In the context of global change, aquatic ecosystems face multiple stressors that may endanger their sustainability and associated ecosystem services. It is therefore crucial to better understand how these pressures interact and modulate the responses and further the tolerance of aquatic organisms, in order to gain knowledge on the resilience of these ecosystems. In particular, regarding the increasing aquatic pollution, there is a need to establish the causality link between the exposure to chemicals and the effect(s) in order to propose monitoring and remediation solutions [1]. To tackle this challenge, the (xeno)metabolomic approach aims to characterize all (chemical) exposures (i.e. xeno-metabolome, xenobiotics as well as their products from phase I and/or II metabolism) in addition to exposure-induced effects on cellular biomolecules (i.e. endo-metabolome) [2,3]. In this global change context, stream biofilms -as a complex assemblage of algae, fungi, bacteria, protozoa - are increasingly used because of their relevance to investigate the impact of multiple environmental stressors at the community level (function and structure), as they also play a key role in aquatic ecosystems (e.g.primary production) [4]. Technological and methodological innovation By using a (xeno)metabolomic approach, this study aims to investigate the influence of environmental confounding factors (temperature, seasonality, flow, photoperiod) on the impact of the chemical stress on stream biofilms. To this end, stream biofilms colonized at a reference site were exposed to the herbicide diuron - a model compound for photosynthesis inhibition- at the laboratory under different controlled conditions. The metabolomics responses were assessed for the different tested conditions through organic extraction by using accelerated solvent extraction followed by injection on UPLC-ToF system (Ultra-Performance Liquid Chromatography — Time of Flight Mass Spectrometry, Xevo G2-S ToF, Waters). As a first step, data were processed in MzMine2 in order to identify relevant features regarding exposure to diuron. The modulation of these signals by environmental confounding factors will be soon investigated. In addition, the photosynthetic activity was also assessed as a functional response of the biofilms supported by the metabolic changes triggered by diuron. Results and impact The data analysis is still ongoing. Nevertheless, by using the PlantCyc database, the first processing of the data in Mzmine2 allowed the identification of two plant-specific fatty acids, the docosapentaenoic acid and the eicosapentaenoic acid. These are two omega-3 fatty acids that play a major in the sustainability of the trophic chain and so ecossystemic servies since they are difficult to produce by higher trophic level (i.e. high metabolic cost). Although their identities remain to be confirmed through the injection of the standards, our result showed a strong inhibition of the production of the docosapentaenoic acid in the exposed biofilms (500 fold). This study will provide a better understanding of the metabolic alteration associated with the inhibition of the photosynthesis activity, as increasing knowledge on the metabolic responses of heterotrophic communities (bacteria, fungi) following diuron exposure. By investigating confounding factors, it would help to identify metabolic pathways that might be involved in the adaptation/tolerance of autotrophic and heterotrophic aquatic communities. It will finally support the discovery of biomarkers for the monitoring of water quality in the global change context.