Metodo analitikoen garapena disruptore endokrinoen biokontzentrazioa arrainetan determinatzeko

Autoretza:

Oihana Ros Ibarretxe

Zuzendaria:

Ailette Prieto Sobrino, Asier Vallejo Ruiz

Unibertsitatea:

Euskal Herriko Unibertsitatea

Fakultatea:

Zientzia eta Teknologia Fakultatea

Saila:

Kimika Analitikoa

Jakintza-arloa:

Kimika

Urtea:

2016

Hizkuntza:

Ingelesa

| Ezaguna da ingurumeneko uretan konposatu endokrinoak (EDC) aurkitzen direla nahiz eta oso kontzentrazio baxuetan (ng/L mailan) egon. Araztegien (WWTP) irteera korronteak izan ohi dira konposatu hauen iturririk nagusienetako bat, araztegiek erabiltzen dituzten mekanismoak ez baitira gai EDCen eliminazio osoa burutzeko. EDC konposatuak ingurumeneko uretan bizi diren arrainen sistema endokrinoan hainbat aldaketa sortzeko gai dira, nahiz eta aipaturiko kontzentrazio baxuetan izan. Aldaketa hauen artean, malformazioak, populazioen aldaketak edo intersexualitatea dira nabarmenenak. Tesi lan honetan bi izan dira proposatu diren erronka nagusienak. Alde batetik, EDC desberdinen (Alkil fenolak (AP), bisfenol-A (BPA), estrogenoak, musk fragantziak, ftalato esterrak (PE) eta pestizidak) analisirako teknika sentikorren, sendoen, doien eta zehatzen garapena burutu da ur laginetan (araztegiko irteerako urak eta estuariokoak) eta arrainen atal desberdinetan (burmuina, gibela, plasma, behazuna eta muskulua). Teknika hauen garapenean metodo klasikoak, fase solidoko erauzketa (SPE) adibidez, edo ingurugiroarekiko errespetagarriagoak diren teknikak, polietersulfonaren (PES) bidezko mikroerauzketa edo fase solidoko erauzketa solido-likidoa (FUSLE), erabili dira. Erabilitako metodo hauek, interesezko konposatuak erauzteaz gain, hainbat interferentzia erauzten dituzte eta analisi kromatografikoaren aurretik garbiketa urrats bat beharrezkoa izaten da. Zentzu honetan, ohiko SPE teknikaz gain, fase solidoko erauzketa dispertsoan (dSPE) oinarritutako garbiketa urratsak garatu dira arrainen atal desberdinetarako. Teknika honek, ohiko SPEak baino fase polimeriko gutxiago erabiltzez gain, disolbatzaile organikoen bolumen txikiagoak behar ditu, ingurumenarekiko errespetagarriagoa izanez. Tesi honen bigarren helburua, garaturiko metodologia guztiak euskal kostalde osoan zehar eta kontrolpeko esperimentuetan erabiltzea izan da arrainek konposatuak metatzeko duten joera aztertzeko. Helburu horiek burutzeko, euskal kostaldean zehar (Santurtzin, Gernikan, Ondarroan, Deban eta Pasaian) jasotako lazunen (Chelon labrosus) behazuna aztertu zen APak, BPA, estrogenoak, musk fragantziak, pestizidak eta PEak determinatzeko. Azkeneko bi familia hauen kasuan, hainbat metabolito ere determinatu ziren. Arrainak arrantzatu ziren egun berdinean ur laginak jaso ziren, arrainek konposatuak metatzeko duten gaitasuna ikusteko asmoz. Ingurumenean jasotako arrainetan zaila izaten da hainbat faktore kontrolpean izatea, arrainen homogeneotasuna adibidez. Arazo horri aurre egiteko, laborategiko esperimentuak burutu daitezke ahalik eta faktorerik gehienak kontrolpean izateko. Lan honetan bi izan dira laborategian burutu diren esposizio esperimentuak. Lehenengoan, heldugabeko lazunak araztegiko irteera korronteetan jasotako uretara esposatuak izan ziren behazunean zeuden EDCak determinatzeko. Bigarren esperimentuan aldiz, estrogenoen, APen eta BPAren kontzentrazio ezaguna (150 ng/L) zuen ontzian lupiak (dicentrarchus labrax) 10 egunez mantendu ziren EDCen metaketa egunetan zehar, eta atal desberdinetan (behazuna, plasma, gibela, muskulua eta burmuina) ikusteko asmoz. It is known that endocrine-disrupting chemical (EDC) are found in environmental waters even if at very low level of concentrations (ng / L levels). Wastewater treatment plants (WWTP) are often among the main sources of these compounds, as the mechanisms used by the treatment plants are not able to completely eliminate EDC. EDC compounds are capable of causing various changes in the endocrine system of fish living in environmental waters even at the aforementioned low levels of concentration. Among these changes malformations, population changes and intersexuality are the most prominent. Two main challenges are proposed in this thesis work. Firstly, sensitive, robust, accurate and precise techniques for the analysis of different EDCs – Alkyl phenols (AP), bisphenol-A (BPA), estrogens, musk fragrances, phthalate esters (PE) and pesticides – were developed in water samples (at waste treatment plant exits and in estuaries) and in different parts of the fish (brain, liver, plasma, bile and muscle). During the development of these techniques classical methods – such as solid phase extraction (SPE) – and more environmentally friendly techniques – such as polyethersulfone (PES) micro-extraction and solid-phase solid-liquid extraction (FUSLE) – were used. These methods, in addition to extracting the compounds of interest, extract various interferences, and require a cleaning phase prior to chromatographic analysis. In this regard, in addition to the usual SPE technique, cleaning steps based on dispersive solid phase extraction (DSPE) were developed for different parts of fish. This technique, in addition to using fewer polymer phases than conventional SPEs, requires smaller volumes of organic solvents, making it more environmentally friendly. The second objective of this thesis was to use all the methodologies developed along the Basque coast and in controlled experiments in order to study the tendency of fish to accumulate compounds. To achieve these objectives, the bile (Chelon labrosus) collected along the Basque coast (Santurtzi, Gernika, Ondarroa, Deba and Pasaia) was analysed to determine APs, BPA, estrogen, musk fragrances, pesticides and PEs. In the case of these last two families, several metabolites were also determined. On the same day that the fish were caught, water samples were collected to see if the fish had the ability to accumulate compounds. With fish caught in the environment several factors were difficult to control, for instance the homogeneity of the fish. To address this problem, laboratory experiments can be performed to control as many factors as possible. In this work there were two exposure experiments carried out in the laboratory. In the first, the immature lizards were exposed to water collected in the treatment plant outlet streams to determine the EDCs in their bile. In the second experiment, sea bass (dicentrarchus labrax) was kept in a vessel with a known concentration of estrogen, AP, and BPA (dicentrarchus labrax) for 10 days during the days of EDC accumulation and in different sections (bile, plasma, liver, muscle, and brain).

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