Study On Degradation And Affecting Factors Of Niclosamide

Oncomelania hupensis is the only intermediate host of Schistosoma japonicum.Snail control has been proven effective to block the transmission of schistosomiasisjaponica. As a widely used mollusicicide in the schistosomiasis endemic areas,niclosamide has played a critical role in the control of schistosomiasis in China due toits high molluscicidal effect and low toxicity to mammals. However, there is littleknowledge regarding the effect on the ecological environment following long-term,large-scale use of nicloasamide in the endemic foci of China. Currently, the widelyused soil pasting and heaping mixed with niclosamide in mountainous regions arereported to achieve high molluscicidal effect, and the residual agent in the soil1month after soil pasting mixed niclosamide remains active against snails, which maycause effect on ecological environment. In addition, niclosamide exhibits a high acutetoxicity to fish and amphibians. The drug used in the marshland may enter into thewater body through the ways of runoff, permeation and flushing, which seriouslythreatens the safety of fish and amphibians. Moreover, China has a vast territory andthe snail habitant is complex. The degradation rate of niclosamide is bound to beaffected by the environmental variation in different endemic foci. Therefore,exploring the degradation of niclosamide in water and soil and the affecting factorscan facilitate the understanding of niclosamide shift and transfer in water and soil,comprehensively evaluate the prognosis and the existent or potential risk of pollutionin environments, and provide scientific evidence for safe and rational use of thismolluscicide.Part ⅠEstablishment and optimization of determination of niclosamide in waterand soil1.1Establishment and optimization of determination of niclosamide in waterAccording to the established solid phase extraction-high performance liquidchromatography (SPE-HPLC) used to determine the contents of niclosamide in water,some factors which affected the recovery of the solid-phase extraction were optimized,including the pH value of water samples, the loading volume of extraction column, and the adding volume of acetonitrile. The effects of the speed through the column,adding speed of ammonia in eluent, volume of elution and the elution speed on therecovery of the solid-phase extraction were assessed using an orthogonal design, so asto improve the recovery of the extraction.The recoveries of niclosamide were96.00%,95.67%,48.33%,45.00%and5.33%at pH values of3.0,5.0,7.0,9.0and11.0and niclosamide concentration of0.10mg/L using SPE-HPLC. The recoveries of niclosamide were99.33%,98.67%,94.67%,74.00%and64.77%at the water samples of3,5,10,15and30ml. If2ml ofwater samples containing niclosamide was added with acetonitrile at volume ratios of0,10%,20%,50%and100%, the recoveries of niclosamide were24.47%,28.81%,46.73%,68.94%,98.35%by SPE-HPLC. The orthogonal experiment revealed that thefactors affecting the recovery involved elution rate, the adding volume of ammonia ineluent, the rate through the column and volume of elution, ranked by the level ofsignificance.The treatment of water samples was optimized as follows. The pH value of watersamples was adjusted to acidity (pH,3-5). Addition of acetonitrile into water samplesat a volume ratio of100%can significantly increase the efficiency of extraction. Thevolume of niclosamide at a concentration of0.3mgL through PEP column was10ml,and the speed through the column was0.5ml/min.5%ethanol was added, and theelution was done at a volume of3ml and a speed of0.5ml/min. Based on the aboveoptimized conditions, the recoveries of water samples containing niclosamide atconcentrations of0.05,0.10and1.00mg/L ranged from96.28%to97.82%withstandard deviation between1.55%and3.03%, using SPE-HPLC. It is concluded thatthe optimized SPE-HPLC is accurate, simple, highly reproducible, which meets therequirements for determination of the niclosamide contents in water samples.1.2Establishment and optimization of determination of niclosamide content insoilSoil samples were pre-treated by means of ultrasonic extraction and solid phaseextraction using methanol, acetonitrile and dichloromethane as the extraction solvent.By comparing the extraction recovery and screening the best extraction condition the method for determination of niclosamide content in soil was established.Therecoveries detected by HPLC were55.33%for the methanol solid phase extraction,46.67%for the acetonitrile solid phase extraction,12%for the dichloromethane solidphase extraction,63.47%for the methanol ultrasonic extraction,80.40%for theacetonitrile ultrasonic extraction, and98.73%for the dichloromethane ultrasonicextraction. Therefore, the determination of niclosamide content in soil consisted ofchloromethane as the extraction solvent, pretreatment with ultrasonic extraction, anda recovery of>95%, which met the requirement of determination. The efficiency