Distinct oxidative and photosynthetic responses to betulin and betulone in the freshwater microalgae Chlamydomonas reinhardtii

Natural products are increasingly used in therapeutic, cosmetic, and eco-friendly applications, yet their impacts on aquatic ecosystems remain understudied. We investigated the effects of two birch-derived triterpenes, betulin and betulone, on the freshwater microalga Chlamydomonas reinhardtii over 24-72 h at environmentally relevant concentrations (0.01-10 µg/L). Both compounds decreased cell mortality under specific conditions, particularly at 48 h, indicating cytoprotective effects. Betulin increased reactive oxygen species (ROS) at low doses, while betulone consistently reduced ROS. Notably, ROS correlated with mortality only for betulin at 48 h, revealing effects not directly mediated by oxidative stress and pointing to other cellular targets. Betulone, but not betulin, reduced chlorophyll fluorescence, suggesting possible inhibition of chlorophyll synthesis, while photosynthetic efficiency showed transient disruptions followed by recovery, highlighting adaptive cellular responses. These compound-specific, time- and dose-dependent responses suggest that betulin and betulone could interfere with primary producers in freshwater environments, potentially destabilizing photosynthetic function and pigment integrity. Such effects are reminiscent of allelopathic interactions described in terrestrial plants, extending the ecological relevance of triterpenoids to aquatic systems. The observed recovery indicates that C. reinhardtii exhibited resilience in our experimental conditions, yet chronic and higher exposures could overwhelm these protective responses, highlighting the importance of monitoring triterpenoid environmental risks. Overall, this study provides the first comparative insight into the differential effects of betulin and betulone in microalgae and underscores the importance of assessing emerging natural compounds for their environmental safety and ecotoxicological implications.