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

## TL;DR

This study shows how TiO2 nanoparticles affect photosynthesis in algae at low concentrations, revealing hidden stress effects not seen in growth rates.

## Contribution

The study introduces photosynthesis-related genes as early biomarkers for nanoparticle stress in aquatic organisms.

## Key findings

- TiO2 NPs caused a hormetic growth response in Chlorella vulgaris with stimulation at intermediate concentrations.
- rbcL gene was repressed at low TiO2 NP concentrations, while psaA and psaD were upregulated at higher concentrations.
- Molecular endpoints revealed sublethal effects not detected by traditional cell count methods.

## Abstract

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 Chlorella vulgaris 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 (atpB, psaA, psaB, psaD, psbA, and rbcL) 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, rbcL was significantly repressed at 1.1–4.4 µg/L, while psaA and psaD 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.