Toxic Effects And Mechanisms Of Typical Pollutants Based On Swimming Behavior Of Daphnia Magna

In recent decades,with the increase of human needs,a large number of synthetic chemicals have been widely used in household articles,textiles,furniture and electronic products,leading to their inevitable entry into the aquatic environment.Toxicological studies have found that these pollutants have many adverse effects on aquatic organisms and even human beings.Thus,it is necessary to establish the evaluation system for carrying out risk assessment or hazard screening of pollutants and provide sufficient data for the standardized usage and environmental management of chemicals.However,the traditional toxicological indicators,such as acute mortality rate,chronic developmental and reproductive indicators,have several disadvantages such as high cost,waste of time and low throughput.Therefore,more sensitive,efficient and comprehensive toxicological evaluation methods are needed.It was found that the behavioral parameters of animals were more sensitive than the traditional toxicological endpoints,and the testing duration was shorter.Therefore,the behavioral tests based on aquatic organisms are of good potential for evaluating the risks of water pollutants.In recent years,with the development of high-resolution monitoring equipment and multifunctional analysis software,behavioral tests are often conducted in toxicological research.However,due to the lack of understanding of basic behavioral patterns and reasonable testing procedures,behavioral test s have not been adopted in the standard procedures for toxicity assessment.Based on that,the toxicological model organism – Daphnia magna was used as the biological subject in this study.First of all,the behavioral parameters of D.magna in the containers of different sizes were compared to select the most appropriate one for formal behavioral tests.And a test program including dark adaptation,the light and dark cycle and vibration stimulation was set up.Then the swimming parameters were determined by using the Danio Vision behavior tracking system(Noldus,Netherlands)to reflect motility,sensitivity and response strength to light stimulation,and response strength to vibration stimulation of D.magna.In this way,the method for behavior measurement based on Daphnia magna neonates was established.On this basis,40 typical pollutants,including heavy metals,flame retardants,insecticides and fungicides,were selected 1)for acute toxicity tests to calculate the half effective concentration(EC50)and the lowest observable effective concentration(LOEC),2)which were used to determine the concentrations(EC5,1/4 EC5,1/16 EC5)for behavioral toxicity test.The toxicity effects of these typical pollutants at sublethal concentrations were evaluated by using behavioral tests to verify the feasibility and high efficiency of our behavioral test methods.The effects of the selected chemicals on gene expression related to neural development,visual perception,muscle composition and signal transduction were analyzed by real time quantitative PCR.Finally,we investigated the toxic effects of subchronic exposure of two insecticides,chlorantraniliprole and fipronil,on the development and behavior of Daphnia magna,and revealed the mechanism of the two insecticides by analyzing the contents of neurotransmitters and the expression of receptor-related genes in D.magna.The main conclusions are as follows:1.By comparing the differences of D.magna behavior in 6-well plates(d=35.14mm),12-well plates(d=22.16mm),24-well plates(d=15.76mm),48-well plates(d=10.82mm)and 96-well plates(d=6.64mm),the optimal test vessel for swimming behavior test is 48-well plate.Analysis of D.magna behavior in 35-minute monitoring procedures(including adaptive phase,light and dark interval and pat vibration stimulation)showed that D.magna behaved actively during light intervals whereas inactively during dark intervals.In addition,D.magna had a short acceleration to both light/dark conversion and pat vibration.The accumulated distance,average speed,light/dark activity,light/dark acceleration and pat distance were used to comprehensively illustrate the movement ability of D.magna,and the baselines of were determined based on the quantitative analysis of these behavioral parameters of a large number of individuals.2.The acute toxicity of 40 typical pollutants,including heavy metals,flame retardants,insecticides and fungicides,to Daphnia magna has been tested according to the OECD 48 h acute test method,and the dose-effect curves between the immobilization rate and the concentrations chemicals have been drawn.The LOEC and EC50 of each chemical were calculated,and it was found that Daphnia magna was the most sensitive to insecticide toxicity.Afterwards,the behavioral toxicity effects of40 pollutants among EC5,1/4 EC5 and 1/16 EC5 concentrations on D.magna were evaluated based on the established behavioral test.The results showed that the behavioral parameters mainly showed 3 types of alterations: concentration-dependent inhibition effect(such as chlorpyrifos),concentration-dependent activation effect(e.g.lead acetate)and no significant effect.This study further analyzes the chloride methyl mercury,lead acetate,BDE-47,bisphenol A,imidacloprid,chlorpyrifos,benzophenone and clotrimazole on gene expression connected to neurodevelopment,visual perception,muscle contractions and nerve signal transduction.As a result,genes related to signal transduction were most significantly affected.This study demonstrated that behavioral parameters of D.magna were more sensitive than acute endpoints,and changes in behavioral parameters could account for the toxic effects of chemicals on different biological tissues or system functions(for example,a decrease in the acceleration of light stimulation suggests impaired visual perception).Moreover,the behavioral test is extremelly effective for screening neurotoxic chemicals or evaluating the neurotoxicity of pollutants.3.On the basis of above studies,chlorantraniliprole(CAP)and fipronil(FIP),two neurotoxic insecticides,were selected as the study objects.The acute toxicity of CAP and FIP were evaluated firstly,and then their LOEC(8 ?g/L and 80 ?g/L)were used as the maximum concentrations for 7-day exposure respectively.The concentrations for 7d exposure concentrations were 1,2,4,8 ?g/L for CAP and 10,20,40,80 ?g/L for FIP,respectively.The mortality rate,cumulative molting number and body length of Daphnia magna were measured every day during the exposure,and the swimming behavior of Daphnia magna was measured at 48 h and 7d,respectively.Furthermore,the content of neurotransmitters and the expression of related receptor genes were analyzed.The mortality,molting number,and body length of D.magna were only significantly affected at the highest concentration of CAP(8?g/L),however,the body length was significantly decreased in a dose-dependent way in the 20,40 and 80 ?g/L groups of FIP exposure.The results of behavioral tests showed that,after 48 hours exposure,both CAP and FIP showed excitability in the low concentration group(1 ?g/L CAP,10 and 20 ?g/L FIP),but inactivity in the high concentration group(8 ?g/L CAP and 80 ?g/L FIP).With the prolongation of exposure time,CAP inhibited the behavior in a dose-dependent manner,while FIP activated the behavior in a dose-dependent manner.The results of UPLC-MS-MS showed that CAP and FIP mainly affected the neurotransmitter system in the early stage of Daphna magna development,and the expressions of receptor-related genes were also mainly affected in the early stage of exposure,indicating that both CAP and FIP had developmental neurotoxicity.Besides,CAP mainly affected the levels of gamma-aminobutyric acid and dopaminergic neurotransmitters,while FIP affects the gamma-aminobutyric acid and cholinergic nervous systems.The significant correlation between behavioral parameters and neurotransmitters,coupled with the change of receptor gene expression proved that behavior was altered by the combined interference of signal transduction of several neurotransmitters.Additionally,KEGG enrichment analysis indicated that CAP and FIP may also interfere with the metabolism of amino acids and lipids in Daphna magna.