Time-dependent responses of earthworms to soil contaminated with low levels of lead as detected using 1H NMR metabolomics

H-1 NMR-based metabolomics was used to profile the time-dependent metabolic responses of earthworms (Eisenia fetida) that were exposed to low-Pb-contaminated-soil (L-Pb-CS) for 28 days using an indoor culture. Earthworms were gathered after days 1, 7, 14, 21 and 28 of the exposure. The earthworm extracts were then analyzed using 500 MHz nuclear magnetic resonance. Spectrum interpretation, statistical analysis and plotting were performed using the R software. The metabolic trajectories, histopathological examination, Pb accumulation, survival rate and mean weights of the earthworms were evaluated to investigate and explain the possible mechanism of action. The results showed that the Pb concentrations of earthworms in the Pb exposure groups increased with longer exposure times. Pb treated groups showed varying degrees of histopathological damage. The metabolic response trajectories in the Pb treated groups were of a similar type but differed in the magnitudes of metabolite responses, which suggested that the responses of the Pb1 and Pb2 groups may involve the same metabolic mechanism. Earthworms may produce a toxic response on days 1-14, whereas a detoxification strategy was initiated during days 14-28 to adapt to the L-Pb-CS, as indicated by the negative maximum distance of metabolic trajectories. Specifically, the most serious damage in the histopathology examination and the most extreme values of metabolites were observed on the 14th day. The metabolic changes involved the amino acid, membrane, and energy metabolism of earthworms. Myo-inositol, 2-hexyl-5-ethyl-3-furansulfonate (HEFS), scyllo-inositol, succinate, alanine and maltose were found to be potential biomarkers for exposure to the L-Pb-CS. This study demonstrated that 1H NMR-based metabolomics along with metal accumulation and histological detection provide a reliable approach for interpreting time-dependent metabolic mechanisms in earthworms.