Dissipation and residue of metalaxyl and cymoxanil in pepperand soil

Xiangyun Liu & Yan Yang & Ying Cui & Huijun Zhu &

Xiong Li & Zhining Li & Kankan Zhang & Deyu Hu

Received: 15 December 2013 /Accepted: 22 April 2014 /Published online: 4 May 2014# Springer International Publishing Switzerland 2014

Abstract A simple and accurate method of determiningmetalaxyl and cymoxanil in pepper and soil was devel-oped by ultra-performance liquid chromatography–pho-todiode array detection. The limits of detection were0.015 mg/kg for metalaxyl and 0.003 mg/kg forcymoxanil. The limits of quantificationwere 0.05mg/kgfor metalaxyl in pepper and soil as well as 0.01 mg/kgfor cymoxanil in pepper and soil. Recoveries of pepperand soil were investigated at three spiking levels andranged within 77.52 to 102.05 % for metalaxyl and87.15 to 103.21 % for cymoxanil, with relative standarddeviations below 9.30 %. For field experiments, thehalf-lives of metalaxyl were 3.2 to 3.9 days in pepperand 4.4 to 9.5 days in soil at the three experimentallocations in China. At harvest, pepper samples werefound to contain metalaxyl and cymoxanil well belowthe maximum residue limit MRLs of the EuropeanUnion (EU) following the recommended dosage andthe interval of 21 days after last application.

Keywords Metalaxyl . Cymoxail . Pepper . Soil .

Residue . Dissipation

Introduction

Pepper is a very important vegetable in China becauseof its heavy consumption, high nutritional value, andprofitability for farmers (Xu et al. 2008). Phytophthorablight, caused byPhytophthora capsicileonian, is one ofthe most serious threats to the production of pepperplants and is widely distributed throughout the world(Hwang and Kim 1995; Ristaino and Johnston 1999).

Me t a l axy l (me thy l -N - ( 2 , 6 -d ime thy l ) -N -methoxyacetyl-D,L-alaninate; Fig. 1) is an N-acylalainepesticide widely used against Peronosporales infectingagricultural and ornamental plants (Kakalíková et al.1996). Cymoxanil (2-cyano-N-[(ethyl amino)-carbon-yl]-2-(methoxyimino) acetamide; Fig. 1) was intro-duced in the late 1970s in response to the growing needfor improved control methods against Phycomycete fun-gal pathogens. Historically, cymoxanil has been used tocontrol grape downy mildew and late blight of tomatoand potato (Meister 1999).Metalaxyl and cymoxanil arejointly used to control the diffusion of phytophthorablight among pepper plants.

Analysis of multiclass pesticides (includingmetalaxyl and cymoxanil) in cabbage using ultra-performance liquid chromatography tandem mass spec-trometry (UPLC–MS/MS) has been reported (Huanget al. 2011). In addition, some papers on the analysisof metalaxyl in vegetables, fruits, and medicinal mate-rials using high-performance liquid chromatographytandem mass spectrometry (HPLC–MS/MS) or high-performance liquid chromatography (HPLC) are avail-able (Liu et al. 2012; Venkateswarlu et al. 2007; Jansson

Environ Monit Assess (2014) 186:5307–5313DOI 10.1007/s10661-014-3779-5

X. Liu :Y. Yang :Y. Cui :H. Zhu :X. Li : Z. Li :K. Zhang (*) :D. Hu (*)State Key Laboratory Breeding Base of Green Pesticide andAgricultural Bioengineering, Key Laboratory of GreenPesticide and Agricultural Bioengineering, Ministry ofEducation, Guizhou University,Guiyang 550025, People’s Republic of Chinae-mail: kankan16@126.come-mail: fcc.dyhu@gzu.edu.cn

et al. 2004; Zheng et al. 2013; Wang et al. 2013). Somepapers had been published for analysis ofcymoxanil using HPLC (Cabizza et al. 2012;Fidente et al. 2005; Hengel and Shibamoto 2001;Li et al. 2008).

