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As lithium intercalating complex metal oxides become more widely used in energy storage devices, there is an increasing need to understand their environmental impact at the end of their life cycle due to the lack of recycling options. In this study, we examine the biological impact of a panel of nanoscale lithium nickel manganese cobalt oxides (LixNiyMnzCo1−y−zO2, 0 < x, y, z < 1, abbreviated as NMCs), to a model Gram-positive bacterium, Bacillus subtilis in terms of cellular respiration and growth. A highly sensitive single-cell gel electrophoresis method is also applied for the first time to understand the genotoxicity of this nanomaterial to bacterial cells. Results from these assays indicate that the free Ni and Co ions released from the incongruent dissolution of the NMC material in B. subtilis growth media induced both hindered growth and cellular respiration. More remarkably, the DNA damage induced by the combination of the two ions in solution is comparble to that induced by the NMC material, which suggests the free Ni and Co ions are responsible for the toxicity observed. A material redesign by enriching Mn is also presented. The combined approaches of evaluating impact on bacterial growth, respiration, DNA damage at a single-cell level, as well as other phenotypical changes allows us to probe the nanomaterial and bacterial cells from a mechanistic prospective, and provides a useful means to an understanding of bacterial response to new potential environmental stressors.