Choose an application

• Reference Manager
• EndNote
• RefWorks (Direct export to RefWorks)

Freshwater systems play a central role in the biogeochemical cycling of carbon (C). They transport organic and inorganic C from the terrestrial biosphere to the oceans, yet this transport of C is not passive, and freshwater systems produce, degrade, or store organic C, and exchange C with the atmosphere. Inland water bodies represent hotspots of C processing: although they cover a limited surface of the Earth, their contribution to the global C cycling is substantial in comparison with marine and terrestrial ecosystems. During the last decade, a new paradigm progressively emerged proposing that respiration of organic matter exceeds autochthonous production in freshwaters ecosystems, meaning that they are predominantly net heterotrophic. This concept seems to hold especially true for oligotrophic, unproductive ecosystems, where the C cycle would be dominated by substantial inputs of allochthonous organic matter of terrestrial origin, which support the growth of heterotrophic organisms. This has important implications, because net heterotrophy has been recognised as one of the main cause for the net emission of CO2 emissions from freshwaters ecosystems to the atmosphere. In contrast, eutrophic ecosystems would tend to be net autotrophic. However, our current knowledge on C dynamics in freshwater ecosystems is still largely of an empirical nature, and hence could be biased because most of the observations were gathered in medium to small systems, located in the temperate and boreal regions of the world. Large (> 250 km²) tropical lakes, especially in Africa, have been consistently undersampled although they differ from temperate lakes in some fundamental characteristics.This study provides evidence that Lake Kivu represents an example of a large tropical lake being net autotrophic, despite the common assumption that oligotrophic freshwater ecosystems are net heterotrophic. The net autotrophic status could be related to specific morphometrical features of Lake Kivu, such as its low catchment-to-surface area ratio that is responsible for the relatively modest allochthonous organic matter inputs from its watershed; but also related to some environmental parameters distinctive of tropical environments, which significantly affect its ecological functioning. This study notably demonstrates that the percentage of phytoplankton production excreted as dissolved organic matter through physiological processes was comparatively higher in oligotrophic tropical lakes than in their temperate counterparts, due to the higher irradiance conditions distinctive of the tropics. The organic molecules freshly excreted by phytoplankton cells (DOCp) were highly labile and rapidly assimilated by heterotrophic prokaryotes. In consequence, the standing stock of phytoplankton-derived organic molecules was very small, and the bulk dissolved organic carbon (DOC) pool was mainly composed of older, more refractory compounds that would reach the mixed layer through vertical advective and diffusive fluxes.Overall, it highlights the ecological importance of C transferbetween the phytoplankton and the bacterioplankton components of the food-web in large tropical lakes. Stable carbon (delta13C) and nitrogen (delta15N) isotope analyses indicate that the phytoplankton-derived autochtonous carbon was channelled up to the zooplankton level of the food-web, throughout the year. The seasonal variability in the delta15N signature of the particulate nitrogen pool in the euphotic zone reflected changes the phytoplankton assemblage structure, with a relatively higher contribution of N2-fixing cyanobacteria during the rainy season. Permanently stratified water bodies, such as Lake Kivu, are characterized by the presence of pelagic gradients in oxygen (oxycline) and redox species (redoxcline), and thus show a clear separation between the oxygenated upper waters rich in electron acceptors (O2, SO42-, NO3-) and the anoxic bottom where electron donors (CH4, H2S, NH4+) are abundant. The pelagic redoxclines are usually an area of intense biogeochemical activities, where chemoautotrophs and methanotrophs derive their energy from the oxidation of reduced species. The results of this study provide evidences for the existence of a biogeochemically active methanotrophic and chemoautotrophic bacterial community in the redoxcline of Lake Kivu. Additionally, phospholipid-derived fatty acid (PLFA) analyses suggest that the bacterial community composition was structured vertically in the water column, with a large dissimilarity between the oxic and oxygen-depleted waters.The vertical variability of the delta13C signature of the particulate organic carbon (POC) pool and methane (CH4) revealed the presence of a consistently abundant methanotrophic bacterial biomass in the oxycline throughout the year. Approximately 4-6% of the vertically-integrated POC pool is derived from CH4-derived carbon on an annual basis, but this contribution locally reached ~50% in the oxycline during the dry season. Indeed, Lake Kivu is well-known for the high amount of CH4 dissolved in its permanently stratified layer of water. However, the emissions of CH4 to the atmosphere was found to be 2 orders of magnitude lower than the estimated upward CH4 flux, suggesting that microbial CH4 oxidation is an important process within the water column. Methanotrophic bacterial production (MBP) rates in Lake Kivu, estimated during the dry and the rainy season, were always the highest at the transition between the oxic and the oxygen-depleted waters. Furthermore, PLFA analyses showed that aerobic methanotrophic bacteria type I (Gammaproteobacteria) dominated the methanotrophic bacterial community. Volumetric chemoautotrophic bacterial production (CBP) rates measured in Lake Kivu were in the same range of values reported from H2S-rich marine redoxclines, such as the Black Sea, the Baltic Sea, and the Cariaco Basin; and as in these systems, the maximal chemoautotrophic activities were observed in sulfidic waters, well below the oxycline. Vertically integrated over the water column, the sum of the measured net MBP and CBP rates (31 - 42mmolC m-2 d-1, depending on season and location) were comparable to the mean phytoplankton particulate primary production (49 mmol C m-2 d-1). This study supports the idea that methanotrophs and chemoautotrophs might play a quantitatively important role in the functioning of permanently stratified tropical lakes. In Lake Kivu, they participate substantially to the O2 consumption in the water column, and hence to the seasonal uplift of the oxycline. They also contribute significantly to the autochtonous primary production, but exert an indirect control on oxygenic photoautotrophs by limiting the vertical nutrient flux to the illuminated surface waters. It is likely that more stable stratification conditions due to climate warming would further reinforce the importance of methanotrophy and chemoautotrophy in the ecosystem functioning of Lake Kivu.

