' LC:'Liquid'chromatography.' LLE:'Liquid>liquid'extraction.' LOD:'Limit'of'detection.' LOQ:'Limit'of'quantification.' MAE:'Microwave'assisted'extraction.' MALDI:'Matrix>assisted'laser'desorption/ionization.' MBR:'Membrane'bioreactor.' MC:'Masa'corporal.' Anexos' ! I.'Acrónimos!255! ' MeOH:'Methanol.' MRM:'Multiple'reaction'monitoring.' MS:'Mass'spectrometry.' MS/MS:'Triple'quadrupole'mass'spectrometry.' NI:'Negative'ionization'mode.' NOG:'Norgestrel.' NP:'Nonylphenol.' NPLC:'Normal>phase'liquid'chromatography.' NPnEOs:'Nonylphenol'polyethoxylates.'' OP:'Octylphenol.' OPnEOs:'Octylphenol'polyethoxylates.'' PA:'Polyacrylate.' PCB:'Polychlorinated'biphenyl.' PCP:'1>(1>phenylcyclohexyl)piperidine).' PDMS:'Polydimethylsiloxane.' PI:'Positive'ionization'mode.' PVC:'Polyvinyl'chloride.' PVPJDVN:'Poly'(n>vinylpyrrolidonedivinylbenzene).' QIT:'Quadrupole'ion'trap.' QTRAP:'Triple'quadrupole/linear'ion'trap'mass'spectrometer.' RPLC:'Reverse'phase'liquid'chromatography.' RSD:'Relative'standard'deviation.' SAESCs:'Sonication>assisted'extraction'on'small'columns.' SBSE:'Stir'bar'sorptive'extraction.' SDB:'Styrene'divinylbenzene.' SFE:'Supercritical'fluid'extraction.' Anexos' ! 262!II.'Lista'de'figuras! ! ultra>resolución' con' espectrometría' de' masas' de' triple' cuadrupolo' para' la' determinación' simultánea' de' compuestos' disruptores' endocrinos' en' muestras' de' aguas'residuales.'' Figure' 1.' Effect' of' the' wash' step' composition' on' the' analyte' retention' into' the' Oasis' HLB' cartridges:' a)' Estriol;' b)' Testosterone;' c)' 17α>ethynilestradiol;' d)' Nonylphenol.' Figure'2.'MRM'chromatograms'of'CAS'final'effluent'sample'spiked'(500'ng'L−1)'with' all' analytes' after' whole' online>SPE' process:' a)' BPA' and' steroids;' b)' Nonylphenol' polyethoxylated'compounds;'c)'Octylphenol'polyethoxylated'compounds.' Supplementay'material:' Figure'1.'Flow'diagram'of' the' On>Line'SPE' system' employed' for' the' parallel' extraction'and'determination'of'all'compounds'from'wastewater'samples.'' Figure'2.'Brief' outline' of' the' entire' process' since' the' sample' is' injected' until' the' cartridge'is're>equilibrated'for'the'next'injection.' ! III.6.'Extracción'asistida'por'microondas'combinada'con'extracción'en'fase'sólida'en' modo'“On>Line”'y'UHPLC>MS/MS'para'la'determinación'de'compuestos'disruptores' endocrinos'en'lodos'de'estaciones'depuradoras'de'aguas'residuales.' Figure'1.'Response'surface' for' the' effect' of'power'and' time' on' the' extraction'of' estriol'(E3).!! Anexos' ! II.'Lista'de'figuras!263! ' Figure'2.'Response'surface' for' the' effect' of'power'and' time' on' the' extraction'of' diethylstilbestrol'(DES).' Supplementary'material:' Figure' 1.' Flow' scheme' of' whole' optimised' MAE>On>Line>SPE>UHPLC>MS/MS' analytical'method.' Figure' 2.' Matrix' suppression' in' the' UHPLC>ESI>MS/MS' analysis' of' norgestrel' in' three'different'matrices'(methanol,'sewage'sludge'and'raw'wastewater'sample)'at' the'same'concentration'level'(50'ng'·∙'g>1).' Figure' 