For roughly thirty years, there is extensive evidence that neither total nor dissolved aqueous metal concentrations are good predictors of metal bioavailability and toxicity. On another side, speciation information has been found fundamental to understand interaction between inorganic contaminants and biomolecules during the transport, incorporation and fixation into a biological cell or tissue. Thus the characterization of biomolecules involved in metabolic pathways and interacting with an element is a key point to elucidate the toxicity of an element. However, the most challenging task is to preserve the integrity of metal-biomolecule complexes from the separation/purification procedure until their identification. This difficulty of the characterization of metal coordination complexes with biomolecules spurs development of new analytical methodologies. The project proposes to develop analytical methodologies allowing the characterization of intact uranium-biomolecules complexes within biological environmental matrices coming from aquatic organisms. This, after exposure of these organisms, dissection of organs of interest and uranium microdistribution analysis in the different fractions of work. New methodologies will allow : (i) A first screening of uranium-protein complexes thanks to the development of uranium imaging in proteins spots of non denaturating 2D-PAGE by Laser Ablation coupled to ICP MS; this will be achieved if an optimized non denaturating separation protocol by 2D gel electrophoresis is developed. Quantitative information will be addressed using unspecific isotopic dilution analysis. (ii) Secondly, the characterization of intact uranium-protein complexes. Using the complementarity of ICP MS and molecular mass spectrometry, a top-down strategy will be developed and include a non denaturating separation of intact uranium-protein complexes by means of preparative gel electrophoresis or multidimensional liquid chromatography (depending on the molecular weight of the uranium-binding proteins) and the identification of the uranium-complexes fragments by high resolution mass spectrometry (FT MS orbitrap). (iii) Thirdly, the characterisation of uranium-metabolites complexes. The developed analytical methodology will combine orthogonal and non denaturating chromatographic mechanisms for the separation of intact uranium-metabolites complexes and high resolution mass spectrometry for their identification. These developments in addition with the realization of a typical uranium metallomics study should allow the investigation of uranium speciation in aquatic organisms such as bivalves (Corbicula fluminea), crayfish (Orconectes limousus) or fish (Danio rerio) which are often used as inorganic pollutants bioindicators. This in vivo characterization of uranium target biomolecules (proteins or metabolites) will be carried out at the basal level and after different condition of exposure of the aquatic organisms (environmental versus incidental). These results will provide insights into the mechanisms by which uranium is sensed, stored, incorporated or released from the aquatic organism organs. In addition, as some toxicity parameters has been previously determined in the conditions of exposure chosen, correlations between uranium speciation and the toxic fraction towards the organisms under study will be investigated. Such work could finally offer interesting prospects for risk assessment linked to uranium exposure (implementation of existing models).