As of this writing, no papers have been pub-lished on the analysis of metalaxyl and cymoxanilresidues in pepper. The residue approaches forvegetables cannot be used to pepper. Those residueapproaches almost use SPE to purify the sample.Sample pretreatments are cockamamie fussy andtime-consuming. UPLC takes advantage of techno-logical advances in particle chemistry performance,system optimization, detector design, and data pro-cessing and control. Using sub-2-μm particles andmobile phases at high linear velocities and instru-mentation that operates at pressures higher thanthose used in HPLC, dramatic increases in resolu-tion, sensitivity, and speed of analysis can beobtained with UPLC. This new category of analyt-ical separation science retains the practicality andprinciples of HPLC while creating a step-functionimprovement in pesticide residue analysis.

According to the Foreign Agricultural Serviceand Pesticide Maximum Residue Limit (MRL)Database from the US Department of Agriculture,the tolerances established for the residues ofmetalaxyl and cymoxanil around the world rangefrom 0.50 to 5.00 and 0.05 to 0.50 mg/kg, respec-tively, which vary for different crops in differentcountries. No systematic research has been con-ducted to date on the dissipation and residues ofmetalaxyl in pepper. Therefore, field dissipationand pesticide residue behavior of metalaxyl andcymoxanil in agricultural fields are needed to en-sure food safety and environmental protection.

In this study, we aimed to establish a simpleand accurate preparation method in combinationwith highly selective UPLC for the analysis of

metalaxyl and cymoxanil in pepper and soil. Thedissipation dynamics and terminal residues ofmetalaxyl and cymoxanil in pepper and soil wereinvestigated in a field ecosystem.

Materials and methods

Chemicals

Standards of metalaxyl (97.8 % purity) and cymoxanil(99.0 % purity) were provided by Shanghai PesticideResearch Institute and Dr. Ehrenstorfer GmbHCompany. HPLC-grade acetonitrile was purchased fromMerck (Darmstadt, Germany). Milli-Q pure water(Watsons, China) was used during the whole analysis.Primary secondary amino (PSA, 40 to 60 μm in size)and octadecyl silane (C18, 40 to 60 μm in size) werepurchased from Agela Technologies (China). Syringefilter (nylon, 0.22 μm) was purchased fromPeakSharp.

Stock standard solution of metalaxyl (502.69 μg/mL)and cymoxanil (249.48 μg/mL) were prepared inmethylalcohol and stored at −20 °C. The stock standard solu-tion was diluted with methyl alcohol as required. Theformulation, a wettable powder (WP) containing 22.5 %metalaxyl and 2.5 % cymoxanil was kindly supplied byZhongxun Agrochemicals Limited Company inGuangdong province, China.

Analytical methods

UPLC conditions

The chromatographic separation was achieved using aWaters H-class UPLC series (Waters technologies,USA) consisting of a PDA detector, a samplemanager-FIN, a quaternary solvent manager, and anACQUITY UPLC BEH C18 1.7-μm (2.1×50 mm) col-umn (Waters technologies, USA). The mobile phasewas acetonitrile-water delivered at a flow rate of0.3 mL/min with a gradient composition. The processwas as follows: from acetonitrile/water (15/85, v/v), heldfor 1.5 min, to acetonitrile-water (21/79, v/v), held for2.5 min, to acetonitrile/water (30/70, v/v), held for3.5 min, and finally a decrease at acetonitrile/water(15/85, v/v) over 2.5 min to stabilize the UPLC systembefore starting the next run. A total run time of 10 minwas obtained. The injection volume was 2 μL. The

Fig. 1 Chemical structures of metalaxyl and cymoxanil

5308 Environ Monit Assess (2014) 186:5307–5313

optimum detection was obtained at 202 and 242 nm formetalaxyl and cymoxanil, respectively, and the columntemperature was maintained at 40 °C.

Sample pretreatment

An aliquot (10 g) of soil sample was weighed into a100-mL Erlenmeyer flask, and 40 mL of extractionsolvent (ethyl acetate) was added. The mixture wasultrasonically extracted for 15 min and filtered by sandcore funnel. The residue was rinsed with 10 mL ethylacetate. The filtrates were combined in a 100-mL pear-shaped flask and dried in the rotary evaporator at 45 °C.The residues were dissolved in 1 mL methyl alcohol,filtered into an autosampler vial with 0.22 μm syringefilters, and analyzed by UPLC without further cleanup.

