Toxicological Responses of Photosynthetic Genes in Chlorella vulgaris Exposed to Environmentally Relevant Concentrations of TiO2 Nanoparticles

Engineered nanoparticles are increasingly released into aquatic environments, raising concerns about their effects on primary producers. Titanium dioxide nanoparticles (TiO₂ NPs), one of the most widely used nanomaterials, are frequently detected at low concentrations in surface waters. Here, we investigated the impact of environmentally relevant TiO₂ NP concentrations (1.1-17.6 µg/L) on the freshwater microalga <i>Chlorella vulgaris</i> by combining standardized growth inhibition bioassays with transcriptional analysis of photosynthesis-related genes. Cultures were exposed for 72 h following OECD TG 201, and cell density, growth factor (GF), and specific growth rate (µ) were determined to validate bioassay reliability. Gene expression of six photosynthetic genes (<i>atpB</i>, <i>psaA</i>, <i>psaB</i>, <i>psaD</i>, <i>psbA</i>, and <i>rbcL</i>) was quantified by RT-qPCR and normalized against 18S rRNA. Statistical analyses included Shapiro-Wilk and Levene's tests, followed by one-way ANOVA with Bonferroni or Dunnett T3 post hoc corrections. The results showed a hormetic growth response, with stimulation at intermediate NP concentrations and no inhibition at the highest dose. At the molecular level, <i>rbcL</i> was significantly repressed at 1.1-4.4 µg/L, while <i>psaA</i> and <i>psaD</i> were upregulated at 8.8-17.6 µg/L, indicating compensatory reinforcement of photosystem I. These divergent transcriptional trajectories demonstrate that molecular endpoints reveal sublethal effects not evident from cell counts alone. Overall, this study highlights the potential of photosynthesis-related genes as early biomarkers for detecting nanoparticle-induced stress in aquatic primary producers.