Academic collection --- Theses

Choose an application

• Reference Manager
• EndNote
• RefWorks (Direct export to RefWorks)

Global urbanization has been happening for decades and will continue in the future, converting substantial amounts of natural land cover types into urban surface types. This human-caused global land cover change has induced environmental issues that can affect the quality of urban life, such as heat waves and air pollution. Accordingly, preventing and regulating these urban environmental problems has become a common concern for scientists and policy makers. To this end, obtaining spatially explicit information on the distribution and change of urban land covers may be the key to solving this problem. With global coverage, free access, and frequent observation intervals, medium spatial resolution multispectral satellite imagery has been one of the primary datasets used for mapping urban land covers at various geographical scales. However, distinguishing between spectrally and spatially heterogeneous urban surface types in these image data is challenging due to their limited spectral and spatial resolutions, thus limiting urban land cover mapping accuracy. For the same reason, all continental-scale land cover products developed from medium spatial resolution satellite imagery cannot characterize the spatial distribution of specific urban land covers, such as the urban green space. In this PhD dissertation, we tried to exploit the potential of medium spatial resolution multispectral satellite imagery in urban remote sensing by (i) improving the capability of the image data to distinguish between urban land covers spatially and spectrally and (ii) producing reliable maps of the spatial distribution of urban green spaces at the Europe continental scale.In Chapter-2, we tried to reduce the presence of mixed pixels in urban areas by increasing the spatial resolution of satellite images, thus improving the capability of the image data to distinguish between urban land covers spatially. In particular, given the spatial textures provided by the four Sentinel-2 10 m bands, we proposed an improved unmixing-based image fusion approach (UnFuSen2) to sharpen the six Sentinel-2 20 m bands to 10 m resolution. Compared to traditional unmixing-based image fusion methods, UnFuSen2 can self-adapt to the spectral variability of varying land covers and improve the image fusion accuracy by constraining the unmixing equations on the basis of spectral mixing models and the correlation between spectral bands of coarse and fine spatial resolution, respectively. In parallel, the objective of Chapter-3 was to improve the capability of satellite imagery to distinguish between urban surface covers spectrally. Given that Fisher Discriminant Analysis (FDA) can enhance the interclass spectral separability between samples of different classes and the spectral similarity of samples of the same category, Chapter-3 integrated FDA and Multiple Endmember Spectral Mixture Analysis (MESMA) (F-MESMA) for more accurate mapping of urban land cover. Our experiments demonstrated that compared to other state-of-art data transformation methods, the ratio of within- vs between-class spectral variability of urban land covers was most strongly reduced after applying the FDA. Consequently, F-MESMA consistently provided the most accurate impervious surface fraction estimates across five urban areas (RMSE F-MESMA = 0.13 vs. RMSE alternative approaches = [0.16-0.17]).Furthermore, in Chapter-4, we analyzed the individual and the combined effect of UnFuSen2, FDA, and multi-temporal observed image data on Sentinel-2-based urban land cover mapping accuracy. Our results demonstrated that the classification accuracy of UnFuSen2-processed single-date imagery, FDA-processed single-date imagery, and Sentinel-2 image time series (ITS) was higher than that of the original single-date Sentinel-2 imagery. The classification of the ITS that consists of UnFuSen2-processed single-date images showed the highest average Kappa coefficient (0.7225) compared to the classifications of other datasets.Finally, in Chapter-5, we used a machine learning-based subpixel classification approach to map urban green spaces across Europe from Landsat images in 1990, 2000, and 2015, filling a gap in the accurate extraction of urban green space information at a continental scale using medium spatial resolution multispectral satellite imagery. Our results showed that the modeled urban green area fractions yielded low RMSE values ranging from 0.09 to 0.16 across ten validation urban areas. Meanwhile, our modeled urban green space maps were validated to outperform other land cover products such as CORINE and the Urban Atlas. Based on the obtained urban green space maps, we found: (i) urban green spaces in Western European countries are more spatially concentrated, while those in Eastern and Southern Europe are relatively sparsely distributed; (ii) the green area in urban core areas (the urbanized areas before 1990) remained almost constant between 1990 and 2000 but started to increase noticeably between 2000 and 2015 throughout Europe; (iii) recent urban expansions (the urbanized areas after 1990) contain more urban green space than the increased urban green area in urban core areas from 1990 to 2015; (iv) the urban green area per capita has been increasing in Western, Eastern, and Northern Europe from 1990 to 2015, but has been declining in Southern Europe.

