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Dissipation and residue of ethephon in maize field
Journal of Integrative Agriculture 2015, 14(1): 106–113 Available online at www.sciencedirect.com
ScienceDirect
RESEARCH ARTICLE
Dissipation and residue of ethephon in maize field DONG Jian-nan1, MA Yong-qiang1, LIU Feng-mao1, JIANG Nai-wen1, JIAN Qiu2 1 2
Department of Applied Chemistry, College of Science, China Agricultural University, Beijing 100193, P.R.China Institute for the Control of Agrochemicals, Ministry of Agriculture, Beijing 100125, P.R.China
Abstract A rapid and reliable method was developed for analysis of ethephon residues in maize, in combination with the investigation of its dissipation in field condition and stabilities during the sample storage. The residue analytical method in maize plant, maize kernel and soil was developed based on the quantification of ethylene produced from the derivatization of ethephon residue by adding the saturated potassium hydroxide solution to the sample. The determination was carried out by using the head space gas chromatography with flame ionization detector (HS-GC-FID). The limit of quantification (LOQ) of the method for maize plant was 0.05, 0.02 mg kg–1 for maize kernel and 0.05 mg kg–1 for soil, respectively. The fortified recoveries of the method were from 84.6–102.6%, with relative standard deviations of 7.9–3.8%. Using the methods, the dissipation of ephethon in maize plant or soil was investigated. The half life of ethephon degradation was from 0.6 to 3.3 d for plant and 0.7 to 5.7 d for soil, respectively. The storage stabilities of ethephon residues were determined in fresh and dry kernels with homogenization and without homogenization process. And the result showed that ethephon residues in maize kernels were stable under –18°C for 6 mon. The results were helpful to monitor the residue dissipation of ethephon in the maize ecosystem for further ecological risk assessment. Keywords: ethephon, residue, maize, degradation, storage stability
1 Introduction Ethephon (2-chlorethylphosphonic acid) was a plant growth regulator commonly used on fruit and vegetable crops (Gao et al. 2009). Its excellent biological action made it to be one of the most important plant growth regulators in agriculture since its discovery in the 1970s. Harm to the environment and non-target organism was reported and acute toxicity,
Received 20 November, 2013 Accepted 27 March, 2014 DONG Jian-nan, E-mail: [email protected]; Correspondence LIU Feng-mao, Tel: +86-10-62731978, Fax: +86-10-62733620, E-mail: [email protected] © 2015, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(14)60768-1
sub-chronic toxicity and mutation (Yu et al. 2006) were demonstrated. The ethephon was also registered on maize crop, and the maximum residue limit (MRL) of ethephon on maize was set at 0.5 mg kg–1 in Japan and China, and 0.05 mg kg–1 in England and European Union. For the analysis of ethephon residue, there were some difficulties to analyze the ethephon residue directly. The transfer rate of ethephon residue to organic solvent from the sample was very low because of its higher water solubility and lower n-octanol/water partition coefficient. Ultraviolet (UV) detector in liquid chromatography (LC) did not fit to the analysis due to its low sensitivity to UV irradiation. GC did not fit to its analysis due to its thermal instability (Marín et al. 2006). From the literatures, the analytical methods of ethephon residue were as follows. Method I: Methylation with diazomethane of ethephon residue in methanol and GC determination with phosphorous-selective detection. This
