Growth Inhibition Effect And Mechanism Of Typical Algae Under Erythromycin Stress

In recent years,the production and use of antibiotics are increasing worldwide,and the presence of antibiotics is often detected in the discharge water of drinking water and sewage treatment plants.High and persistent concentrations of antibiotics have been found in water bodies and soils around the world due to the intensive use and continued release of antibiotics.Such heavy contamination can damage human health and exacerbate the risks associated with drug resistance in organisms.In addition,antibiotics and their metabolites have potential and persistent harm to organisms and humans,which makes the residue of antibiotics in the environment a prominent problem.Erythromycin(ERY)is a common macrolide antibiotic,which is also used a lot in the clinical treatment process.Therefore,the concentration of ERY in the aquatic environment is relatively high.In this study,a simple,fast and accurate method was established to detect the concentration of antibiotics in water.Secondly,according to OECD(1984,2011)guidelines,concentration for 50%of maximal effect(EC₅₀)experiments on four species of algae were carried out by ERY to determine the best algae to assess the risk of ERY.Then,the effects of ERY in different concentrations on the physiological conditions of two kinds of algae were investigated at the macroscopic level.In addition,transcriptomics and metabonomics were used to explore the effects of ERY on the molecular level of Crescent Crescent from the gene and metabolite levels.The research results of this paper are as follows:(1)In this paper,a method for the detection of trace ERY was established by SPE,water bath nitrogen blowing and Agilent 1290 high performance liquid chromatography-6470mass spectrometry.The correlation coefficient(R²)of ERY and ERY-substitute(ERY-13C,d3)was greater than 0.999,and the recovery rate of ERY-13C,d3 was greater than 70%.In order to reduce the error in the detection of ERY,we used the internal standard method to monitor the concentration of ERY,and the internal standard was atrazine-d5.(2)Chlorella vulgaris(C.vulgaris),Raphidocelis subcapitata(R.subcapitata),Anabaena flos-aquae(A.flos-aquae),and Synechococcus sp exposure to different concentrations of ERY 4 days,the EC₅₀of ERY for the four kinds of algae was calculated according to the cell density of ERY on the fourth day.The EC₅₀value measured in this study was C.vulgaris:26.3300 mg/L;R.subcapitata:0.0634 mg/L;A.flos-aquae:0.0102 mg/L;Synechococcus sp:0.0045 mg/L.Therefore,C.vulgaris has the weakest sensitivity to ERY,while Synechococcus sp has the strongest sensitivity to ERY.(3)This study compared the growth inhibition,oxidative stress and changes of photosynthetic pigments of C.vulgaris and R.subcapitata.The concentration of 20,40 and60?g/L ERY promoted the growth of C.vulgaris,however,only the concentration of 20?g/L ERY promoted the growth of R.subcapitata,and the concentration of 40 and 60?g/L ERY seriously inhibited the growth of R.subcapitata.ERY can affect the growth of two kinds of algae by affecting the content of reactive oxygen species(ROS)in algae cells.ERY exposure did not significantly affect the ROS content of C.vulgaris,but significantly increased the ROS content of R.subcapitata.In order to combat the excessive ROS in cells,the activity of antioxidants in R.subcapitata increased significantly.Different concentrations of ERY had no significant effect on the changes of photosynthetic pigment content of the two kinds of algae,but the photosynthetic pigment content of C.vulgaris was significantly higher than that of R.subcapitata.This suggests that C.vulgaris has a stronger ability to deal with ROS than R.subcapitata.(4)The effects of ERY at different concentrations(4,80,120?g/L)on the molecular level of R.subcapitata were studied by transcriptomics.ERY at 4?g/L promoted the growth of R.subcapitata,while ERY at 80 and 120?g/L inhibited the growth of R.subcapitata.218(198 up-regulated,20 down-regulated),950(332 up-regulated,618 down-regulated),and2896(1299 up-regulated,1597 down-regulated)differentially expressed genes(DEGs)were identified in 4,80 and 120?g/L ERY treatment groups.ERY at 4?g/L up-regulated genes related to DNA replication pathway and promoted the growth of R.subcapitata.ERY at 80 and 120?g/L inhibited the growth of R.subcapitata by inhibiting xenobiotic metabolism,lipid metabolism and oxidative stress,DNA replication and repair,carbon fixation,photosynthetic pigment synthesis,vitamins synthesis,and steroid biosynthesis and other pathways.(5)The effects of ERY at different concentrations(4,80,120?g/L)on the molecular level of R.subcapitata were studied by metabonomics.ERY at 4?g/L promoted the growth of R.subcapitata,while ERY at 80 and 120?g/L inhibited the growth of R.subcapitata.Compared with the control group,the 4,80,120?g/L ERY treatment groups respectively in two kinds of ion mode had 72(37 up-regulated,35 down-regulated),178(94up-regulated,84 down-regulated),200(106 up-regulated,94 down-regulated)(positive ion mode)and 232(110 up-regulated,122 down-regulated),772(196 up-regulated,576down-regulated)823(199 up-regulated,624 down-regulated)(negative ion mode)difference metabolites.ERY at 4?g/L promoted the purine metabolism,Fatty acid biosynthesis,Taurine and hypotaurine metabolism,ATP binding family(ABC)transporter,pyruvate metabolism,nicotinate and nicotinamide metabolism,and thus promoted the growth of R.subcapitata.The 80 and 120?g/L ERY inhibited ABC transporter,biosynthesis of amino acids,purine metabolism,2-oxocarboxylic acid metabolism,pyrimidine metabolism pathways of R.subcapitata,and thus inhibited the growth of R.subcapitata.