Choose an application

• Reference Manager
• EndNote
• RefWorks (Direct export to RefWorks)

Iron (Fe) oxyhydroxide minerals are ubiquitous in soil and are major sinks for humic substances, trace metals and oxyanions that sorb strongly to these minerals. A small fraction of the Fe oxyhydroxides can also occur as colloids in the soil solution and may act as a so-called nanovector that mobilizes, rather than immobilizes, the substances and ions associated with them. This explains the colloidal transport of components such as phosphate (PO4), arsenate (AsO4) and lead (Pb) that are strongly bound to the Fe oxyhydroxides. The colloidal transport of these components in soil remains largely unknown at a large scale, mainly because there is a lack of appropriate Fe colloid sampling and characterization methods.This work was set up to evaluate different Fe colloid characterization methods and identify the relationships between soil properties and Fe colloid properties, i.e. their size, concentration and sorption characteristics. The rationale is that understanding the properties of the colloidal carrier will ultimately lead to a better assessment of the fate of the numerous elements that are associated with it.We first explored the potential of two innovative techniques for colloid characterization, single-particle ICP-MS (sp-ICP-MS) and Flow Field Flow Fractionation (FlFFF) to size environmental Fe colloids. The FlFFF allowed distinguishing between Fe-organic carbon (OC) complexes and larger mineral colloids; it has a wide size detection range between 1-2 nm and 300 nm and has acceptable element recoveries in an environmentally relevant background. The size obtained with FlFFF corresponds with the hydrodynamic diameter of particles in their aggregated state. The relatively high size detection limit in sp-ICP-MS analysis (about 30-50 nm) compromised sizing of environmentally relevant Fe colloids. The FlFFF coupled to UV/VIS and ICP-MS was therefore the method selected to characterize the Fe colloids in this work.Colloid stability is a prerequisite for transport and natural organic matter (NOM) provides both electrostatic and steric stability to Fe oxyhydroxide colloids. An experimental study was set up to specifically test the role of NOM on Fe colloid formation by coprecipitation. The Fe colloids were prepared by oxidation of Fe(II) with variable concentrations of NOM. Stable colloids were formed over the range in dissolved organic carbon (DOC) to Fe ratios representative for surface-and groundwaters. Moreover, a striking effect of NOM on colloid size and composition was observed. The Fe colloids size increased consistently with decreasing DOC/Fe ratio in the test solutions. Over a wide molar DOC/Fe range (1400-10), the Fe colloid size was very small (< 10 nm), further decreasing the DOC/Fe ratio yielded larger colloids of approximately 50 nm until the limit for colloid stability was reached at DOC/Fe < 2. The speciation of the colloids changed from Fe-OC mononuclear complexes at high DOC/Fe, to polynuclear Fe-OC complexes or ferrihydrite embedded in NOM at intermediate DOC/Fe and to larger Fe colloids with low NOM content that ultimately settle. The effect of NOM is explained as inhibition of growth and crystallization, which was supported by the higher organic matter loading in the smallest particles. Interestingly, the surface loading of NOM on the colloids increased more than proportionally with decreasing size, suggesting a conformational change of the NOM, i.e. more extended in small colloids, explained by the high electrostatic and/or steric repulsion.These findings were subsequently tested for natural samples. First, processes that govern Fe colloid size and composition were inferred from pore waters of 97 different topsoils covering a wide range in soil physico-chemical properties. Soil solution Fe concentrations are governed by colloid stability because they are highest at low soil solution Ca and high dissolved organic carbon (DOC) concentrations. The colloid size remarkably varied among soils, organic carbon was identified as a dominant factor explaining the variation. The fraction of Fe forming mononuclear Fe-OC complexes (< 5 nm) ranged between 1-36% and increased with an increasing DOC/Fe ratio in pore waters. In addition, small mineral Fe colloids (5─50 nm) prevailed in soils with > 3.5% soil organic carbon, while the larger mineral Fe colloids (50─100 nm) were dominant in soils with low soil organic carbon content. In some