3D tracking and behavioral analysis of Tetraselmis as a bioindicator of copper pollution in marine environment

<i>Tetraselmis</i> is a genus of unicellular, motile green algae that plays a crucial role in regulating aquatic ecosystems. Since the presence of copper in marine environments alters its cellular functions and vital process, this kind of algae can be involved as bioindicators of toxicity in waters. Digital holography (DH) is a valuable technique for monitoring the trajectories of <i>Tetraselmis</i> due to its ability to refocus images. We leveraged this capability to perform precise 3D tracking of motile <i>Tetraselmis</i>. In this study, we characterize <i>Tetraselmis </i>behavior over different copper doses (5, 10, 25 and 50 μmol) across three exposure times: T1 (24 h), T2 (48 h), and T3 (72 h), analyzing both kinematic and morphometric parameters. However, since doses of copper exceed 10 μmol cause <i>Tetraselmis</i> death, the real challenge lies in detecting the presence of copper at lower concentrations (i.e. sub-lethal), where no visible signs of mortality are observed. We demonstrated that <i>Tetraselmis</i> motility and morphology can be involved as bioindicator of copper pollution, achieving an accuracy of over 92% in the binary classification, which serves to discriminate between <i>Tetraselmis</i> exposed to copper and those unexposed. Moreover, in a multiclass classification approach, we obtained over 84% accuracy in classifying different sub-lethal copper doses.