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Dit rapport beschrijft de effecten en milieu-chemie van binaire combinaties van cadmium, zink en koper op de reproduktie van twee wormsoorten (Eisenia andrei en Enchytraeus crypticus) in een standaardtoets met OECD-kunstgrond. Het grote verschil tussen aquatische en terrestrische systemen is de aanwezigheid van de vaste bodemfase, die een belangrijke rol speelt bij sorptieprocessen, die voor een belangrijk deel de biobeschikbare fractie van een stof bepalen. Door de toxiciteitswaarnemingen te interpreteren met totale en extraheerbare metaalconcentraties, wordt de invloed van binding aan de bodemmatrix op de respons van bodemorganismen gekwantificeerd. Tevens zijn extraheerbare concentraties versus totaalconcentraties met Freundlich-isothermen bestudeerd. Effecten op de reproduktie worden met het toxic unit model en het isobologram-model beschreven. Alle toegepaste metaalcombinaties hebben een minder dan concentratie-additief effect op de reproduktie van beide wormsoorten. Dat betekent dat de metalen elkaars toxiciteit deels opheffen of onafhankelijk van elkaar werken. De EC50-waarden voor de toegepaste metaalmengsels hebben een grootte van 1.5 tot 3 Toxic Units. Dat betekent dat er 1.5 tot 3 keer zo veel metaal nodig is om 50 % effect te bereiken als verwacht wordt op grond van de concentratie-effect curves van de individuele metalen. Bij gebruik van extraheerbare concentraties verschuift het combinatie-effect in de richting van concentratie-additie (EC50 = ca. 1 TU). Dit duidt op interacties op het niveau van de bodemmatrix. Milieu-chemisch heeft cadmium -het meest toxische metaal- een hogere beschikbaarheid dan zink en is daardoor in de extracten van het mengsel steeds sterker vertegenwoordigd. Deze verschuiving van de cadmium/zink-ratio kan een veranderende respons teweeg brengen. Voor competitie tussen metalen om sorptieplaatsen in de bodem is geen eenduidig bewijs gevonden. Voor normstelling van mengsels van metalen lijkt concentratie-additie vooralsnog een veilige benadering. De factor 100 voor de afleiding van streefwaarden voldoet hierin. Bij de huidige interventiewaarden wordt geen rekening gehouden met combinatie-effecten, terwijl juist bij hoge concentraties meer competitie op bodem-chemisch niveau verwacht wordt, omdat er verzadiging van sorptieplaatsen optreedt. Door deze verzadiging komt er relatief meer metaal beschikbaar. Door bij interventiewaarden concentratie-additie toe te passen (concentraties van toxicanten worden als fractie van hun interventiewaarde uitgedrukt) kan een evenwichtiger beoordeling van verontreinigde locaties plaatsvinden. In dit rapport is een aanzet voor de te hanteren methodiek gegeven.

This thesis shows the effects that the addition of low molecular weight surfactants (LWMS) to proteins has on the foam stability of the mixture. For this, the bulk, interfacial, thin liquid films and foam properties are determined for different protein-LWMS mixtures at different molar ratios (MR). It was shown that the MR as well as the charge of the protein and LMWS determine the foam stability of the mixtures. For all mixtures it was found that the proteins have a select number of high affinity binding sites. So, the concentration of free LMWS in the solution is 0 until a critical MR (MRcr), at which all high affinity binding sites are saturated. Above this MRcr, part of the LMWS binds to low affinity binding sites of the proteins. The low affinity binding sites have a binding ratio < 1, which determines the concentration of bound and free LMWS. For similarly charged protein-LMWS mixtures (i.e. b-lactoglobulin (BLG) and sodium dodecyl sulphate (SDS) and bovine serum albumin (BSA) and SDS at pH 7) the foam stability typically decreases from the foam stability of the pure protein solution (MR 0) until MRcr is reached. At MR > MRcr the foam stability is dominated by the amount of free LMWS. For oppositely charged protein-LMWS mixtures, the binding of the LMWS to the proteins can be described in a similar way, although the number of high affinity sites and low affinity binding ratio are different. There is also a regime of MRs in which the protein-LMWS complexes form large aggregates. These aggregates were in some cases found to increase foam stability (lysozyme (LYS) and SDS and BLG-SDS at pH 3), while in another case (BLG and cetyltrimethylammonium bromide (CTAB)) they lead to decreased foam stability. Still, in all cases it was found that above MRD the aggregates dissociate and the foam stability becomes dominated by free surfactants, equivalent to what was observed for similarly charged protein-LMWS mixtures. A multi-scale model was developed to describe the stability of thin liquid films in terms of rupture time and thickness. Initially, the model was used to predict the stability of surfactant free films of water and electrolyte solutions. Later, it was used to predict the foam stability in LYS-SDS mixtures. For that purpose, the model was combined with a foam drainage model to provide theoretical estimations of foam stability. This model is the basis to understand coalescence of bubbles in foam. Finally, the concept of the critical MRs and the free LMWS was introduced. Using this, the foam properties of protein-LMWS mixtures can partly be predicted by relative charge of the components and the binding to both high and low affinity binding sites.

