In U.S. Pacific Northwest coho salmon (Oncorhynchus kisutch), stormwater exposure annually causes unexplained acute mortality when adult salmon migrate to urban creeks to reproduce. By investigating this phenomenon, we identified a highly toxic quinone transformation product of N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD), a globally ubiquitous tire rubber antioxidant. Retrospective analysis of representative roadway runoff and stormwater-affected creeks of the U.S. West Coast indicated widespread occurrence of 6PPD-quinone (<0.3 to 19 micrograms per liter) at toxic concentrations (median lethal concentration of 0.8 ± 0.16 micrograms per liter). These results reveal unanticipated risks of 6PPD antioxidants to an aquatic species and imply toxicological relevance for dissipated tire rubber residues.

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.

Nuclear Magnetic Resonance (NMR) spectroscopy is a non-invasive analytical technique which allows for the study of intact samples. Comprehensive Multiphase NMR (CMP-NMR) combines techniques and hardware from solution state and solid state NMR to allow for the holistic analysis of all phases (i.e. solutions, gels and solids) in unaltered samples. This study is the first to apply CMP-NMR to deceased, intact organisms and uses 13 C enriched Daphnia magna (water fleas) as an example. D. magna are commonly used model organisms for environmental toxicology studies. As primary consumers, they are responsible for the transfer of nutrients across trophic levels, and a decline in their population can potentially impact the entire freshwater aquatic ecosystem. Though in vivo research is the ultimate tool to understand an organism’s most biologically relevant state, studies are limited by conditions (i.e. oxygen requirements, limited experiment time and reduced spinning speed) required to keep the organisms alive, which can negatively impact the quality of the data collected. In comparison, ex vivo CMP-NMR is beneficial in that; organisms do not need oxygen (eliminating air holes in rotor caps and subsequent evaporation); samples can be spun faster, leading to improved spectral resolution; more biomass per sample can be analyzed; and experiments can be run for longer. In turn, higher quality ex vivo NMR, can provide more comprehensive NMR assignments, which in many cases could be transferred to better understand less resolved in vivo signals. This manuscript is divided into three sections: 1) multiphase spectral editing techniques, 2) detailed metabolic assignments of 2D NMR of 13 C enriched D. magna and 3) multiphase biological changes over different life stages, ages and generations of D. magna . In summary, ex vivo CMP-NMR proves to be a very powerful approach to study whole organisms in a comprehensive manner and should provide very complementary information to in vivo based research.