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Copper oxide nanoparticles (CuO NPs), used in antifouling paints of boats, are released in the environment and can induce toxicity to aquatic organisms. In this report, we used chlorophyll a fluorescence imaging to evaluate CuO NPs toxicity in Lemna gibba. This approach allowed to evaluate the differential effect of CuO NPs on photosynthesis of whole L. gibba plants. Exposure to 0.1 to 0.4 g/L CuO NPs during 48h induced strong inhibition of photosynthetic processes resulting in a decrease of plant growth. By using fluorescence imaging, different photosynthetic parameters were evaluated simultaneously in microplate conditions. Imaging of F_O fluorescence yield showed the decrease of leaf photosynthetic active surface for whole plants exposed to CuO NPs. This method showed that CuO NPs inhibited photosystem II maximal, photosystem II operational quantum yields, and photochemical quenching of …