Ecotoxicology and Environmental Safety

Anaerobic degradation of chlorothalonil in four paddy soilsHuili Wang a ,Chengjun Wang b ,Fan Chen b ,Xuedong Wang a ,na Department of Environmental Science,School of Environmental Science and Public Health,Wenzhou Medical College,Wenzhou 325035,China bCollege of Chemistry and Materials Engineering,Wenzhou University,Wenzhou 325035,Chinaa r t i c l e i n f oArticle history:Received 6January 2008Received in revised form 25October 2010Accepted 18January 2011Available online 21March 2011Keywords:Chlorothalonil (CTN)Anaerobic degradation 4-OH-chlorothalonil (HTI)Paddy soila b s t r a c tDegradation of Chlorothalonil (CTN)was investigated in four different paddy soils under anaerobic conditions.The CTN biodegradation is strongly affected by the properties of the paddy soils.Soils associating with rich total carbon (TC),repeated CTN application,and neutral pH have shown the high capacity to biodegrade CTN.Additionally,anaerobic CTN biodegradation was accompanied by the methane generation and a drop of oxidation–reduction potential (ORP).The initial CTN concentration had a significant effect on CTN removal efficiency,and increase in the initial CTN concentration resulted in the decreasing of CTN removal percentage.However,it is believed that the inhibitory effect on anaerobic biodegradation of CTN is negligible in natural environment due to the much lower concentration of CTN in natural environment (at ng g À1or pg g À1level)than the one (10m g g À1)investigated in this study.The 4-hydroxy-2,5,6-trichloroisophthalonitrile (HTI),one of the major metabolites of CTN degradation,has shown the significant inhibitation to the anaerobic CTN biodegradation when its residual level is over 0.1m g g À1.&2011Elsevier Inc.All rights reserved.1.IntroductionChlorothalonil (2,4,5,6-tetrachloroisophthalonitrile,CTN),one of the most popular fungicides,belongs to the group of halogenated benzonitriles.It is well known that CTN works by contacting and further inhibiting cell respiration enzymes related to glutathion (Arvanites and Boerth,2001).Over the past two decades,the CTN has been widely applied to control many fungal diseases in agricul-ture (Cox,1997).However,residual CTN has been often detected in vegetables,crops,soils,and environmental water (Andersson and Bergh,1991),and the toxicity of CTN and its effect on human health have been extensively studied (Sherrard et al.,2003).Caux et al.(1996)have reported CTN is highly toxic to fish,birds,and aquatic invertebrates in environment.The CTN has been considered as a moderately persistent fungicide in soil,and the half-life of CTN in soil has been reported in the range from 4days to 6months (Singh et al.,2002).The variation of reported CTN half-life data could be due to the different experimental conditions (Gambacorta et al.,2005).In addition,the repeated application times have a significant effect on degradation of CTN (Walker et al.,1988;Van der Pas et al.,1999).Singh et al.(2002)reported that the half-life of CTN for the first dose in soil was 8.6days and this was extended to 21.5days for the third treatments.In the top layer of soil,4-hydroxy-2,5,6-trichloroisophthalonitrile (HTI)is the primary breakdown metaboliteof CTN in the presence of water,and it is more acutely toxic,persistent,and mobile in soil than the CTN itself (Cox,1997).More-over,the HT1may cause inhibition of the CTN degradation due to its inhibitory effect on microorganisms (Motonaga et al.,1996).Mean-while,Other CTN metabolites,including dechlorinated or substituted forms of CTN,such as 2,5,6-trichloro-4-methoxyisophathalonitrile,3-dicarbamo-yl-2,4,5,6-tetrachlorobenzene,2,4,5-tri-chloroisophtha-lonitrile,1,3-dicyanobenzene,etc.have been reported (Gustavo and Damia,1998).The aerobic metabolism has been considered as the most suitable pathway for CTN microbiological degradation (Katayama et al.,1997).However,some of metabolites from the group of halogenated benzonitriles have been reported (Regitano et al.,2001),and those metabolism pathways might be associated with the mechanisms of sulphate-reduction and reductive dechlor-ination that occur in anoxic environments.Wackett and Hershberger (2001)suggested benzenic ring reduction via carboxilation in anae-robic conditions could be one of the general rules of biodegradation,thus it is necessary to gather more information about CTN anaerobic degradation and metabolism.However,a little information has been reported for CTN degradation under anaerobic conditions.To author’s best knowledge,only Carlo-Rojas et al.