An aliquot (10 g) of pepper sample was weighed intoa 100-mL Erlenmeyer flask, and 60 mL of extractionsolvent (dichloromethane/n-hexane=1/1, v/v) wasadded. The mixture was ultrasonically extracted for15 min and filtered by sand core funnel. The residuewas rinsed with 10 mL ethyl acetate. The filtrates werecombined in a 100-mL pear-shaped flask and dried inthe rotary evaporator (45 °C). The residues were dis-solved in 1 mL methyl alcohol, 100 mg C18 and 50 mgPSA were added, and the solution was vortexed for2 min. The liquid supernatant was filtered into anautosampler vial with 0.22-mm syringe filters and ana-lyzed by UPLC without further cleanup.

Field trials

The field trials, including the dissipation and residueexperiments, were performed at three different loca-tions: Guiyang (26.35°N, 106.42°E, Guizhou province,west of China with subtropical plateau monsoon humidclimate), Changsha (28.12°N, 112.59°E, Hunan prov-ince, south of China with continental humid subtropicalmonsoon climate), and Tianjin (39.10°N, 117.10°E,Tianjin, north of China, with warm, temperate semi-humid continental monsoon climate). Field experimentswere performed from July 2012 to September 2012.

Seven treatments, including six metalaxyl andcymoxanil treatments and one control, were included.Each experimental plot was 30 m2 and each treatmentwas applied three times. No pesticide was used duringthe whole period of pepper growth in the control treat-ment. A buffer area of 30 m2 was used to separate theplots receiving different treatments.

To investigate the dissipation dynamics of metalaxyland cymoxanil in pepper and soil, formulation ofmetalaxyl and cymoxanil (WP, 25 %) was dissolved inwater and sprayed at an active constituent dose of3.75 g/L water (1.5 times the recommended dosage)on the surface of pepper, and 5.00 g/L water (2.0 timesthe recommended dosage) on bare soil with no plants.When the peppers grow to mature individual half hours,it was sprayed. The volume of water supplied to eachexper imenta l p lo t (30 m2) was 1 ,000 mL.Approximately, 2 kg of pepper samples and representa-tive 2 kg of surface soil (from 0 to 10 cm depth) sampleswere collected randomly from several points in eachplot at 2 h (calculated as the original concentration)and at 1, 3, 7,10, 14, 21, 28, 42, and 60 days afterspraying. Samples were placed in a deep freezer at−18 °C for analysis.

For the terminal residue experiment, formulations ofmetalaxyl and cymoxanil (WP, 25 %) were applied at alow dosage of 2.50 g/L water (the recommended dosage)and at a high dosage of 3.75 g/L water (1.5 time therecommended dosage) three and four times. Each treat-ment was applied at root level in pepper plants at 100mL.Approximately, 2 kg of pepper samples and representa-tive 2 kg of surface soil (from 0 to 15 cm depth) sampleswere collected randomly from several points in each plotat 7, 14, and 21 days after last spraying. Samples wereplaced in a deep freezer at −18 °C for analysis.

Results and discussion

Optimization of sample pretreatment

For the soil, we chose several extractant solvents, andthe data are shown in Fig. 2. After a comprehensiveconsideration of the recovery and environmental friend-liness of the process, we chose ethyl acetate as theextraction agent. For pepper, we chose a small amountof polarity solvent to reduce the interference of theimpurity. In the d-SPE procedure, PSA is a weak anionexchange sorbent that retains carboxylic acids, such asfatty acids, from the acetonitrile extracts. C18 is a non-polar sorbent that effectively retains trace amounts oflipids from the extract. Furthermore, GCB has usuallybeen used in cleanup of pigment. Initially, 50 mg PSA,50 mg C18, and 50 mg graphitized carbon black (GCB)were tested in metalaxyl and cymoxanil standard solu-tions, respectively, to determine whether or not they

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affect pesticide recoveries. GCB caused great losses ofcymoxanil, and thus, PSA and C18 were used in thisstudy. After the study, we used 100 mg C18 and 50 mgPSA for purification. The best vortex duration was2 min.