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was a classical and full-fledged method for detection of trace ethephon (Bache and Assoc 1970; Cochrane et al. 1976), and was adopted as the standard method to analyze the ethephon residue in China (NY/T1016, 2006). Its limit of quantification (LOQ) was 0.01 mg kg–1 in fruits and vegetables. However, the most serious problem was its complexity, and the derivatization reagent (diazomethane) was carcinogenic and explosive. Method II: Analysis with ion chromatography coupled plasma mass spectrometry. The ion chromatography was fit to the analysis of ethephon residue in water directly or in organic extractant (Marín et al. 2006; Guo et al. 2007; Ripollés et al. 2011). And the plasma mass spectrometry had excellent selectivity and sensitivity provided by selected reaction monitoring (SRM) mode (Kuster et al. 2009). Its limit of detection (LOD) was 0.1 μg L–1 in water and 0.02 mg kg–1 in vegetables. The method avoided laborious and time-consuming procedures. However, the equipment itself was expensive and not popular in common laboratories on pesticide residue analysis. Method III: Analysis of ethylene instead of ethephon residues by derivatization in alkali condition, which was first published in 1978 (Hurter et al. 1978). It was a rapid method suitable for routine analysis of ethephon residues in several fruits (Li and Zheng 2007), vegetables (Zhou and Wang 2008), juices (Chu et al. 2001), jams (Yao et al. 2008), meat (Kong et al. 2010) and water (Efer et al. 1993). The method was based upon the quantity of ethylene released from ethephon at high pH. Its advantage was using head space of ethylene, which made the clean-up step easier. Most of the matrices studied were water samples or samples with high water content. For solid samples and matrices with low water content, such as soil or plants, seldom researches were reported (Krautz and Hanika 1990). However, the dissipation of ethephon in maize plant and soil has not been reported. Although the storage stability of ethephon in several fruits, vegetables and wheat, cotton was researched (FAO 1994), the stability of ethephon in maize was not reported. In this research, based on method III, a rapid and simple method was developed to determine the residue of ethephon in maize plant, maize kernel and soil
by head space gas chromatography with flame ionization detector (HS-GC-FID) after derivatization with potassium hydroxide. Using the method, the dissipations of ethephon in plant or soil samples, and the storage stability of ethephon in maize kernels were studied.
2. Results 2.1. Recovery and detection limits For the fortified recovery study, the working standard solution of ethephon was spiked to the control samples of maize plant, maize kernel and soil at three levels with five replicates. The results are listed in Table 1. The average recoveries ranged from 84.6 to 99.1% with relative standard deviation (RSD) from 3.8 to 7.9% for maize plant, 86.7 to 93.4% with RSD from 5.0 to 5.5% for maize kernel, and 93.1 to 102.6% with RSD from 5.2 to 7.6% for soil, respectively. The LOQ was set at the lowest spiking level and the LOD was considered to be the concentration that produced a signal-to-noise of 3. From Table 1, this method was more sensitive than the previous report, in which the lowest spiked concentration was 0.1 mg kg–1 in pineapple juices (Chu et al. 2001), 0.5 mg kg–1 in tomato jams (Yao et al. 2008) and 0.1 mg kg–1 in maize (Huang et al. 2012). In the study, it was found that higher injection volume could increase the sensitivity of method, and 50 μL of injection volume was selected.
2.2. Residue dissipation of ethephon in maize plant and soil The dissipation of ethephon in maize plant and soil fit to the first order kinetics, and the results are shown in Table 2. From the result, it could be concluded that the dissipation of ethephon in plant or soil was rapid. The highest initial concentration was 315.4 mg kg–1 in plant and 22.2 mg kg–1 in soil, and the half life was not longer than 3.3 d for plant and 5.4 d for soil. It demonstrated that ethephon was degraded rapidly. The rate of degradation in plants was faster than in soil for the growing diluting factors in plant. No obvious
Table 1 The average recovery, limit of detection (LOD) and limit of quantification (LOQ) of ethephon in three matrices (n=5) Sample Maize plant
Maize kernel
Soil
Spiked level (mg kg–1)
Average recovery (%)
0.05 0.5 1 0.02 0.05 0.5 0.05 0.5 1
91.4 84.6 99.1 93.4 89.5 86.7 93.1 102.6 100.0
Relative standard deviation (RSD, %) LOD (mg kg–1) LOQ (mg kg–1) 7.9 4.5 3.8 5.5 5.1 5.0 7.6 5.4 5.2
0.01
0.05
0.005
0.02
0.003
0.05
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Table 2 The residue dissipation of ephethon in maize plant and soil Sample Plant
Soil
1) 2)
