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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. Scanning electron microscopy (SEM) revealed the presence of large agglomerates (1–10 µm in diameter) in the media prepared by cryogenic grinding with the highest concentration of ZnO-NPs. Neither the cryogenically ground media with lower ZnO-NPs concentrations nor any media prepared from colloidal solutions contained agglomerates exceeding 100 nm, detectable by SEM. Hydrodynamic diameters of particles in the colloids used in the second method of agar preparation were measured using dynamic light scattering (DLS) and ranged between 164 nm and 240 nm. The observed toxicity was thus clearly dependent on the size of ZnO-NPs agglomerates and the technique of exposure media preparation. Experimentally detected LC50 value for dissolved Zn2+ was 37.2 mg kg−1 in agar. The same concentration of Zn induced an approximately 30 % mortality of E. crypticus when administered in form of cryogenically ground ZnO-NPs with agar. No observable effects were found at this ZnO-NPs concentration when the exposure medium was prepared from the colloid solution.