ofultrasonic extraction-HPLC for determination of niclosamide content in soil wasevaluated. Following dichloromethane ultrasonic extraction of theniclosamide-contained soil samples with concentrations of0.5-250.0mg/kg, a goodlinear relationship was detected between the niclosamide concentration and peak area,with a linear regression equation of y=1564.11x-1799.06(R2=0.9999), recoveries of96.16%-101.75%and RSD ranging from2.20%to8.30%. Theniclosamide-contained soil samples with concentrations of0.5,5.0and50.0mg/kgwere repeated three times in parallel, recoveries of95.23%-102.43%and RSD of2.32%-6.99%were detected. It is indicated that this method has a high precision andreproducibility, which meets the requirement of determination of niclosamide contentin soil.It is therefore concluded that the optimized solid-phase extraction is easy toperform, highly precise and highly reproducible for determination of niclosamidecontent in soil, which meets the requirements for determination of the niclosamidecontents in soil in the field.Part ⅡStudy on the factors affecting the degradation of niclosamide in waterThe niclosamide-contained water samples with an initial concentration of0.05mg/L were placed in an incubator (25±1)℃, and exposed to light with intensities of1000and6000lux. Detection was done0,24,48,72,120,168and360days later, toassess the effect of light intensity on niclosamide degradation in water. Theniclosamide-contained water samples with volumes of0.5,1.0and2.0L and initial concentration of0.05mg/L were placed in incubators at (25±1)℃, and exposed tolight with an intensity of6000lux. Detection was done0,24,48,72,120,168and360days later, to assess the effect of water volume on niclosamide degradation inwater. The niclosamide-contained water samples with and initial concentrations of0.05,0.10and0.25mg/L were placed in incubators at (25±1)℃, and exposed to lightwith an intensity of6000lux. Detection was done0,24,48,72,120,168and360days later, to investigate the degradation of niclosamide with various concentrationsin water. The niclosamide-contained water samples with water pH values of5,6,7,8and9and an initial concentration of0.05mg/L were placed in incubators at (25±1)℃,and exposed to light with an intensity of6000lux. Detection was done0,24,48,72,120,168and360days later, to assess the effect of water pH value on niclosamidedegradation in water. The niclosamide-contained water samples with an initialconcentration of0.05mg/L were placed in incubators at (15±1),(25±1) and (35±1)℃,respectively, and exposed to light with an intensity of6000lux. Detection was done0,24,48,72,120,168and360days later, to assess the effect of water temperature onniclosamide degradation in water. The niclosamide-contained water samples with aninitial concentration of0.05mg/L and water:soil ratios of20:1,15:1,10:1and5:1were placed in incubators at (25±1)℃, and exposed to light with an intensity of6000lux. Detection was done0,24,48,72,120,168and360days later, to assess the effectof water:soil ratio on niclosamide degradation in water.The contents of niclosamide gradually reduced in soil with the prolongation oftime. If the light intensity increased from1000lux to6000lux, the degradationhalf-life of niclosamide reduced from7.20days to6.17days; if the water volumeincreased from0.5L to2.0L, the degradation half-life of niclosamide increased from5.83days to6.96days; if the initial concentration of niclosamide increased from0.05mg/L to0.25mg/L, the degradation half-life of niclosamide increased from6.19daysto7.20days; if the water pH value increased from5to9, the degradation half-lifeincreased from5.71to7.13days; if the water temperature increased from (15±1) to(35±1)℃, the degradation half-life reduced from6.89to5.86days; if the water:soilratio increased from5:1to20:1, the degradation half-life of niclosamide increased from4.47days to5.76days. Multiple regression analysis showed that allaforementioned factors had statistical effects on the degradation half-life ofniclosamide (P<0.05).The results showed that the degradation of niclosamide in water accorded withfirst order kinetics equation. The elevation of water volume and initial drugconcentration can cause the reduction in niclosamide degradation, while the increasesin temperature, light intensity and existence of acid environment and soil facilitate thedegradation of niclosamide in water.Part Ⅲ Study on the factors affecting the degradation of niclosamide in soilThe niclosamide-contained soil samples with water contents of10%,30%,50%,70%and90%and initial concentration of5.0mg/kg were stored in an incubator indarkness at (25±1)℃, and detection was done0,1,3,7,10,15,20and30days laterto assess the effect of water content on niclosamide degradation in soil. Theniclosamide-contained soil samples with a water content of30%and initialconcentration of5.00mg/kg were placed in incubators in darkness at (15±1),(25±1),(35±1)℃, respectively, and detection was done0,1,3,7,10,15,20and30days later,to