Formation Of Degradable Micro/Nano Plastics And Heterogeneous Agglomeration And Toxicological Studies On Algae

Micro/nano plastics（MNPs）produced by physical,chemical,and biological aging of plastic products in the environment can be harmful to the ecological environment.As an alternative to conventional plastics,degradable plastics are considered as one of the ways to deal with plastic pollution.In recent years,research on the aging process of plastics has become a hot topic,but research on the aging of degradable plastics and the formation process of MNPs is still slightly insufficient.In addition,NPs may heterogeneously agglomerate with coexisting algal cells in water and produce toxic effects,which in turn affect ecosystem stability.However,studies on the toxic effects of algae at this stage ignore the presence of degradable NPs,which may lead to a biased understanding of the ecological risks of degradable NPs in real environments.In view of this,in this study,polylactic acid（PLA）and poly（butyleneadipate-co-terephthalate）（PBAT）blends were firstly selected to study the aging process of water,UV irradiation,and the combined effect of both for 30 d.Subsequently,Chlorella vulgaris and Microcystis aeruginosa were used as test organisms to elucidate the heterogeneous aggregation behavior of PLA and polystyrene（PS）NPs with algae.Finally,toxicological studies were conducted to clarify the differential response mechanisms of physiological and biochemical indicators of the 2 algae under the stress of PLA and PS NPs.The main results and conclusions of this study are as follows:（1）In this study,different exposure conditions were able to affect the aging of PLA/PBAT films and the production process of MNPs.Compared with air medium,the surface morphology of PLA/PBAT films in water was rougher and the oxygen-containing functional groups,carbonyl index（CI）,and oxygen-to-carbon ratio（O/C）were significantly decreased after 30 d of aging.The analysis showed that this was due to the difference in the degradation rates of PLA and PBAT during the hydrolysis process due to their different structures.A large number of cracks and pores were generated on the surface of PLA/PBAT films,which increased the area of the samples exposed to UV irradiation.While accelerating the chemical bond breakage,the free radicals generated from photochemical reactions also increased accordingly,thus accelerating the aging process.After 30 d of aging,a large number of MNPs with particle sizes ranging from 0.19 to 4.85μm were attached to the surface of the UV irradiated PLA/PBAT films in water,and it is known that these MNPs may be shed with the prolonged aging time based on the porosity of 7.63%on their surfaces.（2）The results of co-sedimentation experiments showed that heterogeneous agglomeration could occur between NPs and algal cells.Except for theΔODr_educed<0 for PLA NPs and extracellular polymeric substances-contain（EPS-C）of Microcystis aeruginosa,theΔOD_reducedwas greater than 0 in all experimental groups.TheΔOD_reduced range of PLA NPs and algal cells was between-0.199 and 0.495,while theΔOD_reduced range of PS NPs and algal cells was between 0.009 and 0.741.The results indicated that the heterogeneous agglomeration ability of PS NPs was stronger than that of PLA NPs.DLVO theoretical calculations revealed that the total potential energy of interaction between PLA NPs and algal cells was lower than that of PS NPs,which was theoretically more prone to heterogeneous agglomeration.This is inconsistent with the results of co-sedimentation experiments.It was speculated that EPS secreted by algal cells might influence the heterogeneous agglomeration process in addition to the DLVO effect.Further study revealed that the co-sedimentation rate of algae with NPs and EPS-C was higher compared to algae without EPS（EPS-F）,indicating that EPS can indeed promote the heterogeneous agglomeration process.In addition,3-dimensional excitation emission matrix fluorescence spectroscopy（3D-EEM）results showed that the intensity of fluorescence peaks corresponding to proteins increased significantly during the sedimentation process,indicating that algae secrete large amounts of protein-rich EPS and NPs to undergo agglomeration.During the agglomeration process,the intensity of the fluorescence peak corresponding to tryptophan was lower compared to that of tyrosine,probably due to the fluorescence burst from the interaction with NPs,suggesting that tryptophan may have an important role in the promotion of heterogeneous agglomeration by EPS.（3）High concentrations of PLA and PS NPs produced similar effects on algal density and growth inhibition rates of algae.The growth inhibition rate of Chlorella vulgaris continued to increase with time under PLA exposure,from 8.28%to 30.18%at 24 h to 13.07%to 37.45%at96 h.In contrast,the growth inhibition rate of Microcystis aeruginosa showed a temporal trend of increasing and then decreasing,reaching a peak of 16.90%～33.85%at 72 h.Except for Chlorella vulgaris exposed to PLA NPs,the trends of algal photosynthetic pigment content and algal density in the other experimental groups were consistent.This may be due to the shading effect caused by the formation of agglomerates of EPS and NPs,resulting in higher photosynthetic pigment content of Chlorella vulgaris in EPS-F compared to Chlorella vulgaris in EPS-C,which had higher algal density.Oxidative stress indicators showed that the superoxide dismutase（SOD）activity of algae showed a significant increase when they were exposed to different NPs,and the SOD activity of algae exposed to PLA NPs was higher than that of PS NPs.The malondialdehyde（MDA）content of algae exposed to PS NPs increased significantly,while that of those exposed to PLA NPs did not change significantly,which may be related to the type of NPs.Further analysis by 3D-EEM revealed that the EPS content of algae increased with increasing concentration of PLA NPs exposure,while increasing concentration of PS NPs decreased the EPS content.The presence of EPS was able to reduce the damage caused by NPs,and the decrease in EPS content indicated that PS NPs might cause more severe damage to algal cells.The integrated biomarker responses（IBR）results suggest that since Microcystis aeruginosa is a prokaryotic cell,it does not have a nucleus or complex cell structure.In contrast to Chlorella vulgaris,when the exposure concentration of PLA NPs is increased,Microcystis aeruginosa,which lacks EPS protection,may undergo lipid peroxidation and consequently lead to cell damage.