Circadian Rhythm Analysis Of Zebrafish Behavior Under Pollutant Exposure

With the rapid development of industrialization,many environmental problems such as water pollution,global warming,and land desertification are also increasing.Among these environmental pollutants,water pollution a serious concern worldwide.Water pollution and pollutants could be seriously threatens to the aquatic organisms and ecosystems.Additionally,it is hard to determine the effects of pollutants under water pollution accidents..Therefore,it is mandatory to develop a reliable monitoring system to evaluate the water quality.Available traditional water quality assessment methods main focus on physical and chemical parameters of the water bodies.In recent years,many countries have begun to establish online bio-monitoring systems to more effectively assess the water quality.With the continuous development and improvement of online bio-monitoring systems,the proportion of its application in water environment monitoring is increasing.Online bio-monitoring systems combined with the physical and chemical monitoring methods thus gradually become an effective way to monitor ecotoxicity of pollutants.Recently,online monitoring systems are adopted in a laboratory to observe the behavior responses of fish.Along with swimming behavior,circadian rhythm was considered as early warning signals in the field of ecotoxicology.In this research,an online biological monitoring system was used to analyse the potential negative impacts of pollutants,and zebrafish was used as the indicator organism to conduct a series of behavioral experiments.The main purpose of the study was to access the impact of environmental stress on circadian rhythm of zebrafish behavior towards water quality monitoring,and to propose circadian rhythm as a potential evaluation index in ecotoxicology.The test chemicals(Deltamethrin,Atrazine,and Thallium)were identified as ubiquitous contaminants.The chemical concentrations chosen in the study were based on Environmental Quality Standard for Surface Water of China.Behavior responses of zebrafish to different concentrations of Deltamethrin,Atrazine,and Thallium(Tl)were conducted for 15 days.The data was processed using MATLAB environment to characterize behavioral and circadian rhythm changes throughSOM,autocorrelation,and time delay analysis.The results of the present study are as follows:First,in the control group,the overall behavioral intensity was 0.80 ± 0.13,and the behavioral intensity of the light period was significantly(p <0.01)higher than the dark period.The differences between AVL and AVD was 23.9%.In 2 μg/L Deltamethrin,20 μg/L Deltamethrin,3 μg/L Atrazine,30 μg/L Atrazine,and 0.1 μg/L Tl treatment groups,the overall behavior strength was 0.76 ± 0.10,0.67 ± 0.11,0.76 ± 0.13,0.66 ± 0.12,and 0.53 ± 0.10,respectively.The differences were 18.8%,21.7%,23.5%,27.6%,and 38.6%,for 2 μg/L Deltamethrin,20 μg/L Deltamethrin,3 μg/L Atrazine,30 μg/L Atrazine,and 0.1 μg/L Tl treatment groups,respectively.The behavior strength of light period was significantly(p <0.001)higher when compared to the dark period.To a certain extent,the results indicate that the circadian rhythm of zebrafish behavior.The behavior strength result indicate that zebrafish are active during light period and relatively calm during dark period.Second,the behavior responses of zebrafish are in line with the stepwise behavior response model(from non-toxic effect stage to stimulation stage,adjustment stage and readjustment stage are clearly reflected).In all conditions a clear circadian rhythm was observed.The peaks in the autocorrelation analysis appear periodically and show good symmetry,reflecting the characteristics of a 24 h periodic cycle.SOM analysis reveals that most of the behavioral intensity matches the time distribution of the light and dark periods.These results explain the circadian rhythm of behavior.Third,the inhibition phenomenon of the behavioral intensity was clearly manifested in all treatment groups.Behavioral intensity in 20 μg/L Deltamethrin,30 μg/L Atrazine,and 0.1 μg/L Tl groups were extremely significant(p <0.001),and the degree of inhibition was more obvious than other treatment groups(p <0.01)of 2 μg/L Deltamethrin,3 μg/L Atrazine.Moreover,the degree of inhibition of behavioral activity appears to increase with increasing environmental stress.Fourth,no significant change in the circadian rhythm periodicity of the control group wasnoted.But in all chemical treatments,the periodicity was abnormal with time delay.The mean time of abnormal behavior periodicity would reach 1.75 h,1.90 h,2.62 h,2.08 h,and 1.77 h for20 μg/L Deltamethrin,2 μg/L Deltamethrin,30 μg/L Atrazine,3 μg/L Atrazine and 0.1 μg/L Tl,respectively.At the same time,the difference of behavior strength between light and dark period was relatively stable in the control group(maintained between 20% and 40%).The difference of behavior strength between light and dark period in treatment groups was more severe(142.5%)than that in the control group.In a word,zebrafish under the experimental conditions in this study showed a clear circadian rhythm.The behavior circadian rhythm was influenced even under exposure to low-concentration pollutants that do not affect its normal survival.Thus,considering the sensitivity,the behavior circadian rhythm could be used as a potential indicator for water quality assessment.But it is difficult to directly apply this theory to water quality monitoring.Hence,more studies are warranted to quantify the phenomenon of behavior circadian rhythm.