Fibrillar structures are important structuring elements for food products. Understanding the behaviour of fibrillar structures in complex food systems is essential for successful industrial applications. This thesis presents the behaviour of two different fibrillar structures, i.e. whey protein isolate (WPI) fibrils and bacterial cellulose (BC) microfibrils in mixtures under various conditions. The WPI fibrils are prepared from WPI and the BC microfibrils are extracted from commercial available ‘Nata de Coco’ by high-energy de-agglomeration. In Chapter 1, a general introduction is given, where we introduce two different fibrillar structures that were studied in this thesis. Also, the aim and the outline of the thesis are presented. In Chapter 2, 3, 4 and 5, the behaviour of mixtures containing WPI and BC microfibrils under different conditions are investigated. By varying the concentration ratios, pH, NaCl concentration and further applying heating treatment, their physico-chemical properties in mixed solutions, mixed solutions after heating and further heat-induced mixed gels are investigated and characterized at both pH 2 and pH 7. In general, both mixing WPI and BC microfibrils without heating and subsequently applying heating treatment lead to stable and homogeneous mixtures at pH 7, as long as BC microfibril concentration is above a critical value. Microscopic images showed that the WPI aggregates and BC microfibrils co-existed in the system. WPI denatured and aggregated in the mixture in the same way as when it is heated alone. Upon gelation, the WPI and BC microfibrils form a duplex gel consisting of two independent and homogeneous networks spanning the whole system. At pH 2, the WPI and BC microfibrils also form stable and homogeneous mixtures in the liquid state, both before and after heating. Microscopic images showed two fibrillar structures that are uniformly and independently present. Upon gelation at higher WPI concentration, a bi-fibrillar gel is formed consisting of a WPI fibrilllar gel and BC microfibrillar gel that co-exist. In Chapter 6 and 7, the behaviour of WPI fibrils at pH 2 in dispersions containing spheres, i.e. emulsions and polystyrene latex dispersions are studied. When WPI and spheres are both positively charged (i.e. WPI-stabilized emulsion), we observed depletion flocculation and depletion stabilization when the WPI fibril concentration increases. When WPI and the spheres are oppositely charged (i.e. polystyrene latex dispersions), bridging flocculation and steric/electrostatic stabilization were observed at low WPI fibril concentration, followed by depletion flocculation and depletion stabilization upon increasing WPI fibril concentrations. In Chapter 8 the stability of emulsions at pH 2 in the presence of only BC microfibrils and in the presence of both BC microfibrils and WPI fibrils was studied. When only BC microfibrils added at a sufficiently high concentration, the emulsions are stabilized by the presence of a yield stress as generated by the BC network. When both WPI fibrils and BC microfibrils are added to the emulsions, the networks they form behave in the same way, as when they are added to the emulsions separately. The WPI fibrils induced depletion flocculation and stabilization of the emulsions, despite the presence of the BC microfibrils. However, at high enough BC microfibril concentrations, the emulsions can be stabilized against depletion flocculation as induced by the WPI fibrils. The competition between stabilization and/or de-stabilization induced by the BC microfibrils and the WPI fibrils can lead to emulsions with different microstructures and rheological properties. A general discussion on the results obtained in this thesis is presented in Chapter 9, which includes recommendations for further research and concluding remarks.

This thesis shows the effects that the addition of low molecular weight surfactants (LWMS) to proteins has on the foam stability of the mixture. For this, the bulk, interfacial, thin liquid films and foam properties are determined for different protein-LWMS mixtures at different molar ratios (MR). It was shown that the MR as well as the charge of the protein and LMWS determine the foam stability of the mixtures. For all mixtures it was found that the proteins have a select number of high affinity binding sites. So, the concentration of free LMWS in the solution is 0 until a critical MR (MRcr), at which all high affinity binding sites are saturated. Above this MRcr, part of the LMWS binds to low affinity binding sites of the proteins. The low affinity binding sites have a binding ratio < 1, which determines the concentration of bound and free LMWS. For similarly charged protein-LMWS mixtures (i.e. b-lactoglobulin (BLG) and sodium dodecyl sulphate (SDS) and bovine serum albumin (BSA) and SDS at pH 7) the foam stability typically decreases from the foam stability of the pure protein solution (MR 0) until MRcr is reached. At MR > MRcr the foam stability is dominated by the amount of free LMWS. For oppositely charged protein-LMWS mixtures, the binding of the LMWS to the proteins can be described in a similar way, although the number of high affinity sites and low affinity binding ratio are different. There is also a regime of MRs in which the protein-LMWS complexes form large aggregates. These aggregates were in some cases found to increase foam stability (lysozyme (LYS) and SDS and BLG-SDS at pH 3), while in another case (BLG and cetyltrimethylammonium bromide (CTAB)) they lead to decreased foam stability. Still, in all cases it was found that above MRD the aggregates dissociate and the foam stability becomes dominated by free surfactants, equivalent to what was observed for similarly charged protein-LMWS mixtures. A multi-scale model was developed to describe the stability of thin liquid films in terms of rupture time and thickness. Initially, the model was used to predict the stability of surfactant free films of water and electrolyte solutions. Later, it was used to predict the foam stability in LYS-SDS mixtures. For that purpose, the model was combined with a foam drainage model to provide theoretical estimations of foam stability. This model is the basis to understand coalescence of bubbles in foam. Finally, the concept of the critical MRs and the free LMWS was introduced. Using this, the foam properties of protein-LMWS mixtures can partly be predicted by relative charge of the components and the binding to both high and low affinity binding sites.

Op welke manier kunnen wiskundige modellen worden ingezet bij het ontwerpen van mengsels, en hoe kan dat op een robuuste manier worden gedaan?