3.' MRM' chromatograms' of' an' spiked' sample' (5' ng' ·∙' g>1)' containing' all' analytes' after' whole' MAE>On>Line>SPE' process' in' both' WWTPs:' a)' Conventional' activated'sludge'samples'(CAS);'b)'Biomembrane'reactor'samples'(MBR).' ! III.7.'Evaluación'de'la'actividad'disruptora'endocrina'en'las'fases'disuelta'y'sólida' de'muestras'de'estaciones'depuradoras'de'aguas'residuales'procedentes'de'la'isla' de'Gran'Canaria'(España).'' Figure'1.'Flow'scheme'of'the'analytical'methodologies'that'have'been'undertaken' for'the'chemical'analysis'of'the'selected'e>EDCs'in'each'fraction.' Figure'2a.'Total'EEQs'obtained'in'the'dissolved'phase'fraction'for'the'three'WWTPs' in'each'sampling'conducted.' Figure'2b.'Contribution'of'each'e>EDCs'to'the'estrogenic'potential'registered'for'the' Station'1'(July'2009'–'July'2010)'in'the'dissolved'fraction.' Anexos' ! 264!II.'Lista'de'figuras! ! Figure'3a.'Total'EEQs'obtained'in'the'sludge'samples'for'the'three'WWTPs'during' the'whole'sampling'period.' Figure'3b.'Contribution'of'each'e>EDCs'to'the'estrogenic'potential'registered'for'the' Station'1'(July'2009'–'July'2010)'in'the'sludge'samples.' Figure' 4a.' Total' EEQs' obtained' in' the' particulate' matter' samples' for' the' three' WWTPs'during'the'whole'sampling'period.' Figure'4b.'Contribution'of'each'e>EDCs'to'the'estrogenic'potential'registered'for'the' Station'1'(July'2009'–'July'2010)'in'the'particulate'matter'samples.' Supplementary'material:' Figure'1.'a)'Canary'Archipelago,'it'is'situated'in'the'Norwest'of'African'Continent.' b)'Locations'of'the'three'WWTPs'under'study'around'Gran'Canaria'Island.' ' ' ' ' ' ' ! ' Anexos' ! III.'Lista'de'tablas'265! ' ' III. Lista'de'tablas' ' Capítulo'I:'Introducción.' ' Tabla! I.1.! Propiedades! físico<químicas! de! varios! EDCs! estudiados! en! esta! Tesis! Doctoral.!a!Coeficientes!de!partición.!b!Solubilidad!a!20º!C.!c!Dato!no!encontrado!en! la!bibliografía.! Tabla! I.2.! Ejemplos! de! concentraciones! de! EDCs! medidas! en! diferentes! tipos! de! matrices!medioambientales.! Tabla!I.3.!Excreciones!diarias!(μg)!de!esteroides!estrogénicos!en!humanos.!a!Calculo! realizado! teniendo! en! cuenta! la! cantidad! 17α<etinilestradiol! presente! en! las! píldoras!anticonceptivas.! Tabla!I.4.!Valores!de!factores!de!equivalencia!de!estradiol!estimados!para!el!17β< estradiol,! estrona,! estriol,! 17α–etinilestradiol,! and! dietilestilbestrol.! a! Factor! de! equivalencia!de!estradiol.! Tabla! I.5.! Diferentes! metodologías! empleadas! para! la! determinación! cuantitativa! de!hormonas!esteroideas!naturales!y!sintéticas.! ! I.2.1.1.' Determinación' de' alquilfenoles' polietoxilados' y' sus' productos' de' degradación'en'muestras'líquidas'y'sólidas.' Anexos' ! 266!III.'Lista'de'tablas! ! Table' 1.' Methods' for' the' determination' of' alkylphenolic' compounds' in' liquid' samples.' Table' 2.' Methods' for' the' determination' of' alkylphenolic' compounds' in' solid' samples.'''''' Table'3.'Methods'for'the'determination'of'alkylphenolic'compounds'by'fluorescence' and'UV'detectors.' ! Tabla! I.6.! Propiedades! físico<químicas! del! bisfenol<a.! a! Valores! medidos! a! temperaturas!de!entre!20<25°!C.! Tabla!I.7.!Parámetros!que!caracterizan!la!partición!del!bisphenol<a.! Table!I.8.!Nivel!de!distribución!medioambiental!haciendo!uso!de!modelo!Mackay! level!I.! Tabla! I.9.! Concentraciones! de! BPA! halladas! en! diferentes! matrices! medioambientales.! Tabla!I.10.!!Diferentes!metodologías!analíticas!empleadas!para!la!determinación!de! BPA! en! varios! tipos! de! matrices! medioambientales.! a! Límite! de! detección.! b! Impacto!electrónico.!c!No!reportado!o!no!encontrado.!e!Estireno<divinilbenceno.!f! Ionización