Method validation

In this study, the external standard method, along withmatrix-matched methods, was chosen for quantitation.Individual calibration graphs were run with mixtures ofmetalaxyl and cymoxanil at concentrations in the range0.50 to 50.00 and 0.10 to 5.00 μg/mL, respectively.Each solution was injected three times. The linear range,intercept, and slope of the curve are shown in Table 1along with the regression coefficient for each pesticide.Satisfactory linear relationships and coefficients of de-termination (R2>0.999) were achieved.

The limits-of-detection (LOD) values, which werebased on a signal/noise ratio of 3 (Miller and Miller2000), were 0.015 and 0.003 mg/kg for metalaxyl andcymoxanil, respectively. The limits of quantification(LOQs) were defined as the concentrations of a compound

at which its signal-to-noise ratios were detected as 10:1(Miller andMiller 2000). The LOQ of metalaxyl in pepperand soil matrices was 0.05 mg/kg, and the LOQ ofcymoxanil in pepper and soil matrices was 0.01 mg/kg.

To evaluate the accuracy and precision of themethod,a recovery experiment was conducted. This experimentwas repeated five times for the precision test. The rela-tive standard deviation (RSD, %) values between mea-sured concentrations showed the precision of the meth-od. Pepper and soil matrices were spiked at three differ-ent levels. A total of five replicate measurements wereperformed for each fortified level. The average recover-ies for metalaxyl were in the range 77.52 to 93.61 %with RSD of 2.31 to 6.43 % in pepper and 87.36 to102.05 % with RSD of 2.84 to 5.81 % in soil. Theaverage recoveries for cymoxanil were in the range87.15 to 101.86%with RSD of 3.58 to 7.26% in pepperand 87.72 to 103.21 % with RSD of 2.90 to 9.30 % insoil. The results are summarized in Table 2. Resultsdemonstrated that the newly established method wassatisfactory and repeatable for the determination ofmetalaxyl and cymoxanil in pepper and soil.

Dissipation dynamics

The dissipation equation was calculated from the first-order rate equation: Ct=C0e

−kt, where Ct is the concen-tration of the pesticide residue at time t, C0 is the initialconcentration, and k is the rate constant in d–1. Half-life(t1/2) was calculated from the k value for each experi-ment (t1/2=ln2/k). The dissipation curves of metalaxylwere fitted with first-order kinetics and were calculated(Table 3).

0

20

40

60

80

100

120

Methyl alcohol Acetonitrile Acetone Dichloromethane Ethyl acetate

Rec

over

y (%

)

MetalaxylCymoxanil

Fig. 2 Metalaxyl and cymoxanil average recovery values (%) andRSDs obtained with different extraction solvents in soil (n=3).Spiking levels used metalaxyl at 0.5 mg/kg and cymoxanil at0.05 mg/kg

Table 1 Linearity, correlation coefficient, and LOD of metalaxyland cymoxanil

Analyte Linearequation

Linearrange(mg/L)

Correlationcoefficient(R2)

LOD(mg/kg)

Metalaxyl y=39,466x+3,707.2

0.50–50.00 1.0000 0.015

Cymoxanil y=14,386x+129.82

0.10–5.00 0.9996 0.003

Table 2 Precision and accuracy of the method for determiningmetalaxyl and cymoxanil in pepper and soil (n=5)

Sample Metalaxyl Cymoxanil

Spikedlevel(mg/kg)

AverageRecovery(%)

RSD(%)

Spikedlevel(mg/kg)

AverageRecovery(%)

RSD(%)