This thesis aims to better understand and further improve the relevance and reliabilityof in vitro bioassaysfor a biobased risk characterisation of complex mixtures, with special focus on persistent organic pollutants (POPs) in sediments. In Chapter 1 the importance of complex mixture characterization in modern society is introduced. The methods available, their current advantages and their disadvantages for complex mixture testing are described. With the shift from policy oriented chemical testing towards the inclusion of in vitro bioanalysis, important challenges have to be overcome to ensure a relevant and reliable quantification of the toxic potency of complex mixtures. These challenges are explained in the introduction, including the status of development and validation of those aspects for reliable testing. One of the main advantages that in vitro bioanalysis has to offer is the possibility to quantify the toxic potency of compounds for which chemical analytical methods have not or hardly been developed, for example because standards do not yet exist. Hydroxylated metabolites of POPs are an example of a toxicologically relevant group of compounds that can exert endocrine disrupting effects, but they cannot yet be routinely analysed. A selection of yet unsolved issues are further studied and discussed in this thesis, as outlined in the “approach and structure of the thesis”. In Chapter 2 a meta-analysis is performed to study the occurrence and relevance of hydroxylated (OH) compounds in humans and wildlife. Reported body burdens of halogenated phenolic contaminants (HPCs), including OH-POP in different tissues from humans and wildlife species, are reviewed in relation to the concentration of their putative parent compounds to be able to reveal relevant exposure routes and sub-populations at risk. Highest OH-POP levels were found in blood plasma, and highly perfused and fetal tissues. Plasma concentrations of analysed known HPCs ranged from 0.1-100 nM in humans and up to 240, 454, 800 and 7650 nM for birds, fish, cetaceans and other mammals, respectively. Reported metabolite blood plasma levels also are compared with relevant toxicological threshold concentrations from toxicological studies, and appeared to fully fall within the in vitro (0.05–10000 nM) and in vivo (3-940 nM) effect concentrations reported for OH-POPs. Given the sensitivity of early developmental stages, and information lacking about the general population, it is advisable to determine HPC background blood levels in children and fetal tissue . Given the toxicological relevance of the OH-POPs, Chapter 3 aims at providing solutions to the long standing problem of the in vitro production and analysis of OH-POP metabolite thyroid hormone disrupting (THD) potency via binding to plasma thyroid hormone binding proteins (THBPs). In sediments and for example seafood, the POPs occur as parent compounds that would only become THD after metabolisation (hydroxylation). Several methods have shown the competitive thyroxine (T4) T4 displacement potency of pure metabolites. However, in vitro metabolization of, among others, polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers(PBDEs) followed by in vitro quantification of their potency has encountered drawbacks related to the co-extraction of compounds disturbing the T4-TTR competitive binding assay. The present study identifies and quantifies the major co-extractants, cholesterol and saturated and non-saturated fatty acids (SFA and NSFA), at levels above 20 μM (20 nmol per mg protein in the incubation mixture) following various extraction methods. A new method is presented to in vitro metabolise parent compounds into OH-metabolites followed by selective extraction of metabolites while four-fold reducing co-extraction of the disturbing compounds. In addition a microplate-format non-radioactive fluorescence displacement assay was developed to quantify the TTR binding potency of the metabolites formed. The effectiveness of the in vitro metabolism and extraction of the OH-metabolites of the model compounds CB 77 and BDE 47 was chemically quantified with a newly developed chromatographic method analyzing silylated derivatives of the OH-metabolites and co-extractants. Due to the mentioned improvements, it is now possible to make a dose-response curve up to 50% inhibition with OH-metabolites extracted from bioactivated CB 77 and BDE 47. Without taking the toxic potencies of bio-activated POPs into account with bioanalysis, the hazard and risk posed by POPs will be seriously underestimated. The chapters 4 and 5 are committed to tackle the issues of supramaximal (SPMX) responses and sample extract concentration which are crucial to reliably quantify of the toxic potencies of complex mixtures with in vitro bioassays. A SPMX effect is the phenomenon that compounds induce a maximum response in an assay that is significantly higher than that of the positive control. As the positive control is used to quantify the toxic potency of a sample, this could result in over-estimation of its toxic potency. As this has been most elaborately reported for in vitro estrogenicity assays, a meta-analysis was performed of such assays, compounds and conditions in which the effect is observed (Chapter 4a).For the 21 natural and industrial chemicals that could be identified as SPMX inducers, the culture and exposure conditions varied greatly among and between the assays. Relevant information on assay characteristics, however, sometimes lacked. Diethylstilbestrol (DES), genistein (GEN) and bisphenol A (BPA) were selected to build a database. The meta-analysis revealed that the occurrence of SPMX effects, could be related to a number of specific assay characteristics: 1) the type and concentration of the serum used to supplement the exposure medium; 2) the endpoint used to quantify the estrogenic potency (endogenous or transfected reporter gene), 3) the number of EREs (estrogen responsive elements) used before the reporter gene, and 4) the nature of the promoter’s. There were no indications that solvent concentration in culture, exposure period or cell model influenced the occurrence of SPMX. It is important to understand the mechanism behind this phenomenon because in vitro assays for estrogenicity are used extensively to characterize and quantify the estrogenic potency of compounds, mixtures and environmental extracts. Several SPMX inducers also have been reported to block cellular efflux pumps in vivo and in vitro (Anselmo et al. 2012; Georgantzopoulou et al. 2013). Therefore it was hypothesized that efflux pump blockers present in environmental matrices could increase the internal concentration of bioassay agonists and thus cause the SPMX. In Chapter 4b this hypothesis was tested by adapting a 96-well plate cellular efflux pump inhibition assay (CEPIA) to the H4IIE rat hepatoma cell line used for the DR.Luc reporter gene assay for dioxin-like compounds. The influence of various environmentally relevant efflux pump inhibitors on the 2,3,7,8-tetrachlorodibenzo-p-dioxine (TCDD) response was tested. Under the DR.Luc assay conditions there was no evidence that P-gp efflux pump inhibitors modified or potentiated the activity of TCDD. Neither genistein nor quercetin, two potent SPMX inducers on ER-mediated assays, induced any signal on the DR.Luc assay, nor influenced the luciferase induction by TCDD. Future work should be focused on testing the consequences of efflux pump inhibition with an AhR-agonist which is a P-gp substrate, as this could result in intracellular accumulation of this AhR-agonist. It is standard practice to use a high single stock concentration of extracts to further dilute test concentrations from and perform the analysis. However, a high contaminant load in an extract may oversaturate the solubility of the extracted compounds in carrier solvents and overload the clean-up columns which may reduce the efficiency of polyaromatic hydrocarbons (PAHs) elimination from the extract. These problems may cause respectively under- or over-estimation of the quantified dioxin-like toxic potency. Therefore Chapter 5 focuses on the effects of initial stock concentrations, including sonication assisted dissolution and exposure time, on the quantified dioxin-like potency of cleaned nonpolar sediment extracts. Indeed, more than 20 g sediment equivalents (SEQ)/mL DMSO) as initial stock concentrations resulted in underestimation of bio-TEQ levels in the sediments as observed for cleaned nonpolar sediment extracts from various locations in Luxembourg. An overload of extract on clean-up columns caused an over-estimation of the dioxin-like potency at 24 hours of exposure, probably due to limited removal of PAHs that can induce false positive responses in the in vitro assays. Sonication assisted dissolution of the stock before serial dilution strongly reduced the standard variation of the outcomes. Taking into account the aspects revealed in this study, in addition to already described important issues for quality control, the in vitro bioassays based bio-TEQs can be applied in a comprehensive monitoring program to determine whether sediments comply with health and safety standards for humans and the environment. For the generally applied sediment quality criteria, advices are given about maximum initial stock concentrations to achieve reliable bioassay outcomes. The methods and concepts developed for metabolic activation of compounds in non-polar sediment extracts and in in vitro analysis of the TTR-competitive binding are applied in Chapter 6 to extracts from highly or less contaminated sediments collected in Luxembourg. Nonpolar fractions of sediment extracts were incubated with S9 rat microsomes, and the metabolites were extracted with a newly developed method that excludes most of the lipids to avoid interference in the non-radioactive 96-well plate transthyretin (TTR) competitive binding assay. Metabolic activation increased the TTR binding potency of nonpolar fractions of POP-polluted sediments up to 100 times, resulting in potencies up to 240 nmol T4 equivalents/g sediment equivalent (nmol T4-Eq/g SEQ). Without bioactivation, medium polar and polar fractions also contained potent TTR-binding compounds with potencies from 1.6 to 17 nmol T4-Eq/g SEQ. This demonstrates that a more realistic in vitro sediment THD risk characterization should also include testing ofboth polar and medium polar sediment extracts for THD, as well as bioactivated nonpolar sediment fractions. Without bioactivation THD potency is not observed in nonpolar sediment extracts, although in in vivo experiments PCBs and PBDEs, and not with dioxins or PAHs, have shown to be thyroid hormone disrupting (THD), demonstrating this bio-activation is toxicologically relevant and therefore required for sediment hazard characterisation. Chapter 7 discusses the implications of our results to improve the relevance and reliability of in vitro bioassay applied for risk characterisation of complex mixtures from sediments and other matrices. The evidence obtained to support the relevance of POP bio-activation is considered both from the exposure perspective as well as the toxicity perspective. Various features of the newly developed methods and knowledge acquired within this PhD project are discussed in relation to in vitro bioassay risk characterization of sediments towards a realistic in vitro bioassay-based risk characterization of complex mixtures. Some important aspects for the inclusion of metabolizing systems within in vitro bioassay are discussed. In addition, alternatives to deal with the SPMX effect and the definition of suitable sample amounts to improve in vitro bioassay reliability are offered. The suitability of the developed approach application is considered for the risk characterization of sediments. Furthermore, an analysis is made to decide whether this thesis have made in vitro bioassays more reliable and relevant for risk characterization of complex mixtures. Finally, it provides some concluding remarks and aspects for further applications and research.

