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Environmental metabolomics provides insight into how anthropogenic activities have an impact on the health of an organism at the molecular level. Within this field, in vivo NMR stands out as a powerful tool for monitoring real-time changes in an organism’s metabolome. Typically, these studies use 2D 13C-1H experiments on 13C-enriched organisms. Daphnia are the most studied species, given their widespread use in toxicity testing. However, with COVID-19 and other geopolitical factors, the cost of isotope enrichment increased ~6–7 fold over the last two years, making 13C-enriched cultures difficult to maintain. Thus, it is essential to revisit proton-only in vivo NMR and ask, “Can any metabolic information be obtained from Daphnia using proton-only experiments?”. Two samples are considered here: living and whole reswollen organisms. A range of filters are tested, including relaxation, lipid suppression, multiple-quantum, J-coupling suppression, 2D 1H-1H experiments, selective experiments, and those exploiting intermolecular single-quantum coherence. While most filters improve the ex vivo spectra, only the most complex filters succeed in vivo. If non-enriched organisms must be used, then, DREAMTIME is recommended for targeted monitoring, while IP-iSQC was the only experiment that allowed non-targeted metabolite identification in vivo. This paper is critically important as it documents not just the experiments that succeed in vivo but also those that fail and demonstrates first-hand the difficulties associated with proton-only in vivo NMR.

Part review, part perspective, this article examines the applications and potential of in-vivo Nuclear Magnetic Resonance (NMR) for understanding environmental toxicity. In-vivo NMR can be applied in high field NMR spectrometers using either magic angle spinning based approaches, or flow systems. Solution-state NMR in combination with a flow system provides a low stress approach to monitor dissolved metabolites, while magic angle spinning NMR allows the detection of all components (solutions, gels and solids), albeit with additional stress caused by the rapid sample spinning. With in-vivo NMR it is possible to use the same organisms for control and exposure studies (controls are the same organisms prior to exposure inside the NMR). As such individual variability can be reduced while continual data collection over time provides the temporal resolution required to discern complex interconnected response pathways. When multidimensional NMR is combined with isotopic labelling, a wide range of metabolites can be identified in-vivo providing a unique window into the living metabolome that is highly complementary to more traditional metabolomics studies employing extracts, tissues, or biofluids.

Toxicity testing of living organisms has been undergoing a paradigm shift over the last decade. Focus has moved from examining apical endpoints such as death or reproduction, to measuring sub-lethal impacts from a biochemical perspective, with future aspirations to automate the approach. One of the ways this is examined is through metabolomics, the study of the impacts to an organism’s metabolic system as a result of an external stressor, such as a potential toxin. Recently, the field of in-vivo nuclear magnetic resonance (NMR) spectroscopy to study metabolomics has provided breakthroughs in toxicity testing information. Much of this work has been done with the aquatic organism Daphnia magna, a model organism for aquatic toxicity studies. While robust, NMR suffers from a lack of sensitivity and heavy spectral overlap in complex systems, such as living organisms. Thus, this thesis introduces novel technologies which help address sensitivity limitations and remove overlap in in-vivo NMR while working towards creating an automated dosing platform in line with the paradigm shift of toxicity testing. First, a new holistic approach to testing is examined which uses three NMR techniques in tandem, providing complementary information of toxin interactions including binding, physical partitioning, and biochemical response inside living organisms. Binding with the organism’s outer shell and metabolic oxidative stress responses were measured, which could not have been examined independently. Additionally, two new NMR pulse sequences are introduced. The first examines new bond formation between different nuclear isotopes (13C-12C), and food incorporation into living biomass of D. magna was examined. In the second sequence, suites of molecules chosen by the user can be isolated from an unchanged matrix, while focusing increases sensitivity over traditional 1H NMR. Four metabolites indicative of oxidative stress were monitored in D. magna simultaneously, while the unselected signals were filtered out. Finally, an automated dosing platform was created by the combination of digital microfluidics (DMF) and NMR to keep D. magna alive for prolonged periods using automated movement of food and water. Moving forward, these four new approaches could be used in tandem to increase sensitivity, reduce overlap, and automate the toxicity testing process.