Growth Inhibition Effect And Mechanism Of Tiamulin On Typical Algae

In recent years,antibiotics,as a new type of contaminants,have been detected in natural water bodies around the world,which has attracted widespread attention.Antibiotics in the aquatic environment negatively affect the normal physiological activities of aquatic organisms,and then destroy the ecosystem stability.In China,tens of thousands of tons of antibiotics are used as veterinary medicine every year.As one of the world's top ten veterinary antibiotics,Tiamulin(TIA)has not received corresponding attention.Until now,the toxic effect and mechanism of this antibiotic on the aquatic ecosystem are still unclear.In this study,a simple,convenient,fast and reliable method was firstly established for the determination of antibiotic concentration in water environment.Secondly,with the purpose of determining the effects of TIA on the growth,oxidative stress,antioxidant mechanism and the toxic mechanism at transcriptional level in the alga,inhibition of algal growth and determination of markers of oxidative stress and gene expression of two typical species of microalga,i.e.,Raphidocelis subcapitata(R.subcapitata)and Chlorella vulgaris(C.vulgaris),were conducted,according to the OECD 201 guideline recommended by the organization for economic cooperation and development.The research results are as follows:(1)A quantitative method for the determination of TIA in water environment was established,by combining with solid phase extraction(SPE)technique,water bath nitrogen blowing and liquid chromatography–mass spectrometry technology.In this solution,atrazine-D5 was chosen as internal standard in internal standard dilution method internal standard dilution method.The coefficient of determination(R²)of the established standard curve of TIA and its substitute(erythromycin-¹³C-D3)were all above 0.995.The recovery of TIA meets the requirements.(2)Comparison of the growth inhibition effect of TIA on the R.subcapitata and C.vulgaris was further conducted.Results showed that the EC₅₀ of Tiamulin on R.subcapitata was0.04495 mg·L^-1,which was much lower than that of C.vulgaris(0.5745 mg·L^-1).It is concluded that R.subcapitata is more sensitive to TIA than C.vulgaris.(3)Based on the growth inhibition experiment of TIA on the algae mentioned above,the content/activity of oxidative stress and antioxidant markers were determined.The results indicated that at the concentration of 0.02 mg·L^-1 and 0.04mg·L^-1,TIA inhibited the growth of R.subcapitata but promoted the growth of C.vulgaris.TIA affects the growth of algae by regulating the content of reactive oxygen species(ROS)in algal cells.R.Subcapitata combats the excessive accumulation of ROS in cells by increasing the activities of superoxide dismutase(SOD),catalase(CAT)and glutathione reductase(GP).However,the ROS content and SOD,CAT and GP content were all lowered in C.vulgaris under the stress of TIA.(4)Based on the growth inhibition experiment of antibiotics on sensitive algal(R.subcapitata),high-throughput sequencing technology was adopted to explore the effect of TIA on the gene expression of this alga.It can be found that environmental concentration of TIA(0.002 mg·L^-1)had no significant effect on R.subcapitata.However,TIA of 0.06 mg·L^-1 could affect the cell activity at transcriptional level.Totally 2028(1074 up-regulated,954down-regulated)genetic variation in 0.06 mg·L^-1 TIA exposed R.subcapitata were identified,in comparison to untreated alga.The concentrations of tiamulin raised related to metabolism of dysplasia and photosynthetic organisms solid carbon way genes,cut and photosynthesis,endoplasmic reticulum protein processing,fatty acid biosynthesis of related genes,which inhibits the growth of algal cells.In this study,the mechanism of TIA's toxic effect on algae was revealed,providing a scientific basis for the evaluation and management of antibiotic chemicals.