This thesis aims to better understand and further improve the relevance and reliabilityof in vitro bioassaysfor a biobased risk characterisation of complex mixtures, with special focus on persistent organic pollutants (POPs) in sediments. In Chapter 1 the importance of complex mixture characterization in modern society is introduced. The methods available, their current advantages and their disadvantages for complex mixture testing are described. With the shift from policy oriented chemical testing towards the inclusion of in vitro bioanalysis, important challenges have to be overcome to ensure a relevant and reliable quantification of the toxic potency of complex mixtures. These challenges are explained in the introduction, including the status of development and validation of those aspects for reliable testing. One of the main advantages that in vitro bioanalysis has to offer is the possibility to quantify the toxic potency of compounds for which chemical analytical methods have not or hardly been developed, for example because standards do not yet exist. Hydroxylated metabolites of POPs are an example of a toxicologically relevant group of compounds that can exert endocrine disrupting effects, but they cannot yet be routinely analysed. A selection of yet unsolved issues are further studied and discussed in this thesis, as outlined in the “approach and structure of the thesis”. In Chapter 2 a meta-analysis is performed to study the occurrence and relevance of hydroxylated (OH) compounds in humans and wildlife. Reported body burdens of halogenated phenolic contaminants (HPCs), including OH-POP in different tissues from humans and wildlife species, are reviewed in relation to the concentration of their putative parent compounds to be able to reveal relevant exposure routes and sub-populations at risk. Highest OH-POP levels were found in blood plasma, and highly perfused and fetal tissues. Plasma concentrations of analysed known HPCs ranged from 0.1-100 nM in humans and up to 240, 454, 800 and 7650 nM for birds, fish, cetaceans and other mammals, respectively. Reported metabolite blood plasma levels also are compared with relevant toxicological threshold concentrations from toxicological studies, and appeared to fully fall within the in vitro (0.05–10000 nM) and in vivo (3-940 nM) effect concentrations reported for OH-POPs. Given the sensitivity of early developmental stages, and information lacking about the general population, it is advisable to determine HPC background blood levels in children and fetal tissue . Given the toxicological relevance of the OH-POPs, Chapter 3 aims at providing solutions to the long standing problem of the in vitro production and analysis of OH-POP metabolite thyroid hormone disrupting (THD) potency via binding to plasma thyroid hormone binding proteins (THBPs). In sediments and for example seafood, the POPs occur as parent compounds that would only become THD after metabolisation (hydroxylation). Several methods have shown the competitive thyroxine (T4) T4 displacement potency of pure metabolites. However, in vitro metabolization of, among others, polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers(PBDEs) followed by in vitro quantification of their potency has encountered drawbacks related to the co-extraction of compounds disturbing the T4-TTR competitive binding assay. The present study identifies and quantifies the major co-extractants, cholesterol and saturated and non-saturated fatty acids (SFA and NSFA), at levels above 20 μM (20 nmol per mg protein in the incubation mixture) following various extraction methods. A new method is presented to in vitro metabolise parent compounds into OH-metabolites followed by selective extraction of metabolites while four-fold reducing co-extraction of the disturbing compounds. In addition a microplate-format non-radioactive fluorescence displacement assay was developed to quantify the TTR binding potency of the metabolites formed. The effectiveness of the in vitro metabolism and extraction of the OH-metabolites of the model compounds CB 77 and BDE 47 was chemically quantified with a newly developed chromatographic method analyzing silylated derivatives of the OH-metabolites and co-extractants. Due to the mentioned improvements, it is now possible to make a dose-response curve up to 50% inhibition with OH-metabolites extracted from bioactivated CB 77 and BDE 47. Without taking the toxic potencies of bio-activated POPs into account with bioanalysis, the hazard and risk posed by POPs will be seriously underestimated. The chapters 4 and 5 are committed to tackle the issues of supramaximal (SPMX) responses and sample extract concentration which are crucial to reliably quantify of the toxic potencies of complex mixtures with in vitro bioassays. A SPMX effect is the phenomenon that compounds induce a maximum response in an assay that is significantly higher than that of the positive control. As the positive control is used to quantify the toxic potency of a sample, this could result in over-estimation of its toxic potency. As this has been most elaborately reported for in vitro estrogenicity assays, a meta-analysis was performed of such assays, compounds and conditions in which the effect is observed (Chapter 4a).For the 21 natural and industrial chemicals that could be identified as SPMX inducers, the culture and exposure conditions varied greatly among and between the assays. Relevant information on assay characteristics, however, sometimes lacked. Diethylstilbestrol (DES), genistein (GEN) and bisphenol A (BPA) were selected to build a database. The meta-analysis revealed that the occurrence of SPMX effects, could be related to a number of specific assay characteristics: 1) the type and concentration of the serum used to supplement the exposure medium; 2) the endpoint used to quantify the estrogenic potency (endogenous or transfected reporter gene), 3) the number of EREs (estrogen responsive elements) used before the reporter gene, and 4) the nature of the promoter’s. There were no indications that solvent concentration in culture, exposure period or cell model influenced the occurrence of SPMX. It is important to understand the mechanism behind this phenomenon because in vitro assays for estrogenicity are used extensively to characterize and quantify the estrogenic potency of compounds, mixtures and environmental extracts. Several SPMX inducers also have been reported to block cellular efflux pumps in vivo and in vitro (Anselmo et al. 2012; Georgantzopoulou et al. 2013). Therefore it was hypothesized that efflux pump blockers present in environmental matrices could increase the internal concentration of bioassay agonists and thus cause the SPMX. In Chapter 4b this hypothesis was tested by adapting a 96-well plate cellular efflux pump inhibition assay (CEPIA) to the H4IIE rat hepatoma cell line used for the DR.Luc reporter gene assay for dioxin-like compounds. The influence of various environmentally relevant efflux pump inhibitors on the 2,3,7,8-tetrachlorodibenzo-p-dioxine (TCDD) response was tested. Under the DR.Luc assay conditions there was no evidence that P-gp efflux pump inhibitors modified or potentiated the activity of TCDD. Neither genistein nor quercetin, two potent SPMX inducers on ER-mediated assays, induced any signal on the DR.Luc assay, nor influenced the luciferase induction by TCDD. Future work should be focused on testing the consequences of efflux pump inhibition with an AhR-agonist which is a P-gp substrate, as this could result in intracellular accumulation of this AhR-agonist. It is standard practice to use a high single stock concentration of extracts to further dilute test concentrations from and perform the analysis. However, a high contaminant load in an extract may oversaturate the solubility of the extracted compounds in carrier solvents and overload the clean-up columns which may reduce the efficiency of polyaromatic hydrocarbons (PAHs) elimination from the extract. These problems may cause respectively under- or over-estimation of the quantified dioxin-like toxic potency. Therefore Chapter 5 focuses on the effects of initial stock concentrations, including sonication assisted dissolution and exposure time, on the quantified dioxin-like potency of cleaned nonpolar sediment extracts. Indeed, more than 20 g sediment equivalents (SEQ)/mL DMSO) as initial stock concentrations resulted in underestimation of bio-TEQ levels in the sediments as observed for cleaned nonpolar sediment extracts from various locations in Luxembourg. An overload of extract on clean-up columns caused an over-estimation of the dioxin-like potency at 24 hours of exposure, probably due to limited removal of PAHs that can induce false positive responses in the in vitro assays. Sonication assisted dissolution of the stock before serial dilution strongly reduced the standard variation of the outcomes. Taking into account the aspects revealed in this study, in addition to already described important issues for quality control, the in vitro bioassays based bio-TEQs can be applied in a comprehensive monitoring program to determine whether sediments comply with health and safety standards for humans and the environment. For the generally applied sediment quality criteria, advices are given about maximum initial stock concentrations to achieve reliable bioassay outcomes. The methods and concepts developed for metabolic activation of compounds in non-polar sediment extracts and in in vitro analysis of the TTR-competitive binding are applied in Chapter 6 to extracts from highly or less contaminated sediments collected in Luxembourg. Nonpolar fractions of sediment extracts were incubated with S9 rat microsomes, and the metabolites were extracted with a newly developed method that excludes most of the lipids to avoid interference in the non-radioactive 96-well plate transthyretin (TTR) competitive binding assay. Metabolic activation increased the TTR binding potency of nonpolar fractions of POP-polluted sediments up to 100 times, resulting in potencies up to 240 nmol T4 equivalents/g sediment equivalent (nmol T4-Eq/g SEQ). Without bioactivation, medium polar and polar fractions also contained potent TTR-binding compounds with potencies from 1.6 to 17 nmol T4-Eq/g SEQ. This demonstrates that a more realistic in vitro sediment THD risk characterization should also include testing ofboth polar and medium polar sediment extracts for THD, as well as bioactivated nonpolar sediment fractions. Without bioactivation THD potency is not observed in nonpolar sediment extracts, although in in vivo experiments PCBs and PBDEs, and not with dioxins or PAHs, have shown to be thyroid hormone disrupting (THD), demonstrating this bio-activation is toxicologically relevant and therefore required for sediment hazard characterisation. Chapter 7 discusses the implications of our results to improve the relevance and reliability of in vitro bioassay applied for risk characterisation of complex mixtures from sediments and other matrices. The evidence obtained to support the relevance of POP bio-activation is considered both from the exposure perspective as well as the toxicity perspective. Various features of the newly developed methods and knowledge acquired within this PhD project are discussed in relation to in vitro bioassay risk characterization of sediments towards a realistic in vitro bioassay-based risk characterization of complex mixtures. Some important aspects for the inclusion of metabolizing systems within in vitro bioassay are discussed. In addition, alternatives to deal with the SPMX effect and the definition of suitable sample amounts to improve in vitro bioassay reliability are offered. The suitability of the developed approach application is considered for the risk characterization of sediments. Furthermore, an analysis is made to decide whether this thesis have made in vitro bioassays more reliable and relevant for risk characterization of complex mixtures. Finally, it provides some concluding remarks and aspects for further applications and research.

