Polypropylene microplastics modify copper associated leaf injury and photosynthetic responses in Nymphaea ‘Black Beauty’

Introduction Microplastics and heavy metals cooccur in freshwater habitats, yet it remains unclear whether microplastics intensify metal toxicity or redirect the dominant mode of plant injury. Methods We exposed the floatingleaved aquatic plant Nymphaea `Black Beauty' for 28 d to control conditions, copper (Cu) alone, polypropylene microplastics (PPMPs) alone, or combined Cu and PPMP stress. Wholeplant phenotype, relative growth rate, leaf chlorosis and necrosis, root scanning electron microscopy, gas exchange, chlorophyll fluorescence, malondialdehyde (MDA), and antioxidant enzymes were integrated to distinguish structural damage from functional inhibition. Results Cu alone produced a necrosisdominated response: relative growth rate decreased by approximately 65%, net photosynthetic rate (Pn) by 54%, effective PSII quantum yield (ΦPSII) by 52%, and electron transport rate (ETR) by 47%, whereas MDA increased by 58% and necrotic area reached 26.4%. PPMPs alone caused moderate growth inhibition and chlorosis, with a numerically higher nonphotochemical quenching (NPQ) mean and little lipid peroxidation. Factorial analysis detected Cu × PP interactions for selected endpoints, including growth, necrosis, Pn, NPQ, ETR, and POD; these endpointwise tests were exploratory. Necrotic area declined to 2.1%, but growth and photosynthesis remained strongly inhibited. The density of morphologically assigned PPlike structures in processed root sections was 4.16fold higher under combined exposure than under PPMPs alone. Under coexposure, Pn declined while no differences in stomatal conductance or transpiration from the control were detected, while ΦPSII, ETR, and NPQ decreased markedly. The lower NPQ mean was consistent with reduced regulated thermal dissipation under the measurement conditions but was not diagnostic of a specific photoprotective defect. POD activity increased by approximately 40%, whereas SOD activity decreased by 35%, showing an enzymespecific antioxidant pattern rather than demonstrating enhanced overall detoxification. Discussion Thus, under the shared 2% Tween20 background, Cu -PP coexposure produced a lownecrosis, highfunctionalinhibition phenotype consistent with an important nonstomatal component. These responses do not establish altered Cu distribution, particle internalization, or detoxification.