Ethidium Bromide Degradation by Cold Atmospheric Plasma in Water and the Assessment of Byproduct Toxicity for Environmental Protection

Ethidium bromide (Et-Br) is a widely used fluorescent dye in molecular biology and biotechnology laboratories for visualizing nucleic acids in agarose gel electrophoresis. However, concerns have been raised about its environmental impact and potential health risks due to its persistence and toxicity. The potential accumulation and long-term effects on the environment necessitate the removal of Et-Br from water. This study investigates the potential of novel cold plasma technology for the degradation of Et-Br. Cold atmospheric plasma (CAP) is an environmentally friendly technology that does not produce secondary pollutants and generates a variety of potent chemical reactive oxidants such as hydroxyl radicals (^•OH), H₂O₂, NO₂, and NO₃. In this study, Et-Br was treated with CAP for 15 min without the addition of any chemicals, resulting in substantial removal of Et-Br. The degradation kinetics revealed that the CAP-treated Et-Br followed a pseudo-first-order reaction, dependent on the treatment time of CAP. The degradation of Et-Br by CAP is distinctly evident through the results obtained from both high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) analyses, providing clear evidence of the occurrence of degradation. Furthermore, toxicity analyses of the degradation products were conducted by evaluating the Et-Br intercalation ability with DNA before and after treatment of Et-Br with CAP. To supplement the assessment, the binding of Et-Br with BSA has also been studied before and after CAP treatment. The impact of CAP-treated Et-Br on the growth and colony-forming unit (CFU) counts of <i>Escherichia coli</i> was also evaluated. Results indicated an increase in bacterial growth with an increase in CAP treatment time, suggesting that the degradation products of Et-Br using CAP were nontoxic. This study highlights the potential of CAP as a clean and efficient technology for the degradation of Et-Br, presenting a promising solution for mitigating its environmental and health risks.