Development of an integrated multi-omic targeted workflow for Gammarus fossarum : a proof-of-concept application to study endocrine disruption in environmental species

The focus of chemical risk assessment is shifting towards mechanistic system biology in order to better capture subtle effects of environmental contaminants such as endocrine disruptors. However, mechanistic studies in ecologically relevant sentinel species often require organ-level investigation, which is hindered by extreme limitations in biomass and high inter-individual variability. In this context, we developed a targeted multi-omic workflow (proteomics, metabolomics, and lipidomics) optimised for low-mass sentinel species such as Gammarus fossarum. A unified ‘metabolomics-first’ co-extraction protocol enabled the simultaneous recovery of proteins, metabolites, and lipids from identical caeca pools. To overcome data processing bottlenecks of high-resolution mass spectrometry (HRMS) and ensure high sensitivity, a fully targeted mass spectrometry strategy was adopted across all omics layers. Specifically, the Scout-MRM approach was implemented via the Scout-MRM Builder tool for metabolomics, providing an efficient alternative to untargeted HRMS. Meanwhile, proteomics and lipidomics utilised targeted MRM. As a proof of concept, the pipeline was applied to caeca exposed to two crustacean endocrine disruptors : fenoxycarb and tributyltin. Integrated analysis revealed consistent molecular shifts across energy, membrane, and detoxification pathways. These findings, serving as mechanistic hypotheses, demonstrate the potential of this single-sample workflow to provide a synchronised view of organismal physiological state. This work establishes a methodological framework for advancing systems ecotoxicology in non-model species.