Pepper 0.05 77.52 6.43 0.01 87.15 6.02

0.5 87.22 4.23 0.05 94.19 7.26

5 93.61 2.31 0.5 101.86 3.58

Soil 0.05 87.36 5.81 0.01 87.72 8.88

0.5 89.87 5.28 0.05 84.22 9.30

5 102.05 2.84 0.5 103.21 2.90

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Pepper samples

The dissipation samples were analyzed and a scatter plotwas constructed. The dissipation dynamics of metalaxylin pepper are shown in Fig. 3. The initial residues inpepper were 1.24 mg/kg in Guiyang, 1.14 mg/kg inChangsha, and 1.57 mg/kg in Tianjin. The initial con-centration in pepper was higher in Tianjin than inGuiyang and in Changsha, which may be due to differ-ent planting densities at the three sites or to unevenspraying. Metalaxyl dissipated fast in pepper 1 day afterthe application, and the degradation rate in pepper at7 days after application was 79.2, 92.8, and 80.9 % inGuiyang, Changsha, and Tianjin, respectively. The

dissipation curve of metalaxyl in pepper was fitted withthe first-order kinetics equations: C=0.6859e−0.1776t

(Guiyang), C=0.6900e−0.2182t (Changsha), and C=1.2904e−0.2083t (Tianjin). The climates in the three ex-periment locations were different, but the t1/2 ofmetalaxyl in pepper calculated from the regressionequations were similar (3.9, 3.2, and 3.3 days inGuiyang, Changsha, and Tianjin, respectively).Because of the stability of metalaxyl on the light anddifferent temperature, the dissipation of metalaxyl inpepper was relatively unaffected by weather. The initialresidues of cymoxanil in pepper were 0.45 mg/kg inGuiyang, 0.26 mg/kg in Changsha, and 0.37 mg/kg inTianjin. However, 1 day after application, the residuesof cymoxanil in pepper samples were lower than0.01 mg/kg in the three locations.

Soil samples

The dissipation dynamics of metalaxyl in soil are shownin Fig. 3. The initial metalaxyl residues in soil were0.83 mg/kg in Guiyang, 0.59 mg/kg in Changsha, and0.65mg/kg in Tianjin. The dissipation curve of metalaxylin soil was fitted with the first-order kinetics equations:C=0.7979e−0.0727t (Guiyang), C=0.5044e−0.1163t

(Changsha), and C=0.4513e−0.1573t (Tianjin). The organ-ic matter content of Changsha soil was higher thanGuiyang soil, but lower than Tianjin soil, which mayhave contributed to the different degradation rates ofmetalaxyl in different soils (t1/2 was 9.5, 6.0, and 4.4 daysin Guiyang, Changsha, and Tianjin, respectively). Resultsalso showed that metalaxyl dissipated slower in soil thanin pepper, which might be attributed to its binding withorganic matter in soil and rainfall. The initial cymoxanilresidues in soil were 0.73 mg/kg in Guiyang, 0.65 mg/kgin Changsha, and 0.71 mg/kg in Tianjin. However, theresidues of cymoxanil were undetectable in the soil sam-ples 1 day after application.

Table 3 Dissipation equationsand half-lives of metalaxyl inpepper and soil at the three sites

Sample Sites Dissipation equation Correlationcoefficient (R)

t1/2 (days)

Pepper Guiyang C=0.6859e−0.1776t 0.9094 3.9

Changsha C=0.6900e−0.2182t 0.9353 3.2

Tianjin C=1.2904e−0.2083t 0.9934 3.3

Soil Guiyang C=0.7979e−0.0727t 0.9848 9.5

Changsha C=0.5044e−0.1163t 0.9547 6.0

Tianjin C=0.4513e−0.1573t 0.9672 4.4

Fig. 3 Dissipation of metalaxyl in soil (a) and pepper (b) atGuiyang, Changsha, and Tianjin in 2012

Environ Monit Assess (2014) 186:5307–5313 5311

Terminal residues

In the terminal residue experiments, the formulation wasroot-irrigated to the pepper plants and terminal residuesamples were analyzed (Table 4). The results showedthat the metalaxyl residues of pepper from Guiyang,Changsha, and Tianjin were lower than 0.50 mg/kg(the MRL of metalaxyl in pepper set by the EuropeanUnion, EU) at 21 days after the last spraying for the lowdosage level (used three times). The residues ofcymoxanil in pepper from Guiyang, Changsha, andTianjin were undetectable (lower than the MRL of0.05mg/kg in pepper set by the EU) at 7, 14, and 21 daysafter spraying for the two dosage levels. The residues ofmetalaxyl in the soil of Guiyang, Changsha, and Tianjinwere lower than 0.93mg/kg at 21 days after spraying forthe two dosage levels, and the residues of cymoxanil inthe soil of Guiyang, Changsha, and Tianjin were unde-tectable at 7, 14, and 21 days after spraying for the twodosage levels.

