Evaluating cell surface extraction methods for improved assessment of silver nanoparticle bioaccumulation

Methods to differentiate surface-bound and internalized Ag in phytoplankton are essential for understanding bioaccumulation but remain limited, especially for studies involving silver nanoparticles (AgNPs). To address this, we assessed biologically relevant ligands for extracting adsorbed Ag from Chlamydomonas reinhardtii and Cyclotella meneghiniana exposed to Ag⁺ or AgNPs, focusing on ligand type, concentration, and extraction time. Additionally, we examined the impact of these agents on AgNP stability and microalgal membrane integrity. The ligands l-histidine, l-glutamine, l-cysteine, and d-penicillamine were tested. The thiol-containing d-penicillamine, tested for the first time, showed the highest efficiency in desorbing Ag⁺ from Ag⁺-treated cells, followed by l-cysteine. Optimal conditions were 1 mmol L⁻¹ ligand concentration and 5 min contact time. For AgNP-exposed cells, two sequential cell resuspensions in Ag-free medium were sufficient to detach loosely bound AgNPs. Then, hydrogen peroxide (H₂O₂), a biologically relevant oxidant, was introduced for the first time in the extraction procedure to facilitate the oxidative dissolution of nanoparticles. A combination of 0.5 mmol L^-1 H₂O₂ with a 1 mmol L^-1l-cysteine or d-penicillamine effectively removed Ag from the cell walls of C. meneghiniana and C. reinhardtii. The tested extraction agents did not affect unexposed phytoplankton cells. However, AgNP-exposed cells treated with H₂O₂ showed slightly increased cell membrane damage in diatoms. A dissolution of AgNP was found in the presence of l-histidine and d-penicillamine and H₂O₂. These findings highlight the need to develop species-specific washing methodologies in AgNPs bioaccumulation studies and provide insight into optimized extraction methods for assessing AgNP adsorption, internalization, and toxicity.