Interpreting the dynamic association of nanoplastics with Chlorella vulgaris: insight from single-cell analysis and Gaussian mixture modelling

Abstract

Nanoplastics (NPls) have been recognized as emerging persistent toxic particulates in aquatic environments and their interactions with biota pose growing ecological concerns. Yet, the mechanisms governing their cellular association and resulting toxicity remain poorly resolved because population-level assays obscure the inherent variability among individual cells. Here, we integrate single-cell inductively coupled plasma mass spectrometry (SC-ICP-MS) with Gaussian mixture modelling (GMM) to quantify the dynamic heterogeneous association of Eu-doped polystyrene nanoplastics (Eu-doped NPls) with the model microalga Chlorella vulgaris. The GMM analysis identified distinct subpopulations exhibiting variable particle association that shift dynamically with exposure time and concentration. Across exposures of 5–20 mg L⁻¹, GMM analysis revealed that the majority of microalgal cells (35–65%) belonged to low-association clusters, whereas only a small fraction (0–10%) exhibited high NPls association, with pronounced temporal and concentration-dependent shifts in subpopulation structure. A generalized linear model (GLM) further demonstrated that these high-burden subpopulations disproportionately account for observed growth inhibition, increasing from 12.75% at 5 mg L⁻¹ to 39.34% and 43.05% at 10 and 20 mg L⁻¹, respectively. Synchrotron-based nano X-ray fluorescence (nano-XRF) provided spatial evidence consistent with particle localization within or closely associated with algal cell, while physiological endpoints (chlorophyll-a content, CO₂ fixation, ROS, and MDA levels) validated the toxicity trends. This integrative single-cell and unsupervised machine-learning modelling framework provides quantitative evidence that stochastic, heterogeneous interactions underpin NPls toxicity in microalgae. The approach offers a transferable analytical paradigm for elucidating the fate and effects of persistent plastic pollutants in aquatic ecosystems.