Contamination with heavy metals in soils worldwide is of pressing concern as they may degrade soil and water quality and have a negative impact on wildlife and human health. In agricultural soils, cu and zn are commonly found due to their presence in organic waste and fertilizers applied to the soils during agricultural practices. Cu and zn are used frequently as growth promoting agents and for preventing diarrhea in piglets. As the majority of cu and zn is excreted via faeces or urine, pig manure contains high concentrations of these metals. Hence soils receiving pig manure accumulate cu and zn when long term amendment with pig manure is applied. Cu and zn are essential trace elements for living organisms, but can be toxic at elevated concentrations. Consequently, cu and zn pose a risk to soil biota and may impair important ecosystem services usually existing in healthy soils. Additionally, cu and zn are also linked to development of antibiotic resistance in bacteria in agricultural environments. When evaluating soil quality, the total (or pseudo-total) soil metal concentration is often used. However, it is the bioavailable fraction that determines the environmental impact and not necessarily the soil total metal concentration. Bioavailability is closely linked to metal speciation as it is the speciation that in part determines the toxic effect and potential for uptake by organisms. Free metal ions have often been shown to be the important species with respect to bioavailability. However, it is now well recognized that under certain conditions, some metal complexes may contribute to bioavailability. More knowledge on bioavailability of different cu and zn species are still required to progress our understanding of the link between speciation and bioavailability under environmentally realistic conditions. The overall aim of this phd project was to progress the understanding of the relationship between cu and zn speciation and bioavailability in manure amended soils. Diffusive gradients in thin films (dgt), hollow fiber permeation liquid membranes (hf plm), ion selective electrode (ise) and extractions with cacl2 were used for determining labile species, free cu2+ and zn2+ and total dissolved species in agricultural soils amended with pig manure. The following research objectives were addressed: 1) evaluation and optimization of a bacterial bioreporter assay for bioavailability determination, 2) evaluation and optimization of hf plm for cu2+ and zn2+ determination. 3) investigation of the relationship between cu and zn species (determined with dgt, hf plm and ise) and bioavailability (determined with cu and zn specific bioreporters) in soils amended with manure, 4) examination of changes in cu and zn iii bioavailability following manure amendment. Furthermore, different chemical speciation techniques ability to determine cu and zn bioavailability was evaluated through experimental research and thorough review of existing literature. Evaluation of a cu specific whole-cell bioreporter demonstrated that by applying the conventional procedure of using a single set-point ph, irrespective of sample ph and range, a ph dependent bias may be introduced. Consequently, this affected the determined bioavailability. Instead it is recommended to use an optimized procedure where a flexible ph-assay is implemented and it is especially important to avoid such speciation artifacts when linking bioavailability with speciation. The hf plm technique was not able to determine free cu2+ and zn2+ ions simultaneously, but free zn2+ ions could be determined in solutions containing high levels of complexing agents. Free cu2+ was instead determined with a cu-ise, which is a robust speciation technique. Alternative techniques are required for zn as no commercial ise exist for zn. In this phd study hf plm was applied for the first time for determination of free zn2+ ions in aqueous extracts of soils with and without manure. Two different soils were amended with various levels of two different manures. Overall the relative bioavailability (percent of total cu and zn concentration) of both cu and zn increased following manure amendment and the level of increase primarily depended on the type of manure. The optimized bioreporter assay was incorporated for both cu and zn bioreporters and bioavailability was determined in the manure amended soils. Subsequently, the determined bioavailability was compared to free ions and labile species of cu and zn as determined with hf plm, dgt and cu-ise. This demonstrated that for both cu and zn, the bioavailable concentrations exceeded the concentrations of free metal ions and labile species determined with dgt. Accordingly, complexed species contribute to bioavailability. Further research is encouraged to investigate if the complexes metals dissociate and hence only free ions are taken up, or whether intact complexes are taken up or react with membrane surfaces of the organism. For the investigated manure amended soils, total dissolved cu and zn in aqueous extractions with cacl2 was significantly and linearly correlated with bioavailability. By combining chemical and biological techniques the knowledge of which cu and zn species contributes to bioavailability was advanced. In that way it was shown that in manure amended agricultural soils, complexed species of cu and zn were bioavailable to the applied bacterial iv bioreporters. This indicates that in systems containing high concentrations of cu and zn combined with high levels of organic matter, bioavailability will be underestimated if models based on free ions are be applied for risk assessment and modelling of the impact of metals. However, research where the link between in situ bioavailability and speciation is investigated is still lacking.