(2004)have studied the anaerobic degradation of CTN in a banana plantation and their response to simulation by different carbon/nitrogen ratios.In most area of East Asia,CTN is often applied to control rice fungal diseases in paddy fields,where anaerobic-like conditions dominantly occur.Thus,it is important to study the anaerobic degradation of CTN in soil.In this study,we investigated the CTN degradation behaviors in four different paddy soils under laboratory anaerobic conditions,andContents lists available at ScienceDirectjournal homepage:/locate/ecoenvEcotoxicology and Environmental Safety0147-6513/$-see front matter &2011Elsevier Inc.All rights reserved.doi:10.1016/j.ecoenv.2011.01.011nCorresponding author.Fax:8657786689733.E-mail address:zjuwxd@ (X.Wang).Ecotoxicology and Environmental Safety 74(2011)1000–1005evaluated the effects of the soil properties,initial CTN concentrations,and the main intermediate (HTI)on anaerobic degradation of CTN.The information on the anaerobic degradation behaviors of CTN in paddy soils obtained in this study is useful for the assessment of CTN contamination in environment and mechanism study of its bioreme-diation under anaerobic conditions.2.Materials and methods 2.1.ChemicalsChlorothalonil (CTN,purity 99.4%)was purchased from Promochem (Wesel,Germany)and 4-OH-chlorothalonil (HTI,purity 99.5%)was obtained from ISK-Biotech (New York,USA).The structures of CTN and HTI are shown in Fig.1.The solubility of CTN in water is only 0.6mg L À1,while it reaches as many as 2000mg L À1in acetone.The standard stock solutions of CTN and HTI were separately prepared in acetone at a concentration of 50mg L À1.The stock solutions were prepared freshly every two month and stored in amber bottles at À201C until use.Except where noted,all solvents were of HPLC-grade and all reagents were of reagent grade.The distilled water was purified with a Mill-Q-Plus system (Millipore,Molsheim,France)before use.2.2.SoilsTwo of the studied paddy soils (designated as HB1and HB2),without CTN application history,were collected from rice fields of two places in Huaibin County,Henan Province,China.The other two paddy soils (named as QJ1and QJ2),which were applied CTN twice yearly at 375g a.i ha À1each time in the middle of May and August of past three years (2004–2006),were collected from two rice fields in Qianjiang City,Hubei Province,China.In the four paddy soils,CTN was not detected in HB1and HB2soil samples,meanwhile CTN was found at 0.89and 0.71ng g À1d.w.in QJ1and QJ2soil samples,respectively.The major CTN metabolite (HTI)was also detected (0.45ng g À1d.w.)in QJ2soil.All paddy soil samples were collected in October 2006,and their physical–chemical character-istics and contamination status are summarized in Table 1.The collected soil samples were air-dried,ground,sieved (o 2mm mesh),and stored submerged under water at room temperature.Before starting the anaerobic degradation experiment,the soil samples were equilibrated under submerged conditions at 221C for two weeks (Shibata et al.,2007).The experimental constant temperature was guaranteed because the operation was conducted under the thermostatic incubation room.2.3.Anaerobic CTN degradationAll experiments were performed triplicately in 125-mL bottles,in which 10g of soils,30ml of deionized water,and 5m g g À1of CTN were added under a gentle nitrogen flow.The small amount of acetone solvent,which was used for dissolving CTN,in soil sample was proved to be no adverse effect on anaerobic microbial activity by our previous study.All bottles were flushed with pure nitrogen gas (99.999%)for 15min to remove any trace of oxygen in containers,tightly capped with butyl rubber stoppers,sealed with aluminum crimps,and wrapped in aluminum foil to prevent from photolysis.All the sample bottles were incubated for two months at 301C,and the residual CTN concentrations,oxidation–reduction potential (ORP)values,and methane concentrations in samples were measured at intervals (0,5,10,20,40and 60days)of incubation.All experiments were conducted in an anaerobic glove box (Forma Scientific,model 1025S/N,USA).In order to study the contribution of anaerobic microorganisms to CTN degradation,the sterilized (ST)control samples