In de normstelling wordt voor bepaalde groepen verbindingen rekening gehouden met de totale toxiciteit van een mengsel van stoffen in het milieu. De huidige concepten om hier normen voor af te leiden zijn geevalueerd en getoetst aan de huidige praktijk. Het rapport bevat aanbevelingen om op een consistente manier om te gaan met mengseltoxiciteit in de normstelling.

De eenvoudigste manier om effecten van mengsels van bestrijdingsmiddelen te beoordelen is om de effecten van de individuele stoffen bij elkaar op te tellen (concentratieadditie). In het algemeen laten experimenten zien dat de stoffen elkaars werking niet versterken (geen synergisme). Als er toch sprake is van versterking, is dat effect doorgaans gering. Het concept concentratieadditie is daarom geschikt om de schadelijke effecten van mengsels van bestrijdingsmiddelen te schatten. Dit blijkt uit een overzicht van recente literatuur over de toxiciteit van mengsels van bestrijdingsmiddelen dat het RIVM met het kennisinstituut Alterra heeft gemaakt. De inventarisatie is een update van een analyse uit 2000 en bevestigt het beeld van toen. Het ministerie van VROM wilde als opdrachtgever in kaart brengen welke ontwikkelingen spelen op het gebied van het beoordelen van mengsels van bestrijdingsmiddelen. De studie beschrijft daarom ook methodologische vernieuwingen die de risicoschatting van mengsels kunnen verfijnen.

