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Aquatic organisms, such as aquatic invertebrates, are exposed to anthropogenic pollutants through their environment. These pollutants, despite their low levels, can adversely affect exposed individuals or even entire ecosystems, especially when present in complex mixtures. The aim of this thesis was to assess the effects of a specific group of complex mixtures of pollutants, wastewater effluent, on damselfly larvae, a common, ecologically relevant invertebrate species. Metabolomics, i.e., the comprehensive analysis of an organism’s metabolites, was explored as a tool to show the sub-lethal effects of wastewater effluent exposure. A set of multi-platform mass spectrometry-based metabolomics methods was developed. These methods were used to measure and identify which damselfly metabolites are responsive to wastewater exposure and thus could potentially be used as early warning tools for anthropogenic pollution. In addition, key behavioural traits of damselfly larvae were assessed after wastewater exposure to investigate whether a change of metabolites would also be reflected at a higher level of biological organisation.

The effects of wastewater effluents treated with different treatment methods on the fatty acid metabolites (oxylipins) of exposed damselfly larvae were assessed (paper I). Oxylipins were affected by wastewater exposure and these effects depended on the degree of wastewater treatment. Using a similar set-up, the effect-based removal of a conventional wastewater treatment plant and an additional ozonation step was evaluated on-site at a wastewater treatment facility (paper II). Oxylipins were affected by the exposure in this study, however fewer effects were observed when compared to the previous paper. In a separate series of experiments, damselflies were lab-reared to different developmental stages and a subset of the larvae were exposed to wastewater effluent. In these larvae, oxylipins (paper III) as well as other metabolites (paper IV) were measured to establish metabolite baseline levels and developmental variations as well as variation in their responses to the exposure. Metabolite variations as well as the metabolites affected by the exposure depended greatly on the life stage of the damselflies. In another study, damselfly larvae were exposed to dilutions of conventionally treated effluent and behavioural alterations and metabolite profiles were investigated in the larvae (paper V). Individual metabolites as well as behavioural traits important for damselfly survival and reproduction were altered by exposure to undiluted effluent; however, few effects were observed in the diluted effluents.

In conclusion, both metabolomic endpoints and behavioural traits measured on the damselfly larvae were responsive to wastewater effluent exposure. The metabolites affected by exposure mainly play a role in fatty acid metabolism, including oxylipins, and in amino acid metabolism. The individual metabolites that were affected differed across the studies. These observed variations might be due to differences in exposure conditions or differences in larval stages across the studies. The studies presented in this thesis pave the way for metabolomics to be used as a novel tool to monitor sub-lethal effects of anthropogenic pollution in the environment. However, more research is needed on, for example, the ecological implications of the affected metabolites for both the individual and the population before it can be implemented in environmental risk assessments.