Untargeted NMR-based metabolomics for field-scale monitoring: Temporal reproducibility and biomarker discovery in mosquitofish (Gambusia holbrooki) from a metal(loid)-contaminated wetland

There is considerable interest in applying omics techniques, which have proven extremely valuable for laboratory-based toxicology studies, towards field-scale ecotoxicology and environmental monitoring. Concerns that confounding factors in natural ecosystems may exacerbate variability in omics datasets must be addressed to validate the transition from laboratory to field. This study explores how temporal variability related to seasonal and climatic trends influence qualitative and quantitative metabolomics outcomes, in fish from reference and metal(loid)-polluted wetlands in Australia. Female mosquitofish (Gambusia holbrooki) were sampled on two separate occasions, from a rehabilitated tailings wetland at the site of historic antimony (Sb) processing and a reference wetland with comparable water quality. The first sampling coincided with greater monthly rainfall and colder water temperature, whereas the second sampling was drier and water was warmer. Despite temporal changes and associated differences in metal(loid) concentrations, site differences in metabolite profiles were qualitatively very similar between sampling events. However, quantitative differences were observed, with a greater number of significantly altered metabolites identified during the second sampling event, which coincided with greater metal(loid) concentrations in both water and fish. The majority of identified metabolites were elevated in fish from the contaminated wetland, but with notable decreases in several metabolites that are known to play a role in various aspects of metal(loid) binding, detoxification and excretion. Specifically, decreased aspartate, histidine, myo-inositol, taurine and choline were observed in fish from the contaminated wetland, and may therefore represent a metabolite suite that is broadly indicative of metal toxicity. Quantitative differences between sampling events are suggestive of a dose-response relationship observable at the cellular level which, if harnessed, may be useful for assigning levels of concern based on the degree of change in a multi-parameter set of metabolite biomarkers

NMR-based lipidomics of fish from a metal(loid) contaminated wetland show differences consistent with effects on cellular membranes and energy storage

Metals and metalloids are priority contaminants due to their non-degradable and bioaccumulative nature, and their ability to regulate and perturb diverse physiological processes in various species. Metal(loid)s are known to cause oxidative stress through production of reactive oxygen species (ROS), thus related endpoints like lipid peroxidation (LPO) have received considerable attention as biomarkers of exposure. However, the implications of metal(loid) toxicity including LPO on actual lipid profiles of species inhabiting contaminated systems are poorly understood. Here we applied Nuclear Magnetic Resonance (NMR) spectroscopy for untargeted lipidomics of mosquitofish (Gambusia holbrooki) collected from reference and metal(loid)-contaminated wetlands. We measured a range of trace elements in water and fish using inductively coupled plasma – mass spectrometry (ICP-MS), and interpreted site differences in the lipid profiles of mosquitofish in the context of known physiological responses to sub-lethal metal(loid) exposure. Results indicate deregulation of cellular membrane lipids (i.e., glycerophospholipids, cholesterol and sphingolipids) and increased energy storage molecules (i.e., triacylglycerols and fatty acids) in fish from the contaminated wetland. These responses are consistent with the recognised induction of oxidative stress pathways in organisms exposed to metal(loid)s and could also be symptomatic of mitochondrial dysfunction and endocrine disruption. It is difficult to attribute metal(loid)s as the sole factor causing differences between wetlands, and a more controlled experimental approach is therefore warranted to further explore mechanistic pathways. Nevertheless, our study highlights the benefits of untargeted 1H NMR-based lipidomics as a relatively fast and simple approach for field-scale assessment and monitoring of organisms inhabiting metal(loid) contaminated environments

Effects of naphthenic acid exposure on development and liver metabolic processes in anuran tadpoles

Naphthenic acids (NA) are used in a variety of commercial and industrial applications, and are primary toxic components of oil sands wastewater. We investigated developmental and metabolic responses of tadpoles exposed to sub-lethal concentrations of a commercial NA blend throughout development. We exposed Lithobates pipiens tadpoles to 1 and 2 mg/L NA for 75 days and monitored growth and development, condition factor, gonad and liver sizes, and levels of liver glucose, glycogen, lipids and cholesterol following exposure. NA decreased growth and development, significantly reduced glycogen stores and increased triglycerides, indicating disruption to processes associated with energy metabolism and hepatic glycolysis. Effects on liver function may explain reduced growth and delayed development observed in this and previous studies. Our data highlight the need for greater understanding of the mechanisms leading to hepatotoxicity in NA-exposed organisms, and indicate that strict guidelines may be needed for the release of NA into aquatic environments