De eenvoudigste manier om effecten van mengsels van bestrijdingsmiddelen te beoordelen is om de effecten van de individuele stoffen bij elkaar op te tellen (concentratieadditie). In het algemeen laten experimenten zien dat de stoffen elkaars werking niet versterken (geen synergisme). Als er toch sprake is van versterking, is dat effect doorgaans gering. Het concept concentratieadditie is daarom geschikt om de schadelijke effecten van mengsels van bestrijdingsmiddelen te schatten. Dit blijkt uit een overzicht van recente literatuur over de toxiciteit van mengsels van bestrijdingsmiddelen dat het RIVM met het kennisinstituut Alterra heeft gemaakt. De inventarisatie is een update van een analyse uit 2000 en bevestigt het beeld van toen. Het ministerie van VROM wilde als opdrachtgever in kaart brengen welke ontwikkelingen spelen op het gebied van het beoordelen van mengsels van bestrijdingsmiddelen. De studie beschrijft daarom ook methodologische vernieuwingen die de risicoschatting van mengsels kunnen verfijnen.

Hoe behoud je structuurvariatie en menging door kleinschalige ingrepen en natuurlijke verjonging in een oud en zeer gevarieerd bos. Verschillende soorten opstanden in het Zeisterbosch werden bezocht en bediscussieerd

Om na de inzaai een optimaal grasland te verkrijgen verdient het aanbeveling om vooraf een bewuste keuze te maken voor een grasmengsel en de daarin voorkomende soorten, typen en rassen.

De voorspelling die in het kader van klimaatverandering gedaan worden laten zien dat we vaker te maken zullen krijgen met extreme weersomstandigheden. In de zomer worden vaker dan voorheen periodes met hoge temperaturen en kans op watertekorten verwacht, maar ook met extreme neerslag. Voor melkveehouders op droogtegevoelige zandgronden zullen warmere en drogere zomerperioden onrustig zijn om hun graslandproductie op peil te houden, zeker als vanuit waterschappen, rijk of provincie beregeningsverboden worden opgelegd. Het jaar 2006 was een goed voorbeeld. Als gevolg van de droogte mochten boeren in diverse provincies, waaronder Brabant, in juli en augustus niet meer met oppervlaktewater beregenen. Ook het beregenen met grondwater wordt steeds verder aan banden gelegd en zal wellicht in de toekomst in sommige gebieden die kwetsbaar zijn voor wateronttrekking geheel verboden worden. Door deze ontwikkelingen zal het vaker voorkomen dat minder water beschikbaar is dan optimaal voor productiegrasland. Deze nota geeft de eerste resultaten weer van een onderzoek naar het functioneren van ‘alternatieve’ grasmengels op melkveebedrijf ‘De Kleijne’, een bedrijf op droogtegevoelige zandgrond waar geen beregening meer wordt toegepast

Onderzoekers van Plant Research International en PPO-fruit ontdekten combinaties van geurstoffen die door de mannetjes van roze appelluis worden gebruikt om de vrouwtjes te vinden. Geuren spelen een belangrijke rol in het leven van bladluizen, met deze geurmengsels konden de mannetjes in de val worden gelokt

De voorspelling die in het kader van klimaatverandering gedaan worden laten zien dat we vaker te maken zullen krijgen met extreme weersomstandigheden. In de zomer worden vaker dan voorheen periodes met hoge temperaturen en kans op watertekorten verwacht, maar ook met extreme neerslag. Voor melkveehouders op droogtegevoelige zandgronden zullen warmere en drogere zomerperioden onrustig zijn om hun graslandproductie op peil te houden, zeker als vanuit waterschappen, rijk of provincie beregeningsverboden worden opgelegd. Het jaar 2006 was een goed voorbeeld. Als gevolg van de droogte mochten boeren in diverse provincies, waaronder Brabant, in juli en augustus niet meer met oppervlaktewater beregenen. Ook het beregenen met grondwater wordt steeds verder aan banden gelegd en zal wellicht in de toekomst in sommige gebieden die kwetsbaar zijn voor wateronttrekking geheel verboden worden. Door deze ontwikkelingen zal het vaker voorkomen dat minder water beschikbaar is dan optimaal voor productiegrasland. Deze nota geeft de eerste resultaten weer van een onderzoek naar het functioneren van ‘alternatieve’ grasmengels op melkveebedrijf ‘De Kleijne’, een bedrijf op droogtegevoelige zandgrond waar geen beregening meer wordt toegepast

In de normstelling wordt voor bepaalde groepen verbindingen rekening gehouden met de totale toxiciteit van een mengsel van stoffen in het milieu. De huidige concepten om hier normen voor af te leiden zijn geevalueerd en getoetst aan de huidige praktijk. Het rapport bevat aanbevelingen om op een consistente manier om te gaan met mengseltoxiciteit in de normstelling.

Toxicity of mixtures can be taken into account when deriving environmental risk limits. Current concepts for deriving risk limits for mixtures of toxic substances are evaluated. Recommendations are given for a consistent use of mixture toxicity theory in deriving risk limits.

In this thesis the combustion properties of methane / hydrogen mixtures are investigated by measering autoignition delay times in methane/hydrogen mixtures under conditions relevant for gasengines. Moreover HCN and C2H2 measurements have been performed in fuel-rich one dimensional laminar CH4/H2/air flames. All measurements have been compared with numerical calculations.

Op welke manier kunnen wiskundige modellen worden ingezet bij het ontwerpen van mengsels, en hoe kan dat op een robuuste manier worden gedaan?

In this thesis the combustion properties of methane / hydrogen mixtures are investigated by measering autoignition delay times in methane/hydrogen mixtures under conditions relevant for gasengines. Moreover HCN and C2H2 measurements have been performed in fuel-rich one dimensional laminar CH4/H2/air flames. All measurements have been compared with numerical calculations.

In this last chapter I will review and integrate the findings of the previous chapters and give suggestions for further research on this topic.In chapter 2 measurements of depletion interactions by means of colloidal probe atomic force microscopy (CP-AFM) are described. We found that the behaviour of the range of the depletion interaction is roughly in agreement with predictions of Fleer et aI. The strength of the depletion interaction is not in agreement with of predictions of Tuinier et al.2 and Louis3 and Bolhuis.4 It is much smaller. We explain this by the fact that the segment-surface interaction for polydimethylsiloxane (PDMS) and a stearylated silica surface is non zero. Comparison of our experimental values with values obtained by Scheutjens Fleer Self Consisted Field theory for different values of Xs leads to a reduced segment surface interaction parameter of 0.41. This corresponds to a decrease to about one order of magnitude of the maximal depletion interaction (see figure 2.13). This strong weakening is in agreement with experimental findings that significantly more PDMS is needed to reach the colloidal liquid-gas binodal than predicted, even if non ideality of the polymers is taken into account.5· 6In chapter 3 we present observations of wetting phenomena in depletion interaction driven, phase-separated colloidal dispersions. These dispersions consist of stearyl coated silica spheres in cyclohexane with PDMS as non-adsorbing polymer. The dynamic contact angle was determined by moving a thin fibre upward or downward through the colloidal liquid gas interface and drawing tangents to the meniscus at the fibre in the three phase contact line. The dynamic contact angle was measured as a function of the velocity of the fibre. From these dynamic contact angles the static contact angle was determined by interpolation to zero velocity. We observed no noticeable hysteresis. The contact angle of the colloidal liquid-gas interface at a solid substrate (coated glass) was determined for a series of compositions. We were able to locate a Cahn wetting transition7 from partial to complete wetting upon approaching the critical point. By this we have tested successfully the well known Cahn prediction regarding wetting transitions for coloid-polyrner mixtures.In the same way we determined the contact angle of the colloidal liquid-gas interface at a soft solid substrate for a series of compositions. A soft solid substrate was obtained by heating a glass fibre in a melt of PDMS. In this way we manipulated the colloidal particle-substrate interaction. Upon approach to the critical point, a transition occurs from partial to complete drying. The region where the wetting transition occurs was compared with that of density functional calculations of Brader et al. and Monte Carlo simulations of Dijkstra et al.8' 9