were prepared in four paddy soils.All 125-mL vials containing 10g soil were capped slightly,wrapped in aluminum foil,and autoclaved for 3h (three separate 1h treatments at 1211C).Then,50m g of CTN was added,followed by adding 30ml of the ST deionized water.The vapor-phase in vials was removed by nitrogen gas to produce the anaerobic conditions and the samples were incubated as described above.2.4.Effect of metabolite HTI on CTN degradationHTI standards were spiked into the samples at a series of concentrations (0.1,0.5or 1m g g À1)to test the effects of HTI on anaerobic CTN degradation.The tested concentrations of HTI were referred to the report by Lu et al.(2008).The non-sterile (NST)controls were prepared without addition of HTI and incubated without shaking at 301C in darkness.2.5.Extraction and clean-up of soil samplesBecause the solubility of CTN in water is relative low,we did not measure its water equilibrium concentration.Referring to the report by Kwon and Armbrust (2006),soil suspension samples (10ml)were collected at intervals,adjusted pH to 3by HCl because the acidic conditions contribute to the high recoveries for most of analytes,and followed by adding 30ml of water/dichloromethane (1/3,v/v)solution.After shaking for 30min,the samples were centrifuged for 5min at 4000rpm,and the supernatants were pipetted into a 100-mL vial.The soil samples were extracted twice following the procedure described above,and the combined supernatants were filtered through a polytetrafluoroethylene (PTFE)filter membrane (30mm diameter,0.2m m pore size)to remove any soil particles.And then the dichloromethane layer was collected and evaporated to dryness under a gentle nitrogen flow.The residue was redissolved in 2ml of water/acetonitrile solution (1/1,v/v)before injected into high-performance liquid chromatography (HPLC).2.6.HPLC analysisAn Agilent 1100model HPLC equipped with photodiode array detector was used for all experiments.The analytical column used was a YWG-C 18reversed-phase column (250Â4.6mm ID,5-m m particle size)and column temperature was controlled at 301C.The mobile phase was made up of acetonitrile (ACN)and 0.5%phosphoric acid in water.The gradient elution started with 20%ACN for 3min,linearly increased to 90%ACN within 20min and then remained at 90%ACN for an additional 10min.The injection volume was 20m l and the flow rate was 0.8ml min À1.The detection wavelength was set at 232nm.2.7.Recovery studyA recovery study was performed by spiking four paddy soils with standard CTN stock solution at a series of concentrations (0.1,1,5and 10m g g À1).The residual extraction and analysis were conducted as the method described above.The average recoveries for CTN in anaerobic NST soils were from 81.2%to 94.5%and the relative standard deviations (RSDs)were from 4.6%to 8.9%.In the case of anaerobic ST soils,the average recovery was in the range of 83.7–91.2%and RSDs was from 3.5%to 9.2%.As a result,the adopted method could meet the requirements for residual analyses of pesticides (the required recovery ranged from 80%to 120%).2.8.Determination of ORP (oxidation–reduction potential)and methane production rateORP value of supernatant was measured by Ultrameter II TM6P (Myron L Company,USA)to ensure the anaerobic conditions.The pH value was measured with a pH meter (PHS-3C model,Shanghai Aiyite CO.,Ltd.,Shanghai,China).Fig.1.Structural formulas of chlorothalonil (CTN)and 4-hydroxychlorothalonil (HTI).Table 1Physical–chemical characteristics of the four paddy soils investigated in this study.Kind of sediment HB1HB2QJ1QJ2pH4.75.16.6 6.3Water content (%)22.619.326.917.4TC (%) 1.37 1.53 3.27 4.12TN (%)0.110.050.040.19SO 42À(mg/kg)9.6725.6813.7614.26NO 3À(mg/kg)0.891.412.173.12Soil textureLight clay Silty loam Silty loam Sandy loam Chlorothalonil (ng g À1.d.w)ND ND 0.890.71Metabolite A (ng g À1.d.w)NDNDND0.45ND indicates the abbreviation of ‘‘not detectable’’.TC and TN indicate the abbreviation of ‘‘total carbon’’and ‘‘total nitrogen’’,respectively.H.Wang et al./Ecotoxicology and Environmental Safety 74(2011)1000–10051001Methane in sample vials was collected periodically and determined by using a gas chromatography (Hewlett Packard,HP 6890series GC system)equipped with capillary column (30mm Â0.53mm ID,0.25um film thickness;Agilent Technol-ogies,USA)and a flame ionization detector.High-purity nitrogen gas was used as carrier gas.2.9.Calculation