Metalaxyl residues in soil were dramatically lower inGuiyang and Tianjin than in Changsha. Residues in soilwere higher than those detected in pepper, and this maybe the result of the fact that metalaxyl dissipated moreslowly in soil than in pepper and the application methodof root irrigation. Furthermore, previous studies haveshown growth rates of vegetables and fruits affect

dissipation rates of applied pesticides (Valverde-Garciaet al. 1993; Walash et al. 1993). This increase in fruitweight may result in a faster dilution of pesticide in thefruit even without pesticide degradation (Metwally andOsman 1997). On the other hand, comparatively lowresidues in pepper suggested that metalaxyl andcymoxanil may otherwise cause no problems in termsof food safety.

Conclusions

The simple and accurate method for the analysis ofmetalaxyl and cymoxanil in pepper and soil was qualita-tively and quantitatively satisfactory. The dissipation ofmetalaxyl and cymoxanil residues was detected to ensurethe safe use of pesticides and the protection of consumerhealth. Metalaxyl dissipated easily in pepper with the half-life ranging from 3.2 to 3.9 days. The data also showedmetalaxyl and cymoxanil residue values measured in pep-per were clearly below the MRL values of metalaxyl andcymoxanil in EU (0.50 and 0.05 mg/kg, respectively) withthe interval of 21 days at the recommend dosage after lastapplication. According to the results of this study, pepperplant should be safe with metalaxyl and cymoxanil (WP,25 %) not more than three times at the recommend dosage

Table 4 Terminal residues of metalaxyl and cymoxanil in pepper and soil at Guizhou, Hunan, and Tianjin in 2012

Dosage (mg/L) Low dosage (2.50) High dosage (3.75)

Number of applications 3 4 3 4

Pre-harvest interval (days) 7 14 21 7 14 21 7 14 21 7 14 21

Residue (mg/kg)

Pepper Guiyang Metalaxyl 0.74 0.66 0.44 1.09 0.85 0.84 1.02 0.92 0.83 1.36 1.30 1.20

Changsha 0.77 0.65 0.33 1.35 1.21 0.62 1.12 1.04 0.50 1.82 1.38 1.31

Tianjin 0.10 ND ND 0.13 ND ND 0.10 0.07 ND 0.11 0.07 ND

Guiyang Cymoxnail ND ND ND ND ND ND ND ND ND ND ND ND

Changsha ND ND ND ND ND ND ND ND ND ND ND ND

Tianjin ND ND ND ND ND ND ND ND ND ND ND ND

Soil Guiyang Metalaxyl 1.22 0.15 ND 1.30 0.44 0.26 1.14 0.94 0.49 1.78 1.12 0.85

Changsha 3.32 1.18 0.32 2.73 1.26 0.32 3.16 1.31 0.46 3.58 1.72 0.68

Tianjin 1.76 1.18 ND 1.96 1.30 0.35 2.91 1.77 0.78 3.50 1.95 0.93

Guiyang Cymoxnail ND ND ND ND ND ND ND ND ND ND ND ND

Changsha ND ND ND ND ND ND ND ND ND ND ND ND

Tianjin ND ND ND ND ND ND ND ND ND ND ND ND

ND Not detected, lower than LOD

5312 Environ Monit Assess (2014) 186:5307–5313

and with an interval of at least 7 days between eachapplication.

Acknowledgements The authors thank the Special Fund forAgro-scientific Research in the Public Interest (No. 201203022)and the program of agricultural research of Guizhou province(20103070).

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