In this last chapter I will review and integrate the findings of the previous chapters and give suggestions for further research on this topic.In chapter 2 measurements of depletion interactions by means of colloidal probe atomic force microscopy (CP-AFM) are described. We found that the behaviour of the range of the depletion interaction is roughly in agreement with predictions of Fleer et aI. The strength of the depletion interaction is not in agreement with of predictions of Tuinier et al.2 and Louis3 and Bolhuis.4 It is much smaller. We explain this by the fact that the segment-surface interaction for polydimethylsiloxane (PDMS) and a stearylated silica surface is non zero. Comparison of our experimental values with values obtained by Scheutjens Fleer Self Consisted Field theory for different values of Xs leads to a reduced segment surface interaction parameter of 0.41. This corresponds to a decrease to about one order of magnitude of the maximal depletion interaction (see figure 2.13). This strong weakening is in agreement with experimental findings that significantly more PDMS is needed to reach the colloidal liquid-gas binodal than predicted, even if non ideality of the polymers is taken into account.5· 6In chapter 3 we present observations of wetting phenomena in depletion interaction driven, phase-separated colloidal dispersions. These dispersions consist of stearyl coated silica spheres in cyclohexane with PDMS as non-adsorbing polymer. The dynamic contact angle was determined by moving a thin fibre upward or downward through the colloidal liquid gas interface and drawing tangents to the meniscus at the fibre in the three phase contact line. The dynamic contact angle was measured as a function of the velocity of the fibre. From these dynamic contact angles the static contact angle was determined by interpolation to zero velocity. We observed no noticeable hysteresis. The contact angle of the colloidal liquid-gas interface at a solid substrate (coated glass) was determined for a series of compositions. We were able to locate a Cahn wetting transition7 from partial to complete wetting upon approaching the critical point. By this we have tested successfully the well known Cahn prediction regarding wetting transitions for coloid-polyrner mixtures.In the same way we determined the contact angle of the colloidal liquid-gas interface at a soft solid substrate for a series of compositions. A soft solid substrate was obtained by heating a glass fibre in a melt of PDMS. In this way we manipulated the colloidal particle-substrate interaction. Upon approach to the critical point, a transition occurs from partial to complete drying. The region where the wetting transition occurs was compared with that of density functional calculations of Brader et al. and Monte Carlo simulations of Dijkstra et al.8' 9

In polluted areas organisms are generally exposed to mixtures of toxic chemicals rather than a single toxicant only. Since the number of mixture toxicity studies with regard to soil systems is limited, the research in this thesis was focused on investigating ecotoxicological consequences of combined exposure for soil invertebrates. Two topics were studied: i) population-level consequences of chronic combined exposure of individual organisms, and ii) the influence of interactions between the toxic compounds in the soil matrix on their joint toxicity. The soil dwelling, free living nematode Caenorhabditis elegans was chosen as test species.A quantitative description procedure was proposed for interpreting the joint toxicity of chemical mixtures, compared to the toxicity of the individual components. It enabled the identification of four biologically relevant deviation patterns from either the additive or the independent reference model, by means of likelihood analysis: i) no deviation, ii) synergism/antagonism, iii) toxicant ratio- and iiii) effect level dependent deviation. Using these models, sublethal effects of chronic stress of binary mixtures of copper and cadmium, and copper and carbendazim on life cycle events of the nematode C. elegans were studied. The cadmium-copper effect on reproduction was transient: it changed from synergistic, to a toxicant ratio dependent deviation from additivity. The effect of copper-carbendazim was synergistic at low effect levels and antagonistic at high effect levels. The juvenile period was a relatively non-sensitive parameter, whereas the length of the reproductive period was relatively more sensitive. In conclusion, it should be realised that mixture toxicity may be transient and that interactions may differ among life history traits.It was discussed that the effect translation of combined effects to the population level depended on three factors: i) the sensitivity of each life history trait to each of the toxicants, ii) the combination effect of the toxicants on each life history trait, iii) the sensitivity of to changes in each life history trait. A detailed analysis of mixture effects on the life history of nematodeCaenorhabditis elegans showed that synergistic effects on reproduction were transferred to the population level, despite the low sensitivity of λ to changes in this trait.Subsequently single and combined toxicity of copper-zinc, copper-cadmium, cadmium-lead, copper-carbendazim and copper-carbendazim-iprodione to the nematode Caenorhabditis elegans were studied in LUFA 2.2 soil , and the one-week population increase was estimated as toxicity endpoint. Metals with the highest partition coefficient affected the sorption of metals with the lowest partition coefficient when both were combined. However, comparing soil sorption characteristics with joint toxicity patterns did not give general results. Nevertheless, it was discussed that for identifying fundamental principles of joint toxicity in ecotoxicology, and for developing predictive models, ecological "mechanisms" should be investigated.

