These studies were undertaken in order to propose and test new methods for the assessment of the acute hazard of ZnO nanoparticles (NPs) to the sediment dwelling oligochaete worm Lumbriculus variegatus. In order to support the developing nanotechnology sector, comprehensive studies must be conducted to assess the toxicity of nanomaterials (NMs) using environmentally relevant organisms. An important part of such studies will entail characterising and understanding the physicochemical properties of these NMs. In this study NMs were characterised using a range of techniques, in order to assess agglomeration/aggregation and dissolution. Toxicology studies included a behavioural assay and the measurement of oxidative stress. When considering the toxicology results from all experiments using L. variegatus within this paper ZnO NPs (0-10 mg/l) were found to cause acute toxicity in terms of behavioural response, but not to cause acute oxidative stress in terms of glutathione (GSH) depletion. It was also concluded that the behavioural assay and the GSH assay were both suitable techniques for assessing the acute hazard of NMs to L. variegatus.