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 Azolla pinnata 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 A. pinnata, demonstrating its high sensitivity to phthalate toxicity and validating chlorophyll fluorescence as a rapid, non-invasive biomonitoring tool for assessing aquatic pollutant stress.