Toxicity of zinc oxide nanoparticles in the earthworm, Eisenia fetida and subcellular fractionation of Zn.

The extensive use of nanoparticles (NPs) in a variety of applications has raised great concerns about their environmental fate and biological effects. This study examined the impact of dissolved organic matter (DOM) and salts on ZnO NP dispersion/solubility and toxicity to the earthworm Eisenia fetida. To be able to better evaluate the toxicity of NPs, exposure in agar and on filter paper was proposed for enabling a comparison of the importance of different uptake routes. A dose-related increase in mortality was observed in earthworms exposed in agar with almost 100% mortality after 96 h exposure to the highest concentration (1000 mg ZnO/kg agar). Scanning electron microscopy (SEM) showed that the addition of salts enhanced the aggregation of ZnO NPs in agar and consequently affected the dissolution behavior and biological availability of the particles. On filter paper, mortality was the highest at the lowest exposure concentration (50 mg ZnO/L) and seemed to decrease with increasing exposure levels. TEM images of ZnO showed that the solubility and morphology of NPs were changed dramatically upon the addition of humic acids (HA). The subcellular distribution pattern of Zn in earthworms after 96 h exposure in agar and on filter paper showed that the Zn taken up via dietary ZnO particles (from agar) was mainly found in organelles and the cytosol while the Zn accumulated as soluble Zn from filter paper was mainly distributed in cell membranes and tissues. Antioxidant enzymatic activities (SOD, CAT, and GSH-px) were investigated in the worms surviving the toxicity tests. A slight increase of SOD activities was observed at the lowest exposure dose of ZnO (50mg/kg), followed by a decrease at 100mg/kg in the agar cubes. Activities of both CAT and GSH-Px enzymes were not significantly influenced in the worms exposed to agar, although a slight decrease at 500 and 1000 mg ZnO/kg agar was observed. A similar change trend of SOD activities was observed for the earthworms on filter paper, but a significant decrease began at a higher ZnO NP concentration of 500 mg ZnO/L. The use of soil extracts instead of deionized water (DW) to simulate a realistic exposure system significantly reduced the toxicity of the ZnO NPs on filter paper, which increases the predictive power of filter paper toxicity tests for the environmental risk assessment of NPs.