and statistical analysisThe anaerobic degradation of CTN in the investigated soils was found to follow the pseudo-first-order model.Therefore,the half-life value was calculated by the following mathematical model expressed as:C t ¼C 0Âe Àktð1Þwhere C 0is the initial concentration (m g g À1soil)of CTN,C t is the concentration (m g g À1soil)at time t ,t is the incubation time (days)and k is the degradation rate constant (d À1).The half-life was expressed by ln(2/k )and k was determined by using regression of ln(C t /C 0).The kinetic parameters of biodegradation were calculated according to the following steps:(1)The residual concentration of CTN by biodegradation at time t was calculatedby the following equation:C t =BD ¼C 0-C t =ST þC t =NSTð2Þwhere C t /BD is the residual concentration by biodegradation at time t ;C 0is the initial concentration of CTN;C t /ST is the concentration at time t under ST conditions;C t /NST is the concentration at time t under NST conditions.(2)Based on the residual concentrations (C t /BD )by biodegradation at differenttime t ,the biodegradation half-life and rate constant were calculated using the previous pseudo-first-order Eq.(1).(3)The biodegradation rate at 60days after treatment (DAT)was calculated bythe following equation:BDR 60¼½ðC 60=ST -C 60=NST Þ=C 0 100%ð3Þwhere BDR 60is the biodegradation rate at 60DAT;C 60/ST is the concentration at 60DAT under ST conditions;C 60/NST is the concentration at 60DAT under NST conditions;C 0is the initial concentration of CTN.Microsoft Excel 2003and Origin 6.0graphing software were used to fit the data to the model.Analysis of variance (ANOVA)and Duncan’s multiple range tests were used to determine significant difference at p o 0.05among each treatment using statistical analysis software (SAS Version 8).3.Results and discussion3.1.Anaerobic CTN degradation in soilsAs shown in Table 2,at 60DAT,the remaining CTN was in the range from 27.3%to 33.1%in anaerobic ST samples,and no significant difference was observed among four paddy soils.It indicates that around 70%of the original CTN was degraded in ST soils and abiotic processes might play an important role in the depletion of the fungicide.Photolysis can be negligible since all experiments were carried out in the darkness.Therefore,the loss under ST conditions might result from hydrolysis and formation of soil-bound residue,volatilization,uptake,etc.Especially,the soil binding might have contributed to these high abiotic degradation efficiencies (Regitano et al.,2001).Carlo-Rojas et al.(2004)reported that only 64%recovery after spiking a soil sample with CTN and extracting the fungicide with acetone solvents.And the high soil-bound CTN residue forms in the first day of microcosm incubation were found by Regitano et al.(2001).The binding process varies widely due to the diversity of the mineral components,the nature and content of organic matter,the proportion and size of the particles in soil and,particularly,the clay content of the soil (Gamble et al.,2001).Soil aggregates have not only physical–chemical effect but also relate to biological activity since they can provide micro spaces allowing microbial diversity (Van eaden et al.,2000).Under NST conditions,the remaining CTN at 60DAT was in the range from 0.4%to 28.5%in four paddy soils.Under ST conditions,four soils had similar half-lives of CTN in the range of 33.2–39.6days (Table 2)due to the abiotic degradation.However,CTN degradation,involving in combination of abiotic and biotic action under NST conditions,in QJ1and QJ2soils was much faster than that in HB1and HB2soils.It indicates that there was a higher microbial CTN-degrading activity in QJ1and QJ2soils than that in HB1and HB2soils.In QJ2soil,CTN degradation at 60DAT was 66.9%under ST conditions,whereas it was almost completely degraded (99.6%)under NST conditions.In QJ1soil,the CTN degradation increased by 23.4%in NST sample comparing with that in ST sample.The observation of similar degradation trend in both soils suggests that anaerobic microbial action was respon-sible for the additional degradation.In contrast,no significant difference was observed of the remaining CTN in HB1and HB2soils under either condition.It indicated that no biodegrada-tion occurred in those two soils.Briefly,the remaining CTN at 60DAT under NST conditions were in the following order:HB24HB14QJ14QJ2.As a result,CTN