The earthworm Eisenia fetida is a potential protein source for animals feeding including cultured fish species and also is a suitable organism for toxicity tests. In present study, the reproductive toxicity of zinc oxide nanoparticles in the earthworm E. andrei was investigated following exposure for 40 days to 0.01, 0.1, and 0.5 ppm zinc oxide nanoparticles (ZnO NPs) in well-rotted cow manure. Zinc oxide nanoparticles (nanopowder, 17.32 ± 5.80 nm particle size) were dispersed in double distilled water using bath sonicator. Cow manure was purchased from a local gardening store and washed several times with water before use. Before exposure, earthworms were acclimatised for one week in untreated cow manure. There were three replicate jars per treatment group. Each jar contained approximately 0.4 kg dry cow manure and ten adult earthworms. Sonicated ZnO NPs from each concentration was sprayed onto each jar and control jars were treated with distilled water. At the end of exposure period, the numbers of produced cocoons were counted and recorded. The results showed that when E. fetida were exposed to nano-sized ZnO, cocoon production significantly reduced in a concentration-dependent manner. The numbers of produced cocoons was highest in the control group (565.33±80.53) and then decreased to 449.66±18.17, 284.66±50.21, and 214.66±10.69 in 0.01, 0.1, and 0.5 ppm zinc oxide nanoparticles treatments, respectively. It seems that cocoon production is a very sensitive endpoint of ZnO nanoparticle exposure and may be used as a suitable biomarker of earthworm's reproduction to assess toxicity of other nanomaterials.