samples with low DOC content and low Ca concentration, the elemental ratios in mineral colloids suggested the presence of phyllosilicates. In these pore waters, Fe might be associated with clay minerals and are therefore distributed to the larger size range of clays.Second, Fe colloids were characterized in pore waters from a podzol profile to identify the factors that cause the pronounced vertical Fe mobilization and subsequent immobilization in such soils. The pore water Fe concentration increased from the A to the E (eluvial) horizon and peaked in the E horizon, which is remarkable given the low soil Fe concentration in the E horizon. The pore water Fe concentration was positively related to the DOC concentration within the profile. The colloid characterization analysis revealed that the Fe colloids (<100 nm) not only consisted of Fe-OC complexes, the generally accepted cheluvation theory, but also of mineral Fe colloids, presumably Fe oxyhydroxides coated with organic matter. The smallest Fe-OC complexes dominated in the A horizon while the larger mineral colloids raised most importantly with increasing depth, explaining the rise in total Fe towards the Bh (illuvial) horizon. The adsorption of the negatively charged OM at the top of the Bs horizon is likely the main mechanism of DOC retention in the Bh. Below this depth, the DOC was very low resulting in low pore water Fe concentration. Straining is unlikely a significant mechanism for colloid immobilization as judged from the pore size distribution and the colloidal size. This study demonstrates that natural organic matter plays a key role in the transport of Fe colloids in acid, sandy soils with low Ca.These findings highlight the key role of NOM in Fe colloid transport, but also raised the question on the effect of NOM on the reactivity. With decreasing colloid size, the specific surface area (SSA) available for sorption increases and likely also the colloid mobility. However, adsorbed humic substances might lower the affinity of the colloids for binding oxyanions. The aim was therefore to determine the implication of this organic matter coating on the affinity of the Fe colloids to sorb trace metals and oxyanions. This was first addressed in an experimental study by measuring PO4 adsorption and coprecipitation to Fe colloids with varying size due to varying amounts of NOM. In a second step, that interaction was also verified in an observational study on pore water colloids of different soils, thereby analysing colloid size-dependent fractions of compounds with high affinity to either humic substances (Cu) or to Fe oxyhydroxides (VO4 and AsO4). Both studies indicated that the PO4 loading on the colloids (experimental study) and the fractions of colloidal VO4 and AsO4 (pore water observational study) increased with increasing Fe colloid size. These results contradict the notion that higher colloidal size reduces the SSA and sorption. The results point to a size-dependent competition between NOM and oxyanions on the Fe colloid surfaces. The NOM over oxyanion selectivity is highest in the smaller colloids, which we explained by steric and/or electrostatic interactions resulting from the extended conformation of the adsorbed humic substances in the smallest colloids. The Cu loading on the Fe colloids decreased with size together with the NOM, which suggests Cu binding to the Fe colloids via surface adsorbed NOM.Taken together, recent advances in analytical chemistry allow to study environmental colloids down to the low nanometer range with a size resolution that was not possible to attain before. This thesis has provided deeper insight into Fe colloid size and composition and the environmental conditions that govern them. The importance of DOC on increasing soil solution Fe and decreasing Fe colloid size was highlighted several times. These results add to the growing body of research that indicates that NOM inhibites Fe oxyhydroxide colloid formation by forming Fe-OC complexes and retards Fe oxyhydroxide growth and crystallization. Nevertheless, this is the first study to show the marked effect of NOM on Fe colloid size measured in suspension. In addition, this study is first in quantifying the competition between oxyanions and humic substances in suspended colloids. A conceptual model explaining the paradoxal size dependent oxyanion binding was presented. This new understanding can help to improve predictions on the impact of colloidal Fe as a nanovector in the environment.

