It is crucial to understand the effects caused by experimental parameters, such as temperature; light availability; and the use of a substrate, that can interfere when identifying a dose-response in toxicology exposures. Limiting variability can be especially crucial when developing key metabolite biomarkers of contaminant exposure. Environmental metabolomics is influenced by internal and external surroundings, that can affect an organism’s overall health. For this reason, metabolomics can be an added tool for sensitive environmental assessments. The initial step to improve ways of testing the subtle biochemical effect of contaminants on aquatic invertebrates is to account for experimental parameters that can cause a metabolomic shift within an organism. Here we investigate the metabolomic response when the freshwater crustacean, the amphipod, Austrochiltonia subtenuis, is exposed to three common alternative substrates, gauze; toilet paper; and cellulose. There were a large number of disaccharides, monosaccharides, fatty acid and conjugates, and TCA acids identified using Gas Chromatography-Mass Spectrometry (GC-MS). The responses caused by the substrates alone resulted in different sensitivities occurring in the amphipods. Amphipods were then exposed to a contaminant to measure if a dose- response changes due to the substrates used. The results of this metabolomic method validation showed different metabolomic responses occurring from experimental design decisions. Here we have highlighted an area for future investigations into dose-response shifts when experimental parameters are altered such as light availability, food type and temperature.