Measurements of released Al3+ in the culture media and plants

The amount of released aluminum ion in the culture media, treated with nano and bulk alumina particles, were measured by GBC 932 AA (Australia) flame atomic absorption spectrometer, equipped with a hollow cathode lamp and a deuterium background collector at 396.2 nm using nitrous oxide flame. The suspension was ultrasonicated and the pH was adjusted on 2.0 prior to atomic absorption spectroscopy (AAS). Samples of plants were oven-dried to quantify the Al concentration in nanoparticle treated groups (n = 3) and bulk alumina-treated groups (n = 3). Microwave acid digestion was performed by the addition of a 4:1 (v/v) ratio of HNO₃:H₂O₂ to 500 mg of dried samples in closed-microwave vessels. Vessels were remained at 25 °C for 10 min after shaking. Digestion parameters for the microwave oven (Milestone Ethos Up, SK-15 high pressure rotor) included heating at 200 °C for 15 min at a total power of 1800 W, followed by cooling. Digested samples were dissolved in 50 ml of Millipore water and were analyzed for the presence of aluminum by inductively coupled plasma-optical emission spectroscopy (ICP-OES, Spectro Arcos, SPECTRO, Germany) according to Fathabad et al. (2018).