showed the much higher degradation rate in QJ2soil than that in other three soils under NST conditions.The degradation of organic chemicals under NST conditions is a result of biological and chemical transformation,while only chemical or abiotic process occurs under ST conditions.Difference of CTN degradation in soils under NST and ST conditions is that the contribution of biological transformation.The rate constant and half-life of anaerobic CTN biodegradation are listed in Table 2.The highest biodegradation rate was observed in QJ2soil (k ¼0.03746d À1),and followed by QJ1soil (k ¼0.02987d À1).The corresponding biodegradation half-lives of CTN were 23.2and 18.5days in QJ2and QJ1soil,respectively (Table 2).It is noteworthy that no CTN biodegradation occurred in HB1and HB2soils throughout the entire incubation period.These results in QJ1and QJ2samples are consistent with the report by Carlo-Rojas et al.(2004),who found that a high CTN degradation (56–95%)was observed in biologically active microorganisms although abiotic loss in a sterile blank was also notable (37%).Table 2Percentage of CTN remaining after 60days of incubation,and the degradation kinetic parameters in four paddy soils.SamplePercentage remaining (%)Rate constant (d À1)/,Correlation coefficient (R 2)Half-life (d)STNST ST BDNST ST BD NST HB127.372.7a 26.771.5a 0.02087(0.7457)–0.02063(0.9231)33.2–33.6HB229.672.1a 28.571.7a 0.01904(0.8933)–0.01963(0.9746)36.4–35.3QJ130.674.8a 7.270.2b 0.01853(0.8517)0.02987(0.7711)0.06188(0.8019)37.423.211.2QJ233.172.4a0.470.01c0.01750(0.7841)0.03746(0.8232)0.09118(0.8248)39.618.57.6All values are means 7SD of triplicate samples;Incubation time is 60days.Different lower cases within a column denote the significant difference at p o 0.05.BD,ST and NST indicate the abbreviation of biodegradation,sterile and non-sterile,respectively.‘‘–’’means no biodegradation was detected.H.Wang et al./Ecotoxicology and Environmental Safety 74(2011)1000–10051002The organic pollutants can be mineralized under anaerobic conditions,and the process of anaerobic mineralization includes two steps(Chang et al.,2005):(1)organic pollutants are decom-posed to organic acid and alcoholate and(2)mineralized to gas forms such as CH4(methane),H2S,CO2and so on in concomitant with the drop of ORP(oxidation–reduction potential).Therefore, methane generation and drop of ORP are commonly employed as indicators of anaerobic biodegradation of organic contaminants (Chen et al.,2004).As shown in Table3,methane only generated in QJ1and QJ2soils under NST conditions,whereas no methane was found in control samples under ST conditions(data not shown).The high-to-low order of methane generation rate under NST conditions was QJ24QJ14HB2or HB1,which was the same as the order of biodegradation rates of CTN.However,the contrary trend was found for the ORP values in the four soils i.e.,HB14HB24QJ14QJ2,suggesting that higher methane pro-duction rate was in concomitant with sharply decreasing of ORP (Table3).These evidences strongly suggest that the difference of CTN degradation in four paddy soils may result from the diversity of anaerobic microbial activity.Similar observations have been reported in organic chlorinated compounds such as biodegrada-tion of chlordane and hexachlorobenzene,nonylphenol poly-ethoxylates,and phthalate esters(Hirano et al.,2007;Lu et al., 2008).3.2.Properties of soils in relation to anaerobic CTN biodegradation o empty4Thefinal biodegraded CTN was80.6%and89.8%in QJ1and QJ2 soils in this study,respectively.Meanwhile,no CTN biodegrada-tion took place in HB1and HB2soils,as shown in Table3.The CTN residues in QJ1and QJ2soils and the major metabolite HTI in QJ2 samples were detected at ng gÀ1d.w level,as presented in Table1.The CTN application at different time may have different inhibitory effects on soil microorganisms.After thefirst application of CTN,the soil bacteria and actinomyces could be significantly reduced and the most marked inhibition on soil microorganisms took place after the second application.However, soil microorganisms gradually adapted to CTN after initial varia-tions and the negative effects became transient and weak after the third and fourth application(Yu et al.,2006).In this research, CTN had been applied many times in QJ1and QJ2soils prior to the soil-collected date.The anaerobes were able to biodegrade CTN since their acclimation