Nano-scale zero-valent iron (nZVI) began attracting research attention in remediation practice in recent decades as a prospective nanomaterial applicable to various contaminated matrices. Despite concerns about the negative effects of nanomaterials on ecosystems, the number of reliable toxicity tests is limited. We have developed a test based on the evaluation of oxidative stress (OS). The test employed the analysis of a typical OS marker (malondialdehyde, MDA), after exposure of six bacterial strains to the tested nanomaterial. We also attempted to use other OS and cell membrane damage assays, including the determination of glutathione and lactate dehydrogenase, respectively. However, we found that the components of these assays interfered with nZVI; therefore, these tests were not applicable. The MDA assay was tested using nZVI and three newly engineered oxide shell nZVI materials with different oxide thicknesses. Six different bacterial species were employed, and the results showed that the test was fully applicable for the concentrations of nanomaterials used in remediation practice (0.1-10 g/L). MDA was produced in a dose-response manner, and the bacteria showed a similar response toward pure pyrophoric nZVI, reaching EC50 values of 0.3-1.1 g/L. We observed different responses in the absolute production of MDA; however, the MDA concentrations were correlated with the cell membrane surfaces of the individual strains (R > 0.75; P < 0.09). Additionally, the EC50 values correlated with the thickness of the oxide shells (except for Escherichia coli: R > 0.95; P < 0.05), documenting the reliability of the assay, where reactivity was confirmed to be an important factor for reactive oxygen species production.

Institute of Microbiology Czech Academy of Sciences v v i Vídeňská 1083 CZ 142 20 Prague 4 Czech Republic

Institute of Microbiology Czech Academy of Sciences v v i Vídeňská 1083 CZ 142 20 Prague 4 Czech Republic; Institute for Environmental Studies Faculty of Science Charles University Benátská 2 CZ 128 01 Prague 2 Czech Republic

Regional Centre of Advanced Technologies and Materials Faculty of Science Palacký University 17 listopadu 1192 12 CZ 771 46 Olomouc Czech Republic

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In Vitro Study of the Toxicity Mechanisms of Nanoscale Zero-Valent Iron (nZVI) and Released Iron Ions Using Earthworm Cells

Oxidative stress in microbes after exposure to iron nanoparticles: analysis of aldehydes as oxidative damage products of lipids and proteins