Unprecedented levels of chemicals of anthropogenic origin are currently released into surface waters globally. Wastewater treatment plant effluent has been identified as a major source, containing a broad mixture of pharmaceuticals and consumer chemicals. Therefore, there is a need for implementation of advanced wastewater treatment techniques, such as ozonation and adsorption methods, to reduce the contamination. However, there are conflicting findings on the toxicity of treated effluent and only limited possibilities for assessing the effect-based removal efficiency (EBRE) of different treatment techniques. Here, we describe a metabolomics approach to detect perturbations in fatty acid catabolic pathways as a proxy for biological effects. Metabolites in three fatty acid pathways were analyzed in a common damselfly larva (Coenagrion hastulatum) by liquid chromatography coupled to mass spectrometry. The larvae were exposed for one week to either conventionally treated effluent (activated sludge treatment), effluent additionally treated with ozone or effluent additionally treated with biochar filtration and results were compared with those from tap water control exposure. Five lipoxygenase-derived oxylipins (9,10,13-TriHOME, 9,12,13-TriHOME, 9-HODE, 9-HOTrE, and 13-HOTrE) decreased in response to conventionally treated effluent exposure. By using an additional treatment step, oxylipin levels were restored with exception of 9,10,13-TriHOME (ozonated effluent), and 9-HOTrE and 13-HOTrE (effluent filtered with biochar). In conclusion, exposure to wastewater effluent affected fatty acid metabolite levels in damselfly larvae, and a subset of the analyzed metabolites may serve as indicators for biological effects in biota in response to effluent exposure. To that effect, our findings suggest a new metabolomics protocol for assessing EBRE.

Animal behaviour is remarkably sensitive to disruption by chemical pollution, with widespread implications for ecological and evolutionary processes in contaminated wildlife populations. However, conventional approaches applied to study the impacts of chemical pollutants on wildlife behaviour seldom address the complexity of natural environments in which contamination occurs. The aim of this review is to guide the rapidly developing field of behavioural ecotoxicology towards increased environmental realism, ecological complexity, and mechanistic understanding. We identify research areas in ecology that to date have been largely overlooked within behavioural ecotoxicology but which promise to yield valuable insights, including within- and among-individual variation, social networks and collective behaviour, and multi-stressor interactions. Further, we feature methodological and technological innovations that enable the collection of data on pollutant-induced behavioural changes at an unprecedented resolution and scale in the laboratory and the field. In an era of rapid environmental change, there is an urgent need to advance our understanding of the real-world impacts of chemical pollution on wildlife behaviour. This review therefore provides a roadmap of the major outstanding questions in behavioural ecotoxicology and highlights the need for increased cross-talk with other disciplines in order to find the answers.