period,during which almost no biodegra-dation occurred,was completed.On the contrary,HB1and HB2 samples were collected from the places without contamination of CTN,and the anaerobes required a long time tofinish acclimation period and thus were not able to biodegrade the CTN during the incubation period.As a result,no CTN biodegradation was observed in HB1and HB2soils.The high-to-low order of CTN biodegradation rates was QJ24QJ14HB1(or HB2),which was the same as the order of total carbon(TC)content,methane generation rate,and the drop in ORP,as shown in Table3.Especially,the high-to-low order of TC in four paddy soils was in concomitant with the same order of biodegradation rate,suggesting that the high TC contributed to high biodegradation of CTN.QJ2sample,having original CTN residue for a long time and the highest TC content,had the highest biodegradation rate among the four paddy soils.Mean-while,the HB1and HB2samples had very poor TC and no CTN, and no biodegradation occurred within the60-day of incubation period.Therefore,it is possible that the original TC and CTN residue in the soils contributed the CTN degradation.Hirano et al. (2007)investigated the biodegradation of chlordane and hexa-chlorobenzene in river sediments,and found that high carbon content and contamination by the target chemicals can enrich microorganisms such as sulfate-reducing bacteria,methanogen, and eubacteria,which are responsible for degrading organic pollutants.In addition,the neutral pH of QJ2and QJ1soils might be another important factor to affect their biodegradation capa-cities.As reported by other researchers previously,anaerobic microorganisms can be inhibited at pH values below6or above 9(Widdel,1988).Chang et al.(2002)also concluded that the optimal pH for the anaerobic biodegradation of PAHs by soil culture was pH8.0.The fact that no CTN biodegradation occurred in HB1and HB2soils over a60-day incubation period may be due to the poor organic carbon,absence of the CTN residue,and acidic pH values.These observations suggest that the paddy soils with rich TC, CTN,and neutral pH may have a high CTN biodegradation potential.The TC and CTN can induce the growth of microorgan-isms,which are responsible for biodegradation of these chemicals and the neutral pH can produce suitable conditions for biodegradation.3.3.Effect of the initial concentration on anaerobic degradation of CTN in QJ2soilAs listed in Table4,the different initial concentration had a significant effect on CTN removal efficiency.Obviously,there is a decreasing linear relationship between initial concentration and the removal percentage(y¼À0.8597x+100.54,r2¼0.7649).How-ever,no significant difference between the removal percentages at5and10-m g gÀ1treatments were observed,and the removal percentage was more than90%in both cases,as shown in Table4. The results suggest that there was no inhibitory effect even at initial level of10m g gÀ1.However,a sharp decreasing tendency in removal efficiency occurred when the initial concentration further increased from10to20m g gÀ1.It indicated that the inhibitory effect occurred on anaerobic biodegradation of CTN at the high initial level.Especially,the removal percentage(70.4%)at initial CTN concentration of40m g gÀ1was not significant(at p o0.05)different from that(66.9%)at5m g gÀ1under ST condi-tions,which demonstrated that the microorganisms were wholly inhibited and the CTN removal was due to abiotic contribution.In fact,the concentration of CTN in natural environment usually maintains at a very low level(at ng gÀ1or pg gÀ1level),and is much less than the one(10m g gÀ1)investigated in thisTable3Biodegradation rate of CTN,ORP and TC under NST conditions in four paddy soils.Kind of soil HB1HB2QJ1QJ2TC(%)before treatment 1.37 1.53 3.27 4.12Biodegradation rate(%)at60DAT0080.689.8ORP(mV)at60DATÀ104À109À267À294Methane generation rate(m mol/g d.w./day)under NST conditions00 1.76 2.42TC and ORP indicate total carbon and oxidation–reduction potential,respectively.Table4Effect of initial concentration on anaerobic CTN degradation in NST QJ2paddy soil.Initial concentration (m g gÀ1)Removalpercentage(60d)Half-life(days)Rate constant(dÀ1)R2599.672.4a7.60.091180.82481095.473.5a9.40.073720.71812072.373.9b27.90.024840.82464070.474.9b31.60.021930.9077All values are means7SD of triplicate samples;Incubation time is60days.Different lower cases within a column denote the significant difference at p o0.05.H.Wang et al./Ecotoxicology and Environmental Safety74(2011)1000–10051003experiment.Therefore,it is possible that the inhibitory effect on anaerobic biodegradation of CTN is negligible in most natural environment.3.4.Effect of major metabolite on anaerobic degradationIn soils,plants,and animals,CTN can be metabolized into the 4-hydroxy-2,5,6-trichloro-isophthalonitrile (HTI).HTI is 30times more acutely toxic than CTN itself,and it is more persistent and mobile in soil (Cox,1997).Motonaga et al.