Chlorophyll Fluorescence and Biochemical Biomarkers Reveal Plasticizer Di-n-Butyl Phthalate-Induced Stress in Azolla pinnata

Phthalates, particularly di-n-butyl phthalate (DBP), are persistent plasticizers that pose serious ecological risks to aquatic environments. This study evaluated the phytotoxic effects of DBP on the aquatic fern <i>Azolla pinnata</i> through morphological, biochemical, and photosynthetic responses. Plants were exposed to graded DBP concentrations (0-10 mg/L) for 4 and 8 days. Increasing DBP levels caused visible symptoms including frond chlorosis, necrosis, and root inhibition. Biochemically, total chlorophyll content declined by up to 95%, while malondialdehyde (MDA) levels increased by approximately 300% at 10 mg/L, confirming severe lipid peroxidation. Antioxidant enzymes showed biphasic trends: superoxide dismutase (SOD) and catalase (CAT) activities rose under moderate stress but declined by ~73% and ~78%, respectively, at the highest concentration, indicating oxidative enzyme suppression. Chlorophyll fluorescence analysis revealed strong, dose-dependent inhibition of photosystem II efficiency, characterized by reduced performance indices (PIabs, PIcs) and quantum yields (фPo, фEo), alongside increased фDo and Fo/Fm, indicating enhanced energy dissipation and photoinhibition. Overall, DBP exposure disrupted oxidative balance and PSII function in <i>A. pinnata</i>, demonstrating its high sensitivity to phthalate toxicity and validating chlorophyll fluorescence as a rapid, non-invasive biomonitoring tool for assessing aquatic pollutant stress.