Comprehensive metagenomic and enzyme activity analysis reveals the negatively influential and potentially toxic mechanism of polystyrene nanoparticles on nitrogen transformation in constructed wetlands

The widespread use of nanoplastics inevitably leads to their increasing emission into constructed wetlands (CWs). However, little is known about the impacts of nanoplastics on nitrogen transformation in CWs. In this study, the influence of polystyrene nanoparticles (PS NPs), one of the most widely used plastics, on the nitrogen transformation in CWs was comprehensively investigated, and the influential and toxic mechanism was evaluated through metagenomic analysis (DNA level) and key enzyme activities (protein level) related to N-transformation metabolism and antioxidant systems. The results showed that over 97% of PS NPs were retained in CWs, and the biofilm of sand was the main sink of PS NPs. Exposure to 1 and 10 mg/L PS NPs suppressed the nitrogen transformation, causing a certain degree of inhibition in TN removal, especially in the relatively short term of the exposure experiment (p < 0.05). At the protein level, 1 and 10 mg/L PS NPs negatively affect enzyme activities involved in denitrification (nitrate reductase and nitrite reductase) and electron transport system activity (ETSA). In contrast, 10 mg/L of PS NPs significantly suppressed the activities of nitrifying enzymes (ammonia monooxygenase, hydroxylamine dehydrogenase and nitrite oxidoreductase), whereas 1 mg/L PS NPs showed no impacts on nitrifying enzymes. Metagenomic analysis further certified that PS NPs restrained the relative abundances of genes involved in nitrogen transformation including nitrification and denitrification biochemical metabolisms (the electron production, electron transport and electron consumption processes). It also indicated that PS NPs could affect nitrogen transformation by reducing the abundance of genes for electron donor and ATP production involved in carbon metabolism (glycolysis and tricarboxylic acid cycle metabolism). In our study, the potential toxic mechanisms of PS NPs attributed to over production of reactive oxygen species and variations of antioxidant systems in macrophytes and microorganisms. These results provided valuable information for evaluating the impacts of PS NPs on CWs and arouse more attention to their impacts on the global geochemical nitrogen and carbon cycles.

Automated summary

This study found that polystyrene nanoparticles (PS NPs) had a significant inhibitory effect on nitrogen transformation in constructed wetlands, particularly in short-term exposure experiments. PS NPs mainly accumulated in the biofilm of wetlands, affecting a range of enzyme activities related to nitrogen metabolism and antioxidant systems. The toxic mechanisms of PS NPs may involve altering the abundance of nitrogen transformation genes and key genes in carbon metabolism.