! química! en! negativo.! g! Extracción! asistida! por! microondas.! h! “Stir' bar' sorptive'extraction”.!!!!!!!! Tabla!I.11.!Diseño!factorial!23!empleado!en!la!optimización!de!la!metodología!de! MAE!presentada!en!el!apartado!III.4!de!esta!Tesis!Doctoral.! Anexos' ! III.'Lista'de'tablas'267! ' Tabla!I.12.!Diseño!factorial!32!empleado!en!la!optimización!de!la!metodología!de! MAE!presentada!en!el!apartado!III.6!de!esta!Tesis!Doctoral.! ' Capítulo'III:'Parte'experimental'y'resultados.' ' III.1.' Determinación' de' alquilfenoles' polietoxilados,' bisphenol>A,' 17α> ethinylestradiol,'17β>estradiol'y'sus'metabolitos' en'muestras'de'aguas'residuales' mediante'SPE'y'LC>MS/MS.' Table'1.'Physiochemical'properties'of'the'compounds'studied'[4,6].' Table'2.'Characteristic'of'ESI/MS/MS'parameters'for'each'compound'studied.' Table'3.'Evaluation'of'the'analyte'signal'suppression'in'the'primary'and'secondary' treatments'and'final'effluent.'a'Mean'of'three'determinations.' Table'4.'Method'detection'limits'obtained'in'the'different'matrices:'MDLw'in'Milli>Q' water,'MDLpt'in'primary'treatment,'MDLst' in' secondary' treatment,'and'MDLpm'in' particulate'matter'(primary'treatment).' Table'5.'Recovery'percentages'and'RSD'(%)'(n'='3)'for'different'matrices'spiked'with' 500' ng' mL−1'(dissolved' phase)' or' 10' μg' g−1' (particulate' phase' from' primary' treatment'samples)'of'each'analyte.' Table' 6.' Concentrations' of' each' analyte' in' sewage' from' WWTP:' Cpt' in' primary' treatment,'Cst'in'secondary'treatment,' Cfe' in'final'effluent,'and'Cpm'in'particulate' Anexos' ! 268!III.'Lista'de'tablas! ! matter' (primary' treatment).'a' Mean' of' three' determinations.' b' Not' detected.' c'x' 10−3.' ! III.2.'Determinación'simultánea'de'compuestos'disruptores'endocrinos'en'muestras' de' aguas' residuales' mediante' cromatografía' líquida' de' alta' resolución' con' detección'por'fluorescencia.'''' Table'1.!Final'concentrations'of'each'analyte'in'sewage'from'WWTP:'Cw'in'Milli>Q' water,'Cpt'in'primary'treatment,'Cst'in'secondary'treatment,'Cfe'in'final'effluent,'and' Cpm' in' particulate' matter' (primary' treatment),' a' mean' of' three' determinations,' b' below'detection'limits.'c'no'detected.' ! III.3.'Determinación'y'evaluación'de'compuestos'mimetizantes'del'estradiol'en'las' fases' disuelta' y' particulada' de' muestras' de' estaciones' depuradoras' de' aguas' residuales.' Table'1.'Physiochemical'properties'of'the'compounds'investigated.'' Table' 2.' Analytical' parameters' obtained' by' SPE>LC>FD' and' UAE>SPE>LC>FD.' a' Retention'time'(Rt).'b'Relative'standard'deviation'(RSD).'c'Method'detection'limit' (MDL).d'Method'quantification'limit'(MQL).' Table' 3.' Concentrations' of' each' analyte' in' sewage' from' WWTP:' Cpt' in' primary' treatment.'Cst'in'secondary'treatment.'Cfe'in'final'effluent.'a'Dissolved'phase'(mean' Anexos' ! III.'Lista'de'tablas'269! ' of'three'determinations).'b'Particulate'phase'(mean'of'three'determinations).'c'No' detected.' Table' 4.' Estradiol' equivalency' factors' values' of' estrone,' estriol,' 17α> ethynylestradiol,'bisphenol>A,'octylphenol'and'nonylphenol.' ' III.4.'Determinación'de'varios'compuestos'mimetizadores'del'estradiol'en'lodos'de' estaciones' depuradoras' de' aguas' residuales' mediante' la' combinación' de' la' extracción' asistida' por' microondas' y' la' cromatografía' líquida' con' detección' de' masas'de'triple'cuadrupolo.!!!! Table' 1.' Physiochemical' properties' of' the' compounds' investigated.! a' Data' taken' from'Refs.'