Responses And Mechanisms Of Chlorella Pyrenoidosa Exposed To Typical Macrolide Antibiotics

In the past few decades,the extensive use of antibiotics has led to their frequent detection in aquatic environments,causing widespread concern about the toxic stress on aquatic organisms.Antibiotic residues in water environment can produce a variety of degradation products through biodegradation and non-biodegradation,which may induce higher ecological risks.Previous studies have extensively conducted research on the acute ecotoxicity effects of antibiotics to aquatic environments.However,the mechanisms of chronic toxicity caused by long-term exposure to antibiotics and the toxicity effects of their degradation products are still not well understood.In this study,two macrolide antibiotics（MLs）,erythromycin（Ery）and roxithromycin（Rox）,which are frequently detected in water environments and have a high ecological risk,were selected as target pollutants.Chlorella pyrenoidosa,a representative green microalga commonly used in toxicological experiments due to its common occurrence and high sensitivity,was used as the test organism.The acute and chronic toxicity responses of C.pyrenoidosa to MLs under short-term（96hours）and long-term（21-day growth period）exposure were investigated,the removal and degradation mechanism of MLs during long-term exposure were clarified,and the toxicity effects of degradation products on C.pyrenoidosa were explored.Finally,based on the gene expression of C.pyrenoidosa cells and the functional response of bacterial communities,the response mechanism of C.pyrenoidosa to typical MLs and their degradation products under long-term stress was revealed,with the aim of providing scientific basis for the ecological risk assessment of antibiotics in water environments and the formulation of relevant environmental quality standards.The main research results are as follows:（1）Ery and Rox exhibited a"low-stimulation-high-inhibition"hormesis phenomenon in terms of growth and chlorophyll of C.pyrenoidosa.Low concentrations of MLs promoted the growth and chlorophyll synthesis ability of microalgae cells,while high concentrations of MLs exhibit significant inhibitory effects,which generally increase with increasing concentration.The 96-h EC₅₀ values of Ery and Rox for C.pyrenoidosa were 11.78 and 0.81 mg/L,respectively.Under high concentration MLs exposure stress,superoxide dismutase（SOD）and catalase（CAT）were insufficient to effectively remove oxidative damage induced by reactive oxygen species（ROS）,resulting in significantly higher malondialdehyde（MDA）content than the control group（p<0.05）.In chronic toxicity experiments,Ery had a greater inhibitory effect on the growth and chlorophyll synthesis of microalgae cells in the first 14 days,and then gradually weakened;while the inhibitory effect of Rox on the growth and chlorophyll synthesis of microalgae cells gradually increased within 21 days.Long-term exposure to MLs resulted in significantly higher oxidative damage to microalgae cells than the control group.Ery caused CAT activity to be insufficient to effectively remove oxidative damage caused by H₂O₂,while Rox caused SOD activity to be insufficient to remove oxidative damage caused by O₂^-.（2）C.pyrenoidosa can accelerate the removal efficiency of MLs from water.After 21 days of exposure to the MLs system,the remaining amounts of Ery and Rox were between 4.04%-23.53%and 22.92%-34.75%,respectively.The removal efficiency of MLs was positively correlated with the biomass and chlorophyll content of microalgae cells.The main mechanisms for MLs removal from water in the exposure system are photodegradation,biodegradation,bioadsorption,and bioaccumulation.Biodegradation and photodegradation contributed 24.82%-67.01%and29.61%-51.22%,respectively,to the removal of different concentrations of Ery,and46.00%-53.30%and 21.37%-27.15%,respectively,to the removal of different concentrations of Rox.They were the main forms of MLs removal from water,while the effects of bioadsorption and bioaccumulation were negligible.