Choose an application

• Reference Manager
• EndNote
• RefWorks (Direct export to RefWorks)

Cadmium (Cd) is a toxic trace metal with widespread occurrence. The risk of Cd is largely affected by its bioavailability in the environment, i.e. the total Cd concentrations in the environment (soil, water, food,…) are poorly explaining uptake and effects in different biota across different compartments. The complexation of ionic Cd2+ by ligands in solution changes its bioavailability. Two common equilibrium models of bioavailability, the free ion activity model (FIAM) and biotic ligand model (BLM), assume mostly that mainly ionic Cd2+ and not its complexes determine the uptake. These models are widely applied to aquatic and terrestrial organisms. However, uptake of Cd in higher plants was recently shown not to obey this mechanism because labile complexes of Cd enhanced Cd uptake. It was demonstrated that this is related to kinetic limitations, more in particular the process by which labile complexes increase the flux of metals over unstirred layers adjacent to cells, only some tens of micrometer thick.The objective of this study was to assess the validity of equilibrium (FIAM) and/or kinetic models to describe the Cd bioavailability, i.e. Cd uptake in cells under contrasting scenarios. More specifically, this work addressed abiotic and biotic boundary conditions at which the FIAM starts to fail and kinetic considerations have to be invoked. In addition, this work assessed the validity of the equilibrium models in the presence of natural dissolved organic matter (DOM) sampled from different sources and with associated differences in Cd2+complexation properties. Experiments were designed with Caco-2 cells, a model systems for human gut cells and with the freshwater algae Pseudokirchneriella subcapitata (Korschikov). The Cd uptake by Caco-2 cells was higher from a solution in the presence of complexes than from a solution with the same free Cd2+ activity but without complexes, illustrating that the FIAM does not apply. The contribution of the complexes decreased with increasing Cd2+ concentration. At low Cd2+ concentration (1 nM), chloride complexation with Cd2+ forming CdCln2-n contributed to the uptake almost to the same extent as the free ion. At large Cd2+ concentration (10 µM), the contribution of the complexes was much smaller. Modelling suggested that these treatment effects were the result of alleviating the diffusion limitation of the free metal ion to the cell surface over an unstirred layer of about 2 mm.The technique of the algal bottle assay to asses metal uptake by algae was refined to better control Cd speciation during algal growth. A resin-buffered nutrient solution was developed and this was applied to test the effect of chloride (Cl-) on cadmium (Cd) uptake. Standard nutrient solution enriched with 40 mM of either NaNO3 or NaCl contained equal Cd2+ but varying dissolved Cd due to the presence of CdCln2-n complexes. The refined algal bottle test was compared to the traditional algal bottle test by growing green algae in the standard nutrient solutions in the absence (designated ‘-R’) or in thepresence (designated ‘+R’) of a cation exchange resin. The Cd concentrations in solution of the –R treatments decreased with 50-58 % of initial values due to Cd uptake. No such changes were found in the +R treatments. Cadmium uptake was unaffected by either NaNO3 or NaCl treatment in the +R treatment, confirming that Cd2+ is the preferred Cd species in line with the FIAM. In contrast, Cd uptake in the –R treatments was two-fold larger in the nutrient solution with NaCl than in the nutrient solution with NaNO3 in contrast with what FIAM would predict. The effect of synthetic ligands and DOM (20 mg C L-1) on the Cd uptake by algae was assessed with the refined algal bottle assay. Long-term (3 days) Cd uptake was measured in resin buffered solutions with or without synthetic ligands and at three different Cd2+ ion activities (pCd 8.2-5.7). Total dissolved Cd increased up to 35-fold by adding the synthetic ligands at constant Cd2+ activity. In contrast, Cd uptake by algae increased maximally 2.8 fold with increasing concentration of the synthetic ligands and the availability of the complexes were maximally 5.2% relative to Cd2+ for NTA and CDTA complexes. It is concluded that synthetic labile Cd complexes do not greatly enhance Cd bioavailability to the green algae and calculations suggest that Cd transport from solution to these small cells is not rate limiting. Hence, Cd uptake by algae in the presence of synthetic ligands generally obeys the FIAM in a test in which Cd2+ is buffered by a resinNatural dissolved organic matter (DOM) can have contrasting effects on metal uptake in algae because of complexation reactions and because of DOM adsorption to algal surfaces, thereby affecting the metal ion uptake process. Six different DOM samples were collected and isolated from natural freshwater systems and isolated by reverse osmosis and one 13C enriched DOM sample was isolated from soil to identify DOM adsorption to algae. In the presence of the resin, Cd uptake was unaffected by the presence of DOM or increased maximally 1.63-fold. In the absence of the resin, Cd uptake increased by DOM up to factors 2.4 but that was mostly due to the lack of buffering of solution Cd. The 13C analysis revealed that 6% of algal C was derived from DOM. Hence, Cd2+ and not DOM-complexed Cd is the main bioavailable form of Cd as predicted by FIAM, however the lack of instantaneous buffering of Cd2+ , as in resin free systems, may result in DOM enhancing Cd bioavailability in natural systems by acting as a mobile metal carrier locally buffering Cd2+.In summary, equilibrium models such as FIAM and BLM are not valid whenever the free ion activity is not constant in time and space. In such conditions, uptake is better related to the total initial concentrations of labile metal than to the initial free metal ion concentration. The FIAM and BLM are valid models at high, toxic metal concentrations where no such gradients in time and space are present. For small cells such a microalgae, the FIAM applies when the Cd2+ ion isbuffered in the medium because concentration gradients to small cells with high specific surface area are generally small.