[6,7].' Table' 2.' Characteristic' of' ESI/MS/MS' parameters' for' each' compound' studied.' a' Capillary' voltage.' b' Collision' energy' in' brackets.' c' Fragment' ion' used' for' quantitation'(MRM).' Table'3.'Methods'for'the'determination'of'endocrine>disrupting'compounds'(EDCs)' in' solid' samples.' Abbreviations:' DAD,' diode' array' detection;' DCM,' dichloromethane;'FD,'fluorescence'detection;'GC,'gas'chromatography;'LDTD,'laser' diode'thermal'desorption;'MeOH,'methanol.' Table'4.'Partial'and'bivariate'correlations'between'the'variables'under'study.'The' maxima'correlations'are'+1'and'−1.' Anexos' ! 270!III.'Lista'de'tablas! ! Table' 5.' Analytical' parameters' for' the' determination' of' EDCs' under' study' using' MAME>SPE' procedure.' a' n' =' 6.' b' Limit' of' detection.' c' Limit' of' quantification.' d' Relative'standard'deviation.' Table'6.'Concentrations'levels'of'each'analyte'in:'(a)'AST1'and'(b)'AST2'and'MBR' treatment' plants.' a' Mean' and' standard' deviation' of' two' determinations.' b' Concentration'below'of'the'limit'of'detection.' ''! III.5.' Desarrollo' y' optimización' de' una' metodología' analítica' basada' en' la' extracción' en' fase' sólida' tipo' “on>line”' acoplada' a' la' cromatografía' líquida' de' ultra>resolución' con' espectrometría' de' masas' de' triple' cuadrupolo' para' la' determinación' simultánea' de' compuestos' disruptores' endocrinos' en' muestras' de' aguas'residuales.'' Table'1.'Gradient'elution'profiles'employed'in'both'binary'and'quaternary'solvent' manager'pumps.' Table' 2.' Details' of' the' target' compounds' and' their' multiple' reaction' monitoring' (MRM)'parameters'in'UHPLC–MS/MS'under'both'positive'and'negative'ionization' modes.'a'm/z'precursor'ion'[M−H]−.'b'm/z'precursor'ion'[M+NH4]+'adducts.'c'm/z' precursor'ion'[M−H]+.' Table'3.'Analytical'parameters'of'the'online>SPE>UHPLC–ESI>MS/MS'methodology.'a' Retention'time.'b'Relative'standard'deviation'(n'='6).'c'Limit'of'detection.'d'Limit'of' quantification.' Anexos' ! III.'Lista'de'tablas'271! ' Table'4.'Recovery'percentages'and'RSD'(%)'(n'='3)'for'different'matrices'spiked'at' two'different'concentration'levels.' Table' 5.' Evaluation' of' the' analyte' matrix' effects' in' the' primary' and' secondary' treatments' and' final' effluent.' a' Mean' of' three' determinations.' b'Signal' enhancement.' Table' 6a.' Concentration' levels' for' each' target' compound' in' both' WWTPs' under' study'and'at'each'sample'conducted'(May'2011).'a'Mean'and'standard'deviation'of' three'determinations.'b'Concentration'below'the'limit'of'detection.'c'Concentration' below'the'limit'of'quantification.'d'Compound'not'detected.' Table' 6b.' Concentration' levels' for' each' target' compound' in' both' WWTPs' under' study'and'at'each'sample'conducted'(July'2011).'a'Mean'and'standard'deviation'of' three'determinations.'b'Concentration'below'the'limit'of'detection.'c'Concentration' below'the'limit'of'quantification.'d'Compound'not'detected.' Table' 6c.' Concentration' levels' for' each' target' compound' in' both' WWTPs' under' study' and' at' each' sample' conducted' (September' 2011).' a' Mean' and' standard' deviation'of' three' determinations.' b' Concentration' below'the'limit' of' detection.' c' Concentration'below'the'limit'of'quantification.'d'Compound'not'detected.' Supplementay'material:' Table'1.'Physiochemical'properties'of'the'compounds'investigated.'a'Octanol/water' partition'coefficients.' Anexos' ! 278! ! ! ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' Anexos' ! V.'Comunicaciones'a'congresos!279! ' ' V. Comunicaciones'a'congresos' ' 1)'' 'Título:' “Optimization' and' development' of' HPLC>ESI>MS/MS' method' for' the' simultaneous' determination' of' alkylphenol' ethoxylates' and' their' degradation' products'in'coast'sewage'water'samples”.' ' Autores:'T.'Vega>Morales,'Z.'Sosa>Ferrera,'J.J.'Santana>Rodríguez.' 'Congreso:'II'International'Symposium'in'marine'sciences'(ISMS'2009).' 'Lugar'de'celebración:'Vigo,'España.'' ' Fecha:'Abril'2009.' ' 2)'' Título:'“Analysis' of' alkylphenol' ethoxylates' and' their' degradation' products' in' coast'sewage'samples'using'SPE>HPLC>ESI>MS/MS'method”.' ' Autores:'T.'Vega>Morales,'Z.'Sosa>Ferrera,'J.J.'Santana>Rodríguez.' Congreso:'34th' International' Symposium' on' HPLC' and' Related' Techniques' (HPLC'2011).' 'Lugar'de'celebración:'Dresden,'Alemania.' ' Fecha:'Junio'2009.' ' 3)'' Título:'“Determination'of' Alkylphenolic' Ethoxylated'Surfactants' (APEOs),' their' Degradation' Products' (DPs)' and' Bisphenol' A' (BF>A)' by' SPE>HPLC>ESI>MS/MS' method'in'coast'sewage'water'samples”.' ' Autores:'T.'Vega>Morales,'Z.'Sosa>Ferrera,'J.J.'Santana>Rodríguez.' 'Congreso:'EuroAnalysis'2009.' 'Lugar'de'celebración:'Innsbruck,'Austria.'' ' Fecha:'Septiembre'2009.' ! 4)'' Título:'“Determination' of' alkylphenol' polyethoxylates,' bisphenol>a,' 17α> ethynylestradiol' and' 17β>estradiol' and' its' metabolites' in' particulate' matter' Anexos' ! 280!V.'Comunicaciones'a'congresos! ! from' sewage' water' samples' by' Ultrasonic' Assisted' Extraction' and' HPLC>ESI> MS/MS”.' ' Autores:'T.'Vega>Morales,'Z.'Sosa>Ferrera,'J.J.'Santana>Rodríguez.' Congreso:'7º'ANQUE'International'Congress.'Integral'Water:'Present'a'future'a' shared'commitment.' 'Lugar'de'celebración:'Oviedo,'España.'' ' Fecha:'Junio'2010.' ' 5)'' Título:'“Simultaneous' determination' of' endocrine' disrupting' chemicals' in' wastewater' samples' by' High' Performance' Liquid' Chromatography' with' Fluorescence'Detection'(HPLC>FD)”.' ' Autores:'T.'Vega>Morales,'Z.'Sosa>Ferrera,'J.J.'Santana>Rodríguez.' Congreso:'XIV' International' Symposium' on' Luminescence' Spectrometry' (ISLS' 2010).! 'Lugar'de'celebración:'Praga,'República'Checa.' Fecha:'Julio'2010.' ' 6)'' Título:'“Simultaneous' determination' of' endocrine' disrupting' chemicals' in' wastewater'treatment'plant'sludge'samples'by'Microwave'Assisted'Extraction' and'LC>ESI>MS/MS”.' ' Autores:'T.'Vega>Morales,'Z.'Sosa>Ferrera,'J.J.'Santana>Rodríguez.' 'Congreso:'28º'International'Symposium'on'Chromatography'(ISC'2010).' 'Lugar'de'celebración:'Valencia,'España.'' ' Fecha:'Septiembre'2010.' ' 7)'' Título:'“Assesment'of'the'presence'of'some'endocrine>disrupting' chemicals' in' sewage'sludge'samples'using'a'MAE>SPE>LC>MS/MS'method”.' ' Autores:'T.'Vega>Morales,'Z.'Sosa>Ferrera,'J.J.'Santana>Rodríguez.' Anexos' ! V.'Comunicaciones'a'congresos!281! ' 'Congreso:'3rd' International' Congress' Smallwat11:' Wastewater' in' small' communities.' Towards' the' Water' Framework' Directive' (WFD)' and' the' Millennium'Development'Goals'(MDG).' 