（3）In the long-term exposure system,the degradation products of MLs did not significantly increase the overall toxicity of the system.Under biodegradation and photodegradation,MLs produced degradation products through hydroxylation,dehydrogenation,demethylation,dehydroxylation,dehydration,deethylation and side chain shedding.Among the 11 degradation products produced by the biodegradation of Ery,4 of them（DP749,DP747,DP719A,and DP719B）had higher toxicity than the parent antibiotic Ery.The model predicted that the overall toxicity of the mixture increased in the early to middle stages and decreased to the same level as the initial toxicity in the later stage.The toxicity of the 17 degradation products produced by the biodegradation of Rox was lower than that of the parent antibiotic,and the overall toxicity gradually decreased during the 21-day degradation process.Among the 6 degradation products produced by the photodegradation of Ery,3 of them（DP749,DP747,and DP719）had higher toxicity,and the overall toxicity was higher in the early stage of photodegradation and gradually decreased in the later stage.The 19 degradation products produced by the photodegradation of Rox had lower toxicity than the parent antibiotic,and the overall toxicity gradually decreased with the photodegradation time.The results of the toxicity experiments of the photodegradation products of Ery and Rox on C.pyrenoidosa also showed that the inhibitory effect of the photodegradation products on the growth of microalgae cells and chlorophyll synthesis gradually weakened with the photodegradation time.The microalgae cells did not suffer significant oxidative damage relying on the antioxidant defense mechanisms of SOD and CAT.（4）Long-term exposure to Rox and its photodegradation products resulted in differential expression of 2010 and 2988 genes,respectively,in C.pyrenoidosa cells.In the Rox treatment group,the significantly enriched pathways of differentially expressed genes were photosynthesis,DNA replication,porphyrin and chlorophyll metabolism,and mismatch repair,among others,with a total of 13 pathways（p<0.05）;in the photodegradation products treatment group,the significantly enriched pathways were photosynthesis,photosynthesis-antenna proteins,carbon metabolism,and porphyrin and chlorophyll metabolism,among others,with a total of 12 pathways.Rox treatment led to fold changes of-1.10 to-2.10,-1.48 to-2.98,and-2.11 to-2.47 fold in genes related to porphyrin and chlorophyll synthesis,DNA replication,and mismatch repair pathways,respectively,while most genes related to photosynthesis-antenna proteins,photosynthesis,carbon metabolism,and antioxidant enzymes were significantly upregulated,and the differential expression of the mitochondrial inner membrane protein gene MPV17 was-1.23-fold,which may be the fundamental reason for the inhibition of microalgae cells growth and chlorophyll synthesis and the enhancement of oxidative damage caused by long-term MLs stress.In the photodegradation product treatment group,most genes related to photosynthesis,photosynthesis-antenna proteins,carbon metabolism,porphyrin and chlorophyll metabolism,and antioxidant enzymes were significantly upregulated,providing a foundation for alleviating oxidative damage caused by photodegradation product stress.（5）In the long-term exposure system,bacterial communities played a positive role in protecting microalgae against antibiotic stress.A total of 13 bacterial genera were detected in the long-term exposure system of C.pyrenoidosa to MLs（relative abundance greater than 1%）,among which the norank-f-norank-o-Chloroplast（genus）under Cyanobacteria had the highest relative abundance in all treatment groups,ranging from 57.56%-25.13%,63.61%-28.95%,and 85.73%-47.48%,respectively,and gradually decreased with the cultivation time.Other bacterial genera showed different performances in each treatment group,but in the long term,MLs did not cause significant changes in species richness and diversity in the bacterial community,except for the 10mg/L high concentration Ery treatment group.The PICRUSt2 prediction model results showed that MLs promoted metabolic functions in bacterial communities,such as cell population,cell growth and death,carbohydrate metabolism,and amino acid metabolism.The FAPROTAX prediction model results showed that ecological functions related to nitrogen nitrification and denitrification in nitrogen cycle were significantly inhibited in the high concentration treatment group,which prevented the loss of nitrogen in the system and provided effective protection for the defense mechanism of microalgae against antibiotic stress.