Combined Effects And Mechanisms Of Low Concentration Mixed Antibiotics On Chlorella Vulgaris

Pharmaceutical and personal care products(PPCPs)are used as a new class of environmental pollutants,which are complex and widely used.And they have received increasing attention due to the environmental behavior and toxicological effects in recent years.Among them,antibiotics are frequently detected in various environments,and have certain biological activities.The antibiotics have caused potential risks to various types of organisms and human health,while their ecological toxicological data are seriously insufficient.The various antibiotics tend to have "mixed-long-dose-low-dose" exposure characteristics in aquatic environment,but most of the current studies only focus on the single biotoxicity of high-dose antibiotics,and joint toxicity studies are extremely scarce.Therefore,it is an urgent need for a joint toxicity study of low concentrations in mixed antibiotics on aquatic organisms.In this paper,three common antibiotics,erythromycin(ERY),enrofloxacin(ENR)and sulfamethoxazole(SMX),were used as target compounds to study their individual and combined effects on Chlorella vulgaris.Then two antibiotics,ERY and ENR with relatively high toxicity,were selected to explore their combined mechanisms at low concentration from physiological response and gene transcript level in C.vulgaris.The major results are as follows:(1)The basic toxicity test results of three antibiotics against C.vulgaris showed that the 96 h-EC50 of ERY,ENR and SMX to C.vulgaris were 86,125 and 1 673 ?g/L,respectively,indicating that the toxic order was ERY > ENR > SMX.However,these three antibiotics significantly promoted algae growth at low concentrations.The results of the joint toxicity test showed that each binary mixture of these three antibiotics showed a synergistic effect to C.vulgaris.(2)The results on the antioxidant defense system in C.vulgaris showed that the exposure of ERY and ENR increased the content of malondialdehyde(MDA)in C.vulgaris,indicating that both antibiotics caused oxidative damage to C.vulgaris.In addition,the activities of superoxide dismutase(SOD),peroxidase(POD),catalase(CAT),ascorbate peroxidase(APX),and glutathione reductase(GR)in C.vulgaris also increased in various degree.The increase of reduced glutathione(GSH)and oxidized glutathione(GSSG)further proved that C.vulgaris suffered oxidative stress.Combined toxicity experiments showed that the combination of ERY and ENR synergistically increased the activity of antioxidant enzymes and the contents of MDA,GSH and GSSG.(3)We detected the transcript abundance of three photosynthesis-related genes(psaB,psbC and chlB)in C.vulgaris by real-time PCR.The results showed that the transcript abundance of these three genes increased under the exposure of ERY and ENR.And the combination of these two antibiotics could synergistically increase the transcript abundance of the three genes.The chlorophyll content showed a hormesis effect.More specifically,the antibiotics could stimulate synthesis of chlorophyll at low concentration(< 0.03 mg/L),and inhibit synthesis of chlorophyll at high concentration(> 1 mg/L).However,the combination of these two antibiotics with low concentration could significantly inhibit the content of chlorophyll.Based on the above findings,we found that the effects of ERY,ENR and SMX on growth of C.vulgaris all showed a hormesis effect,and they had a significant synergistic effect when they were combined.The mechanism of combined ERY and ENR at low concentration was confirmed by the changes in antioxidant defense system and photosynthetic gene expression of C.vulgaris.It provided a scientific basis for ecological risk assessment of combined antibiotics at low-concentration exposure in freshwater environments.