evaluate the effect of soil temperature on degradation of niclosamide in soil. Theniclosamide-contained soil samples with initial concentration of1.0,5.0and10.0mg/kg and a water content of30%were stored in an incubator in darkness at (25±1)℃, and detection was done0,1,3,7,10,15,20and30days later to assess the effectof soil water content on niclosamide degradation in soil. The niclosamide-containednon-sterilized soil and sterilized soil samples with initial concentration of5.0mg/kgand a water content of30%were stored in an incubator in darkness at (25±1)℃, anddetection was done0,1,3,7,10,15,20and30days later to assess the effect of soilmicrobes on niclosamide degradation in soil.The contents of niclosamide gradually reduced in soil with the prolongation oftime. If the soil water content increased from10%to90%, the degradation half-life ofniclosamide reduced from4.258days to2.412days; if the soil temperature increasedfrom (15±1)℃to (35±1)℃, the degradation half-life of niclosamide reduced from4.398days to2.828days. However, if the initial concentration of niclosamide increased from1.0mg/kg to10.0mg/kg, the degradation half-life of niclosamideincreased from3.209days to3.451days. The degradation half-lives of niclosamdie inthe non-sterilized and sterilized soil were3.273and4.886days, respectively, and thedegradation rate constants were0.0758and0.0151, respectively. Multiple regressionanalysis showed that the initial concentration of niclosamide had no statistical effecton the degradation half-life (P>0.05), while the soil water content, soil temperatureand soil microbe had statistical effects on the degradation half-life of niclosamide(P<0.05).The results showed that the degradation of niclosamide in soil accorded with firstorder kinetics equation. The elevation of soil water content and soil temperature andexistence of soil microbes facilitate the degradation of niclosamide in soil, while thedegradation half-life increases with the increases in the initial concentration of theagent.Part Ⅳ Observation the degradation of niclosamide in water and soil collectedfrom the endemic foci4.1Degradation of niclosamide in waterIn September2011, two pools measuring0.63m3and0.82m3，respectively wereselected from a marshland in Jiangxinzhou, Zhenjiang City. SCN and WPN at dosesof0.25mg/L and0.5mg/L were added into the pools, and stirred evenly. Watersamples were collected every5hours on days0-10and once on days15,20,25and30after the addition of the drugs. Five parallel sampling points were set at each pool,and5ml of water samples were collected and stored at10ml tubes for the subsequentdetermination by SPE-HPLC. The experiment was repeated in May2012, but theconcentrations of SCN and WPN were changed into0.50mg/L and1.00mg/L,respectively, and the observation time was120h, while the other conditions were thesame as described above.With the prolongation of the treatment time, the contents of SCN and WPNshowed a gradual decline tendency in water. In2011, the degradation half-life of SCNin water was5.51days, and the degradation rate constant was0.0116, while thedegradation half-life of WPN in water was5.41days, and the degradation rate constant was0.0089. In2012, the degradation half-life of SCN in water was4.79days,and the degradation rate constant was0.0166, while the degradation half-life of WPNin water was4.77days, and the degradation rate constant was0.0170.It is considered that niclosamide is an agent that easily degrades in water, and hasa short duration of retention in water, which is safe.4.2Distribution and degradation of niclosamide in soilSCN and WPN at a dose of1g/m2were evenly sprayed on two experiment spotswith an area of24m2each. The soil at depths of1,2,3,4,5,6,7,8,9,10,11,12,13,14and15cm was sampled at0,24and48h after the spraying for observation of thedistribution of niclosamide in soil, while the soil at depths of1,5,10and15cm weresampled on days0,1,2,3,4,5,6,7,8,9,10,15,20,25and30after the spraying forevaluating the degradation of niclosamide in soil. At each sampling site, the soil with1m in thickness and25cm2in area were collected, placed into clean bags, and storedat-20oC for the subsequent experiments.Following spraying on the soil, SCN and WPN mainly distributed in the soil atdepths of1-5cm, and they were rarely detected in the soil at depth of5-15cm. Thecontent of niclosamide gradually reduced with the increases in the depth.The degradation half-lives of SCN in soil at depths of1,5,10and15cm were2.23,3.35,3.38,2.40days, respectively, and the degradation rate constants were0.214,0.070,0.068,0.182, respectively. The degradation half-lives of WPN in soil atdepths of1,5,10and15cm were2.42,3.47,3.04,3.38days, respectively, and thedegradation rate constants were0.177,0.062,0.096,0.068, respectively.After spraying on soil, niclosamide mainly distributes in the soil at depths of0-5cm. It is considered that niclosamide is a easily degradable agent, and has a shortduration of retention in soil, which is therefore safe.