Choose an application

• Reference Manager
• EndNote
• RefWorks (Direct export to RefWorks)

The widespread contamination of soils with trace metals such as zinc (Zn), cadmium (Cd), nickel (Ni) or copper (Cu) can have an adverse ecological effect. The evaluation of the toxic effects of metals in soils has almost exclusively been estimated from toxicity studies with single metal dosing. As a rule, metals occur as mixtures in a contaminated environment, e.g. Cd is usually enriched where higher Zn concentrations are found. The premise in the current assessment is that risks can be excluded provided that all metal concentrations are below their corresponding limits. This assumption is questioned and has been the starting point of this work. A mixture of different metals can have a larger effect than each metal individually, e.g. because the effects of each metal add up or because metals can even interact and act synergistically. There is a need to understand how metals act together in producing combination effects, especially at low effect concentrations and at a large number of metals in the mixture. It is predictable that metals do interact since they compete for sorption, uptake in biota, translocation and detoxification.The general objective of this thesis was to analyze low level metal mixture effects in soil grown plants. The scientific questions were (i) to identify which of two reference mixture models to predict the toxic effect of a mixture applies. These models are the concentration addition (CA) or independent action (IA) models, respectively assuming that there is an equal or an independent mode of action of the different metals; (ii) to identify if deviations from additivity (‘interactions’) can be explained based on soil metal bioavailability. The environmental question was to identify if metal mixture effects occur and if they are relevant. More specifically, a relevant mixture effect occurs when metals dosed in isolation are not affecting plant growth, but become toxic when dosed as a mixture at equal concentrations.First, a study was set up to investigate the toxicity of multiple metal mixtures of Cu, Ni, Cd and Zn to plants at metal doses individually causing low level effects. Barley (Hordeum vulgare L.) root elongation toxicity tests were performed in resin buffered nutrient solutions to control metal speciation. Mixtures of different metals at free ion concentrations each causing <10% inhibition, yielded significant mixture effects when dosed in combination at equal concentrations, with inhibition reaching up to 50%. The IA model predicted mixture toxicity statistically better than the CA model, but some synergisms relative to the independent action model were observed. These synergisms relative to IA were most pronounced in quaternary mixtures and when the dose response curves had steep slopes. Generally, antagonistic interactions relative to the CA model were observed. Increasing solution Zn concentrations shifted metal interactions (CA based) from additive or slightly synergistic at background to antagonistic at higher supply, suggesting a protective effect of Zn. Overall, this study showed that the CA model can be used as a conservative model to predict metal mixture toxicity to barley.Second, interactions at biochemical level in plant roots were investigated. Net toxic effects and interactions of mixtures on plant growth may be better explained by biochemical parameters than by exposure information, and therefore effects of mixtures of Zn, Cd and Cu on barley plants were analyzed using antioxidant and oxidative stress parameters and root K+-efflux. Root elongation in Cu+Cd mixtures was well predicted from solution concentrations, using CA or IA reference models. In contrast, Zn acted antagonistically when combined with Cu and/or Cd, relative to both CA and IA. This protective effect of Zn correlated with the biomarkers, i.e. oxidative stress (indicated by e.g. MDA and H2O2 levels) decreased upon addition of Zn. However, external solution metal concentrations, i.e. the exposure, explained mixture effects