In recent years, scientific concern, public debate and media attention has risen about the environmental presence of natural and synthetic compounds due to the observation that environmental concentrations in the low ng.l-1 and ng.g-1 level may affect human and wildlife. The aim of this doctoral thesis was to develop and to evaluate analytical approaches for the detection of estrogens en chlorotriazines, two important groups of endocrine disrupting chemicals (EDCs), in different environmental matrices, e.g. water, sediments, suspended solids and biota. More specifically, the aim was to develop analytical methods for the detection of these compounds in the low ng.l-1 and ng.g-1 level. Environmental concentrations in an estuarine ecosystem like the Scheldt estuary (Belgium-The Netherlands) were established. In chapter I, the issue of endocrine disruption is introduced and the recent developments in environmental chemistry are discussed. This doctoral thesis was carried out within an interdisciplinary research project, Endis-Risks. This 4 year project focussed on the distribution, exposure and effects of endocrine disrupting chemicals in the Scheldt estuary. In this doctoral research, the emphasis is laid on the estrogens and the chlorotriazine herbicides. In order to understand the possible risks of the environmental occurrence of these compounds, their chemistry, their use, their potential for endocrine disruption and their environmental occurrence is described. Also the legislation in relation to their environmental occurrence and their possible abuse in veterinary practice is introduced. In short, the risk assessment procedure is presented. Finally, the conceptual framework and the outline of this doctoral thesis are formulated. In chapter II.1., the development and validation procedure of an analytical method that enables the routine analysis of (estuarine) water samples for estrogens (E1, E2, E3 and EE2) in concentrations in the low ng.l-1 range is described. The method included extraction of water samples using solid phase extraction disks and detection with gas chromatography coupled to multiple mass spectrometry in the electron impact mode (GC-EI-MS-MS). Method performance characteristics (according to 2002/657/EC), e.g. trueness, recovery, calibration, precision, accuracy, limits of detection and quantification and the compound stability are presented for each of the targeted estrogens. Quantification limits of 0.25 ng.l-1 are achieved. Application of this Belac accredited procedure to water samples from the Scheldt estuary revealed that E1 was detected most frequently. In chapter II.2. the results of a 4-year (2002-2005) research on the occurrence of E1, E2 and EE2 in the Scheldt-estuary are presented. Chemical analysis of the water samples was performed using Speedisk extraction as described in chapter II.1.. Suspended matter, sediment and biota samples were extracted with Accelerated Solvent Extraction (ASE). Chromatographic analysis of the extracts was carried out with GC-EI-MS-MS. Detected concentrations were in the low ng.l-1 or ng.g-1 range. In all matrices considered, E1 and E2 were detected, whereas concentrations of E3 and EE2 were below the limit of quantification (LOQ). E1 was observed most frequently and at the highest concentrations. This research demonstrates that estuarine waters and associated sediments and suspended solids are contaminated with the same estrogens which were found within the same concentration range as those reported for freshwater and wastewater. In chapter II.3., a case-study is described concerning the detection of estrogens in an unknown aqueous sample found during a raid. Therefore, different analytical techniques were combined. At first, in vivo techniques (mice trials with anatomical and histological research) were carried out by administration (orally and subcutaneously) of the unknown water solution. To determine the estrogenic potency an in vitro technique, the yeast estrogenic screen, YES was used. After extraction of the water sample (based on the method described in chapter II.1.) and HPLC-fractionation, analysis of the extracts was performed using both Liquid Chromatography and Gas Chromatography coupled to multiple Mass Spectrometry (LC- and GC-MS-MS). The result of this investigation was the detection of both E1 and EE2 at low ng.l-1 concentrations. Chapter II.4. describes the development of an analytical routine approach for the determination of low ng.l-1 levels of EGAs (Estrogens, Gestagens, Androgens) and corticosteroids in aqueous preparations (i.e. drinking water, drinking water supplements) brought on the ‘black’ market. For this, after speedisk extraction of the samples (see chapter II.1.), HPLC fractionation was performed using a methanol:water gradient program (see chapter II.3.). GC-EI-MS-MS screening was used for the EGAs, whereas GC-NCI-MS was used for the screening for corticosteroids. Confirmation of the latter (when dexamethasone (Dxm) or betamethasone (Btm) was suspected) was done by LC-ESI-MS-MS. The combined use of GC and LC coupled to MS enabled the identification and quantification of anabolics and corticosteroids at the low ng.l-1 level. In this chapter 2 case-studies are described of the detection of medroxyprogesterone acetate (MPA) and Btm at low ng.l-1 levels in unknown aqueous solutions. In chapter III the occurrence of the chlorotriazine herbicides atrazine, simazine and terbutylazine in water, sediment and suspended matter over a period of 4 years (2002-2005) is described. Atrazine was found to be the herbicide exhibiting the highest concentration in the different targeted matrices. Simazine and terbutylazine were detected at lower concentrations. Traces of the targeted pesticides were detected in sediments, but these were below the LOQ. As part of an ecotoxicological assessment, the potential acute effect (mortality) and chronic effects (growth, molting) of atrazine on the mysid Neomysis integer (Crustacea:Mysidacea), a resident invertebrate in the Scheldt estuary were studied. This study concluded that atrazine could be toxic to mysids at high concentrations, but at present environmental concentrations (as measured in the Scheldt estuary), acute or chronic effects will probably not result in mortality or decline of the mysid population. In chapter IV, general conclusions and future research perspectives are formulated. Summarising, the major goal of this doctoral thesis was the development of analytical approaches to detect EDCs in different complex matrices at ng.l-1 and ng.g-1 levels. |
