Digital holography unveils sub-lethal copper doses using motility patterns of Tetraselmis microalgae bioprobes

The escalating issue of marine pollution stemming from heavy metals, notably copper, demands urgent attention. To effectively address this concern, the development of reliable techniques is critical to accurately assess the impact of contamination on aquatic ecosystems. Tetraselmis , a genus of unicellular and motile green algae, contributes significantly to the regulation of aquatic ecosystems. However, Tetraselmis vital processes and cellular functions may be altered by the presence of copper in marine environments. Its sensitivity to copper pollution allows exploiting Tetraselmis as a bioindicator of toxicity in waters. In fact, elevated concentrations of copper (exceeding 10 μmol) cause Tetraselmis death, a process that can be easily detected using any imaging technique, as the dead microalgae become motionless. In contrast, at lower copper concentrations, this effect is not observable. Here, we demonstrate that Tetraselmis 3D motility is correlated with copper pollution level in water. We leverage refocusing capability and 3D tracking of Digital Holography (DH) to monitor Tetraselmis trajectories and their morphological parameters. We use morphometric and kinematic information to distinguish between different copper doses over various exposure times. We perform both binary and multiclass classifications to analyze the data. In the binary approach, we achieved an accuracy of over 92 % in differentiating Tetraselmis exposed to copper from those unexposed. In the multiclass approach, we successfully identified and classified different sublethal copper doses, achieving an accuracy of over 84 %. These results show that both Tetraselmis motility and morphology can serve as an effective bioindicator for assessing copper pollution in water samples. • Optical method to identify doses of Cu in water samples. • We use Tetraselmis microalgae as bioprobes for Cu pollution. • Digital Holography as a label-free microscopy method. • Holographic 3D tracking to study the motility patterns of free-swimming Tetraselmis. • Sub-lethal doses identified from machine learning analysis of motility parameters.