Keywords: biopolymer, gelatine, dextran, PEO, phase separation, polydispersity, molar mass distribution, SEC-MALLS, CSLM The temperature-composition phase diagram of aqueous solutions of gelatine and dextran, which show liquid/liquid phase segregation, were explored at temperatures above the gelation temperature of gelatine. It turned out that the compositions of coexisting phases show practically no dependence on temperature between 40oC and 80oC. Also, the total polymer concentration at which phase separation occurred was found to be nearly independent on temperature. These observations suggest an entropy driven phase separation. Phase separation is found to be accompanied by strong fractionation of the molar mass distribution in the two phases. Molar mass distributions in coexisting phases were investigated using Size Exclusion Chromatography with MultiAngle Laser Light Scattering. The molar mass of the native material and concentration appeared to be the only factors that affected the final molar mass distributions, temperature having no effect. The results show that in the molar mass range where fractionation is the strongest, i.e. roughly below the maximum in the distribution, fractionation is governed by a Boltzmann factor , where DG denotes the free energy involved in transferring a polymer with a certain length from the enriched to the depleted phase, and in this case turns out to be proportional to the molar mass. The phase behaviour of aqueous mixtures of poly(ethylene oxide) (PEO) and dextran is also studied. From the temperature dependence we conclude that the phase separation between PEO and dextran is partly caused by sterical interactions. From the equilibrium phase volumes of the phase separated mixture and the shape of the temperature-composition phase diagram of PEO and dextran we conclude that also the decrease of solvent quality of water for PEO at increasing temperatures is involved. It is suggested that the characteristics of the PEO-water interaction can affect the degree of fractionation. This suggestion is based on the observation that the degree of fractionation is not a simple exponential function of the molar mass. Adsorption of high molar mass dextran at the interface between gelatine and dextran during the phase separation of aqueous mixtures of these two polymers is also studied. This adsorption is observed after macroscopic phase separation as well as during the phase separation. In the last case, the system is studied with Confocal Scanning Light Microscopy. For this purpose, the dextran is covalently labelled with fluorescein 5-isothiocyanate (FITC). It turns out that the adsorption of high molar mass dextran is highly affected by the labelling. The adsorption of labelled dextran leads to a stable film between the two coexisting phases, thus preventing dextran droplets to coalesce. If the degree of labelling increases above a certain level, the labelled dextran does not lead to a stable film, but is preferentially present in the gelatine-rich phase.

In polluted areas organisms are generally exposed to mixtures of toxic chemicals rather than a single toxicant only. Since the number of mixture toxicity studies with regard to soil systems is limited, the research in this thesis was focused on investigating ecotoxicological consequences of combined exposure for soil invertebrates. Two topics were studied: i) population-level consequences of chronic combined exposure of individual organisms, and ii) the influence of interactions between the toxic compounds in the soil matrix on their joint toxicity. The soil dwelling, free living nematode Caenorhabditis elegans was chosen as test species.A quantitative description procedure was proposed for interpreting the joint toxicity of chemical mixtures, compared to the toxicity of the individual components. It enabled the identification of four biologically relevant deviation patterns from either the additive or the independent reference model, by means of likelihood analysis: i) no deviation, ii) synergism/antagonism, iii) toxicant ratio- and iiii) effect level dependent deviation. Using these models, sublethal effects of chronic stress of binary mixtures of copper and cadmium, and copper and carbendazim on life cycle events of the nematode C. elegans were studied. The cadmium-copper effect on reproduction was transient: it changed from synergistic, to a toxicant ratio dependent deviation from additivity. The effect of copper-carbendazim was synergistic at low effect levels and antagonistic at high effect levels. The juvenile period was a relatively non-sensitive parameter, whereas the length of the reproductive period was relatively more sensitive. In conclusion, it should be realised that mixture toxicity may be transient and that interactions may differ among life history traits.It was discussed that the effect translation of combined effects to the population level depended on three factors: i) the sensitivity of each life history trait to each of the toxicants, ii) the combination effect of the toxicants on each life history trait, iii) the sensitivity of to changes in each life history trait. A detailed analysis of mixture effects on the life history of nematodeCaenorhabditis elegans showed that synergistic effects on reproduction were transferred to the population level, despite the low sensitivity of λ to changes in this trait.Subsequently single and combined toxicity of copper-zinc, copper-cadmium, cadmium-lead, copper-carbendazim and copper-carbendazim-iprodione to the nematode Caenorhabditis elegans were studied in LUFA 2.2 soil , and the one-week population increase was estimated as toxicity endpoint. Metals with the highest partition coefficient affected the sorption of metals with the lowest partition coefficient when both were combined. However, comparing soil sorption characteristics with joint toxicity patterns did not give general results. Nevertheless, it was discussed that for identifying fundamental principles of joint toxicity in ecotoxicology, and for developing predictive models, ecological "mechanisms" should be investigated.

Keywords: biopolymer, gelatine, dextran, PEO, phase separation, polydispersity, molar mass distribution, SEC-MALLS, CSLM The temperature-composition phase diagram of aqueous solutions of gelatine and dextran, which show liquid/liquid phase segregation, were explored at temperatures above the gelation temperature of gelatine. It turned out that the compositions of coexisting phases show practically no dependence on temperature between 40oC and 80oC. Also, the total polymer concentration at which phase separation occurred was found to be nearly independent on temperature. These observations suggest an entropy driven phase separation. Phase separation is found to be accompanied by strong fractionation of the molar mass distribution in the two phases. Molar mass distributions in coexisting phases were investigated using Size Exclusion Chromatography with MultiAngle Laser Light Scattering. The molar mass of the native material and concentration appeared to be the only factors that affected the final molar mass distributions, temperature having no effect. The results show that in the molar mass range where fractionation is the strongest, i.e. roughly below the maximum in the distribution, fractionation is governed by a Boltzmann factor , where DG denotes the free energy involved in transferring a polymer with a certain length from the enriched to the depleted phase, and in this case turns out to be proportional to the molar mass. The phase behaviour of aqueous mixtures of poly(ethylene oxide) (PEO) and dextran is also studied. From the temperature dependence we conclude that the phase separation between PEO and dextran is partly caused by sterical interactions. From the equilibrium phase volumes of the phase separated mixture and the shape of the temperature-composition phase diagram of PEO and dextran we conclude that also the decrease of solvent quality of water for PEO at increasing temperatures is involved. It is suggested that the characteristics of the PEO-water interaction can affect the degree of fractionation. This suggestion is based on the observation that the degree of fractionation is not a simple exponential function of the molar mass. Adsorption of high molar mass dextran at the interface between gelatine and dextran during the phase separation of aqueous mixtures of these two polymers is also studied. This adsorption is observed after macroscopic phase separation as well as during the phase separation. In the last case, the system is studied with Confocal Scanning Light Microscopy. For this purpose, the dextran is covalently labelled with fluorescein 5-isothiocyanate (FITC). It turns out that the adsorption of high molar mass dextran is highly affected by the labelling. The adsorption of labelled dextran leads to a stable film between the two coexisting phases, thus preventing dextran droplets to coalesce. If the degree of labelling increases above a certain level, the labelled dextran does not lead to a stable film, but is preferentially present in the gelatine-rich phase.