better than any of the 16 antioxidant and oxidative stress biomarkers, i.e. the biochemical effects. It was concluded that the biomarkers are no robust indicators for metal mixture toxicity, potentially because different metals have different parallel modes of action on growth that are insufficiently indexed by the biomarkers.Next, interactions at the exposure and uptake level were investigated by incorporating bioavailability in the interpretation of metal interactions. The Biotic Ligand Model (BLM) and the WHAM-Ftox model that assume that toxicity depends on the concentration of metal bound to a biological binding site (the biotic ligand), were used. That concentration bound to the biotic ligand, is in turn calculated from the speciation of the metals in (soil) solution and the concentrations of ions competing with metal binding. First, Cu2+ and Zn2+ mixture toxicity was tested in resin buffered solutions at three different Ca2+ concentrations. Antagonistic interactions between Cu2+ and Zn2+ were found at low Ca2+ concentrations, but became smaller or insignificant at higher Ca2+, illustrating that mutual competition is eliminated at high concentration of a third competing ion (Ca2+). These effects obeyed the BLM combined with the IA reference mixture toxicity model. In a second test in nutrient solution, the complexity was increased by adding the metal chelator NTA (nitrilotriacetic acid) in solution. Metals compete for binding to NTA and this model molecule represents general complexation of metals in the environment. Mixture toxicity of Cu and Zn was investigated at contrasting NTA supply (-NTA and +NTA). In the +NTA solutions, Cu and Zn acted synergistically (IA based) when evaluation of toxicity was based on total metal concentration in solution. This interaction shifted to antagonism when the toxicity evaluation was based on the solution free ion activities of the metal, thus by accounting for competition effects on the NTA ligand and, hence, by accounting for bioavailability. In a final test, mixture toxicity and interactions of Cu and Zn were investigated in three different soil samples. The toxic effects of Cu and Zn mixtures on barley root elongation were synergistic in soils with high and medium cation exchange capacity (CEC), but antagonistic in a low CEC soil. This was found when expressing the dose as the conventional total soil concentration. In contrast, antagonism was found in all soils when expressing the dose as free ion activities in soil solution, indicating, again, that there is metal ion competition for binding to the plant roots. Neither a CA, nor an IA model fully explained mixture effects, irrespective of the dose expressions. In contrast, a multi-metal BLM model and a WHAM-Ftox model successfully explained the mixture effects from pore water composition across all soils and showed that bioavailability factors mainly explain the interactions in soils.Concluding, metal mixture effects can be predicted from the effects of each metal separately and the CA reference model is more conservative (protective) than the IA model, however the latter was statistically more accurate. This suggests that different metals, in general, act independently for toxicity. Metals can act synergistically in solution-plant or soil-plant systems when considering the total metal concentration, however, that almost always reverted to antagonism when considering the bioavailability, i.e. metal speciation in the exposure medium and competition for uptake. Biomarkers were no robust indicators for physiological effects and offer little opportunities to address mixture effects in the environment. A multi-metal BLM model and a WHAM-Ftox model successfully explained mixture effects in different soil samples. From risk-assessment point of view, this work showed that mixture effects are relevant and that, for metals, something can happen from nothing, especially when individual dose-response curves have steep slopes. Validated chronic mixture toxicity models accounting for bioavailability can be included in tiered risk assessment approaches.