Single-Cell Insights into Size-Dependent Heterogeneous Bioconcentration and Distribution of Metal Species Ranging from Ions to Nanoclusters and Nanoparticles in Chlorella vulgaris

Abstract Particle size is a key factor influencing metal uptake by aquatic unicellular primary producers. To investigate size-dependent bioconcentration ranging from ions to nanoclusters (NCs) and nanoparticles (NPs), we used cytometry by time-of-flight (CyTOF) to quantitatively examine single-cell uptake, retention, and bioconcentration in Chlorella vulgaris exposed to Au3+ ions, three glutathione (GSH)-protected AuNC preparations (Au10–12SG10–12, Au15SG13, and Au25SG18), and four GSH-coated AuNPs (5, 15, 25, 50 nm). Au3+ and AuNCs showed rapid cell-associated accumulation but followed exponential-decay and two-phase clearance kinetics, indicating efficient cellular elimination and a tendency to be released back into the medium. In contrast, AuNPs followed saturation-type bioconcentration kinetics, with larger particles showing more persistent cell association. Single-cell analysis revealed strong size-dependent heterogeneity: under equal-mass exposure, the fraction of Au-positive cells decreased exponentially with increasing atom number, whereas the Au burden per Au-positive cell increased approximately linearly. Larger particles exhibited higher volume concentration factors (VCFs) with a small subset of “high-VCF” cells. These results highlight that both particle size and cellular heterogeneity jointly govern algal bioconcentration patterns and should be accounted for in environmental risk assessment of metal-based nanomaterials.