(1996)reported that HTI could cause inhibition of CTN degradation in fields due to its toxicity to microorganisms.In this investigation,some metabolic byproduct peaks besides the metabolite HTI were detected.For lack of authentic standards,further identification and discussion of these byproduct peaks were not performed except HTI.Standard HTI was added to the QJ2soil to investigate the effect of typical intermediate on anaerobic CTN degradation.As shown in Fig.2,the degradation of the control (no addition of HTI)at 60DAT had no significant difference from that of the sample spiked by 0.1m g g À1of standard.However,it was sharply decreased from 94.8%to 75.6%when HTI was increased from 0.1to 1.0m g g À1,as presented in Table 5.With the further increase of HTI concentration from 1.0to 5.0m g g À1,the degradation percentage still remained at a stable level ($73%),which was not significant form that in ST QJ2soil.The HTI could significantly inhibit the anaerobic biodegradation of its parent compound when the residual level of this metabolite was over 0.1m g g À1.Motonaga et al.(1998)have suggested that the residual toxicity of HTI might be responsible for the suppression of CTN degradation in soils after repeated applications.Chaves et al.(2007)also reported that HTI accounted for as much as 65%of the totalresidues in soils and could reach concentrations as high as 34.7ng g À1(d.w.).In agreement with the previous reports by other researchers,the results in this study indicated that the significant inhibitory effect on CTN degradation could occur when the concen-tration of HTI residue in soil was more than 0.1m g g À1.4.ConclusionsIn this study,all four paddy soils were capable of degrading CTN under anaerobic conditions without any external acceptors.However,only two of them (QJ1and QJ2)could biodegrade CTN.CTN degradation potential in paddy soils varied with soil proper-ties such as TC,pH value,and application times of the CTN.The QJ2soil had the highest CTN biodegradation rate due to its high content of TC,neutral pH value,and CTN application times.However,the HB1and HB2samples had poor TC and no CTN,thus no CTN biodegradation was observed.Additionally,methane was generated and ORP was dropped during the anaerobic CTN biodegradation.The inhibitory effect on anaerobic biodegradation of CTN is negligible in natural environment due to the much lower concentration of CTN in natural environment (at ng g À1or pg g À1level)than the one (10m g g À1)investigated in this study.HTI,one of the major CTN metabolites,could significantly inhibit the anaerobic CTN biodegradation when the residual HTI level in soil was more than 0.1m g g À1.To author’s best knowledge,this is the first report on anaerobic degradation of CTN in paddy soils.AcknowledgmentsThis work was jointly funded by National Natural Science Foundation of China (No.31071115,21077109),and International Cooperation Project of Wenzhou City (H20100053,H20100054).The authors are also grateful to the anonymous reviewers for their reading of the manuscript,and for their suggestions and critical comments.ReferencesAndersson,A.,Bergh,T.,1991.Pesticide residues in fresh fruit and vegetables onthe Swedish market,January 1985–December 1989.Fres.J.Anal.Chem.339,387–389.Arvanites, A.C.,Boerth, D.,2001.Modeling of the mechanism of nucleophilicaromatic substitution of fungicide chlorothalonil by glutathione.J.Mol.Model.7,245–256.Carlo-Rojas,Z.,Bello-Mendoza,M.,Figueroa,M.S.,Sokolov,M.Y.,2004.Chlorotha-lonil degradation under anaerobic conditions in an agricultural tropical soil.Water Air Soil Poll.151,397–409.Caux,P.Y.,Kent,R.A.,Fan,G.T.,Stephenson,G.L.,1996.Environmental fate andeffects of chlorothalonil:a canadian perspective.Crit.Rev.Environ.Sci.Technol.26,45–93.Chang,B.V.,Shiung,L.C.,Yuan,S.Y.,2002.Anaerobic 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