Background: Urban stormwater runoff transports complex contaminant mixtures into freshwater
ecosystems, potentially impairing primary producers and disrupting key physiological functions. Biomarkers
such as photosynthetic pigments, antioxidant enzyme activities, and lipid peroxidation are sensitive tools
for detecting early biochemical stress in aquatic plants. This study aimed to evaluate the effects of urban
stormwater runoff on sub-individual biomarkers in the macrophyte Lemna minor. Methods: Water samples
were collected in autumn 2025, after a rainfall event, from three Portuguese waterbodies (Este River, Sousa
River, Vale das Flores Stream) at upstream, outlet discharge, and downstream sites. Bioassays were
conducted with filtered and unfiltered water samples to distinguish dissolved from particle‑bound
contaminants. L. minor was exposed to a water sample following the standard guidelines for 7 days.
Afterward, biochemical biomarkers were quantified, including total chlorophyll content and carotenoids,
antioxidant enzyme activities (catalase, CAT; glutathione S-transferases, GSTs), and lipid peroxidation
(malondialdehyde, MDA content). Results: A decrease in pigment content was observed in both treated
water samples, indicating a disruption of photosynthetic processes. Overall, antioxidant defense
mechanisms were activated, as shown by the increased activities of CAT and GSTs. The rise in MDA levels
suggests that lipid peroxidation and cellular damage occurred across all water samples. Moreover, these effects
were more pronounced in the unfiltered samples, underscoring the relevance of particulate‑bound
toxicants. Vale das Flores stream exhibited the strongest responses, with an increase in CAT and GSTs
activities and MDA content, as well as a reduction in pigment levels. Additionally, the outlet discharge and
downstream samples induced a more pronounced biochemical response in L. minor. Conclusions: This
integrated biomarker approach proved sensitive for detecting sub-individual effects of urban stormwater
runoff. These findings highlight the value of biochemical indicators for assessing contaminant effects on
primary producers and support their use in water‑quality and ecotoxicological monitoring of urban
freshwater systems.