Toxicity of zinc oxide nanoparticle (ZnO-NPs) powder and water soluble salt of Zn (ZnCl2) to the annelid Enchytraeus crypticus was tested in agarose gel. Influence of the spiking method on the resulting size of nanoparticles and on E. crypticus mortality was studied. Two methods of ZnO-NPs powder (mean particle size diameter of 10 nm) introduction into the exposure media were used. In the first method, the nano-powder was initially cryogenically ground with dry agar followed by an addition of water. The second procedure began with re-suspension of nanoparticles in demineralized water containing a dispersant (sodium pyrophosphate decahydrate). The obtained colloid was subsequently mixed with hot agar gel. Relative mortality in worms observed after 96 h of their exposure to the ZnO-NPs concentrations (all in mg of ZnO-NPs per kg of agar) of 50, 100, 200, 500 and 1000 in the cryogenically ground medium ranged between 28.9 % and 34.4 % and it did not exhibit any concentration dependence. When the second method of exposure media preparation was applied, the relative mortality ranged from 0 % to 66.6 % in the same concentration region depending on the concentration.