'Lugar'de'celebración:'Sevilla,'España.'' ' Fecha:'Abril'2011.' ' 8)'' Título:'“On>Line'SPE>UPLC>MS/MS'methodology'for'the'selective'determination' of'several'endocrine'disrupting'compounds'in'sewage'samples”.' ' Autores:'T.'Vega>Morales,'Z.'Sosa>Ferrera,'J.J.'Santana>Rodríguez.' Congreso:'36th' International' Symposium' on' HPLC' and' Related' Techniques' (HPLC'2011).' 'Lugar'de'celebración:'Budapest,'Hungría.'' ' Fecha:'Junio'2011.' ' 9)'' Título:'“Simultaneous' determination' of' Endocrine' Disrupting' Chemicals' in' Wastewater' Treatment' Plant' samples' by' On' Line>Solid' Phase' Extraction' and' UPLC>ESI>MS/MS”.! ' Autores:'T.'Vega>Morales,'Z.'Sosa>Ferrera,'J.J.'Santana>Rodríguez.' 'Congreso:'13ª'Jornadas'de'Análisis'Instrumental! 'Lugar'de'celebración:'Barcelona,'España.'' ' Fecha:'Noviembre'2011.' ' 10)'' Título:'“Optimisation'and'development'of'a'Microwave'Assisted'Extraction' coupled'to'On>Line'Solid'Phase'Extraction'with'UPLC>MS/MS'methodology'for' the'determination'of'Endocrine>Disrupting'Chemicals'in'sewage'sludge' samples”.' ' Autores:'T.'Vega>Morales,'Z.'Sosa>Ferrera,'J.J.'Santana>Rodríguez.' Congreso:'“Hyphenated'techniques'in'sample'preparation'(HTSP>12)'and' Hyphenated'techniques'in'chromatography'(HTC>12)”.' 'Lugar'de'celebración:''Brujas,'Bélgica.' ' Fecha:'Enero'2012.' Anexos' ! 282!V.'Comunicaciones'a'congresos! ! ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' Anexos' ! V.'Comunicaciones'a'congresos!283! ' ' ' Figura!A.1.!II!International!Symposium!in!Marine!Sciences!(ISMS!2009)!(Vigo,! España,!2009).! Anexos' ! 284!V.'Comunicaciones'a'congresos! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Anexos' ! V.'Comunicaciones'a'congresos!285! ' ! ! Figura!A.2.!HPLC!2009!<!34th!International!Symposium!on!High!Performance!Liquid! Phase!Separations!and!Related!Techniques!(Dresden,!Alemania,!2009).! ! Anexos' ! 286!V.'Comunicaciones'a'congresos! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Anexos' ! V.'Comunicaciones'a'congresos!287! ' ! ! Figura!A.3.!EUROANALYSIS!2009!(Innsbruck,!Austria,!2009).! ! ! Anexos' ! 294!V.'Comunicaciones'a'congresos! ! ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' Anexos' ! V.'Comunicaciones'a'congresos!295! ' ' ' Figura!A.7.!3rd!International!Congress!Smallwat11:!Wastewater!in!small!communities.! Towards!the!Water!Framework!Directive!(WFD)!and!the!Millennium!Development! Goals!(MDG)!!(Sevilla,!España,!2011).! ! Anexos' ! 296!V.'Comunicaciones'a'congresos! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Anexos' ! V.'Comunicaciones'a'congresos!297! ' ! ! Figura!A.8.!HPLC!2011!<!36th!International!Symposium!on!High!Performance!Liquid! Phase!Separations!and!Related!Techniques!(Budapest,!Hungría,!2011).! Anexos' ! 298!V.'Comunicaciones'a'congresos! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Anexos' ! V.'Comunicaciones'a'congresos!299! ' ! ! Figura!A.9.!13as!Jornadas!de!Análisis!Instrumental!(Barcelona,!España,!2011).! ! ! Anexos' ! 300!V.'Comunicaciones'a'congresos! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Anexos' ! V.'Comunicaciones'a'congresos!301! ' ! ! Figura!A.10.!Hyphenated!techniques!in!sample!preparation!(HTSP<12)!and!Hyphenated! techniques!in!chromatography!(HTC<12)!(Brujas,!Bélgica,!2012).! Anexos' ! 302!V.'Comunicaciones'a'congresos! ! ! !