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The use of glyphosate-based herbicides (GBHs) worldwide has increased exponentially over the last two decades increasing the environmental risk to marine and coastal habitats. The present study investigated the effects of GBHs at environmentally relevant concentrations (0, 10, 50, 100, 250, and 500 ug.L-1) on the physiology and biochemistry (photosynthesis, pigment, and lipid composition, antioxidative systems and energy balance) of Ulva lactuca, a cosmopolitan marine macroalgae species. Although GBHs cause deleterious effects such as the inhibition of photosynthetic activity, particularly at 250 ug.L-1, due to the impairment of the electron transport in the chloroplasts, these changes are almost completely reverted at the highest concentration (500 ug.L-1). This could be related to the induction of tolerance mechanisms at a certain threshold or tipping point. While no changes occurred in the energy balance, an increase in the pigment antheraxanthin is observed jointly with an increase in ascorbate peroxidase activity. These mechanisms might have contributed to protecting thylakoids against excess radiation and the increase in reactive oxygen species, associated with stress conditions, as no increase in lipid peroxidation products was observed. Furthermore, changes in the fatty acids profile, usually attributed to the induction of plant stress response mechanisms, demonstrated the high resilience of this macroalgae. Notably, the application of bio-optical tools in ecotoxicology, such as pulse amplitude modulated (PAM) fluorometry and laser-induced fluorescence (LIF), allowed separation of the control samples and those treated by GBHs in different concentrations with a high degree of accuracy, with PAM more accurate in identifying the different treatments.