Study On The Combined Toxicity And Mechanism Of Typical Antibiotics And Disinfectants To Chlorella Pyrenoidosa

Antibiotics are widely used to prevent diseases and develop aquaculture.However,antibiotics cannot be fully utilized,and residual antibiotics will be released into the environment,resulting in serious environmental pollution,and will produce cumulative toxicity to the human body through the amplification of the food chain.In addition,with the outbreak of novel Coronavirus,the use of disinfectants is increasing day by day.However,the excessive use of disinfectants will generate a large amount of residue into the environment,which will greatly damage the aquatic ecosystem,affect the water quality and thus increases ecological and human health risks.Antibiotics and disinfectants entering the water environment are likely to coexist,forming complex mixtures of pollutants that pose greater risks to aquatic life and even human health.Therefore,widely used four tetracycline antibiotics(TCLs),tetracycline(TCL),chlorotracycline(CTC),oxytetracycline(OTC),doxycycline hydrochloride(DHY)and four quaternary ammonium salt disinfectant(QASD),benzalkonium bromide(BKB),didodecyldimethylammonium bromide(DAB),domiphen(DOM)and tetradecyl dimethyl benzyl ammonium chloride(TDC),were selected as research objectives.The binary,ternary and more complex mixture systems of TCLs and QASD were designed by direct equipartition ray(Equ Ray)and uniform design ray(UD-Ray).The single and ternary toxicity effects of TCLs and QASD were investigated by the time-dependent microplate toxicity analysis(t-MTA)based on Chlorella pyrenoidosa(C.pyrenoidosa).The toxicity interactions of mixtures were qualitatively and quantitatively characterized by concentration addition(CA)model and fitting zeroing method,respectively.The toxicity mechanism of TCLs and QASD on C.pyrenoidosa was analyzed by the changes of chlorophyll and protein content determined by acetone method and ultraviolet spectrophotometer method,and the changes of cell morphology of C.pyrenoidosa,observed by scanning electron microscope.The main conclusions are as follows:(1)The toxicity of four TCLs and four QASDs to C.pyrenoidosa showed a good concentration-effect relationship over the whole exposure time,and had time-dependent and concentration-dependent toxic effects.The toxicity of the eight chemicals varied.Taking the exposure time endpoint(96 h)as an example,the order of toxicity was as follows:TDC(p EC₅₀=5.625)>DOM(p EC₅₀=5.493)>BKB(p EC₅₀=5.365)>DAB(p EC₅₀=5.363)>OTC(p EC₅₀=3.750)>CTC(p EC₅₀=3.670)>DHY(p EC₅₀=3.650)>TCL(p EC₅₀=3.470)?(2)Six groups of binary mixture system,four groups of ternary mixture system and one group of quaternary mixture system,altogether 55 rays,of the four TCLs,showed a good concentration-effect relationship to C.pyrenoidosa.And they had time-dependent and concentration-dependent toxicity.Among the binary,ternary and quaternary mixtures of TCLs,only TCL-DHY,OTC-DHY and CTC-DHY systems showed antagonism,while the other three binary,four ternary and one quaternary mixture systems showed no toxicity interaction(addition).In addition,the antagonism appeared in the medium concentration region,the antagonism related to the proportion of components.(3)Six groups of binary mixture system,four groups of ternary mixture system and one group of quaternary mixture system,altogether 55 rays,of the four QASD,showed a good concentration-effect relationship to C.pyrenoidosa.And they had time-dependent and concentration-dependent toxic effects.In binary,ternary and quaternary mixture systems,except TDC-DAB-BKB showed antagonistic effect in medium and high concentration region,the other mixture systems(six binary,three ternary and one quaternary)showed time-dependent synergism which related to concentration and composition.(4)Four quintuple mixture systems and one quintuple mixture system,altogether 38rays,of the TCLs and QASD,showed a good concentration-effect relationship to C.pyrenoidosa.And they had time-dependent and concentration-dependent toxicity.Except for the antagonism of some rays(U7-72 h and U6-96 h)in TDC-DAB-BKB-DOM-TCL system,the synergism of the other five-and eight-element mixture systems was obvious in a specific concentration ratio and concentration range,and the synergism intensity increased first and then decreased with the increase of exposure concentration.(5)Single toxicity of TCLs and QASD and their combined toxicity could destroy the cell morphology of C.pyrenoidosa,such as ablation,lysis,folding and deformation,etc.,so that algae cells could not grow and metabolize normally.In addition,TCLs and QASD can inhibit the production of chlorophyll a,chlorophyll b and protein contents of C.pyrenoidosa,thus inhibiting the growth of C.pyrenoidosa.