Date 2010.08.01 2010.07.07 2011.06.19 2011.07.20 2011.06.19 2011.07.26 2012.06.03 2012.07.30 2010.08.01 2010.07.07 2011.06.19 2011.08.01 2011.07.20 2011.06.19 2011.07.26 2012.06.03 2012.07.14
Region1) Formulation2) Dosage (g.a.i ha–1) Initial concentration (mg kg–1) BJ A 152 8.82 SD A 152 2.57 315.4 BJ A 3 037 50.5 SD A 1 498 BJ B 135 10.4 AH B 135 11.9 BJ B 135 1.73 AH B 135 21.19 BJ A 759 7.81 SD A 15 2.33 3.29 BJ A 3 037 22.2 BJ A 16 200 0.62 SD A 1 498 BJ B 135 0.32 AH B 135 0.43 BJ B 135 0.26 AH B 135 0.58
Kinetic equation C=15e–0.52T C=4.2e–0.97T C=350e–0.55T C=56e–0.24T C=20e–0.69T C=15.5e–0.61T C=1.889e–0.71T C=24e–1.32T C=9.795e–0.23T C=3e–0.96T C=4e–1.32T C=24.5e–0.23T C=0.65e–0.66T C=0.32e–0.23T C=0.43e–0.21T C=0.26e–0.08T C=0.6e–0.22T
R2 Half life (d) 0.943 2.3 0.733 1.2 0.940 1.5 0.982 3.3 0.960 2.0 0.936 1.5 0.980 1.1 0.730 0.6 0.988 3.9 0.894 1.7 0.942 0.7 0.984 3.4 0.319 1.1 0.840 3.0 0.902 3.3 0.727 5.7 0.935 3.2
BJ, Beijing City; SD, Shandong Province; AH, Anhui Province. A, 30% DA-6·ethephon aqueous solution; B, 40% ethephon aqueous solution.
correlation was found between the rate of degradation and dosage applied in plants or soil.
residue of ethephon was also stable in the commodities in high starch content category.
2.3. Storage stability of ethephon residue under laboratory condition
3. Discussion
Results of stability of ethephon in maize, homogenized and unhomogenized in –18°C refrigerator are presented in Table 3. There was almost no variation of ethephon residue level during the storage. The residue of ethephon was stable, in fresh or dry maize, whether it was homogenized or unhomogenized, under the condition of –18°C for 6 mon. The JMPR report (FAO 1994) summarized the stability of ethephon in apples, blackberries, cherries, grapes, pineapples, cantaloupes, peppers, tomatoes, walnuts, wheat grain and wheat straw, cotton seed, but maize was not included. In most matrixes the residue was stable for more than six months in frozen conditions (except walnuts). It showed that although ethephon was not stable from the perspective of physicochemical property, it could remain stable in many agricultural products under the frozen condition for six months in the laboratory. According to the principles of extrapolation in OECD guidelines (OECD 2007), the stability of ethephon in more than three diverse commodities in high water content category (apples, cherries, cantaloupes, peppers, tomatoes) and more than two diverse commodities in high acid content category (blackberries, grapes, pineapples) has been confirmed, so the residue of ethephon of other crops that belong to both categories should be stable. For the high starch content category in OECD, only wheat was mentioned. In this research, the result of maize was a makeup to OECD results. It was concluded that the
3.1. Derivatization The reaction path on the degradation of ethephon at pH>4.0 was expounded particularly by Yang (1969), as shown in Fig.1. This enabled the indirect determination of residue of ethephon by testing the ethylene produced. The chromatogram peaks for ethylene coincide perfectly with the concentration range expected for ethephon, which demonstrated that the percent conversion was stable. This made it feasible to determine the amount of ethephon conveniently. Then the head space volume was decreased to 6 mL, which was less than 10 mL, half of the inner volume of the head space vial, to improve the sensitivity of the method. Deionized water was added to soil or plant sample to increase the volume of liquid-solid phase, and methanol was added to maize sample to prevent the starch from becoming a gelatin, which would make it too viscous to stir. The concentration of ethephon in matrices was so low that the ethylene released by the degradation reaction had no effect on the pressure in head space violently. It was feasible for the manual sampling for small change of the pressure in the sample vial.
3.2. Influence of pH From the previous researches (Efer et al. 1993; Chu et al. 2001; Li and Zheng 2007; Yao et al. 2008), higher concentrations of potassium hydroxide (KOH) solution showed better
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Table 3 The ethephon residue recovery in fresh and dry maize with different status during the storage Sample type1)
Storage time (d)
FM-H-1
RSD (%)
Recovery (%)
Sample type1)
0.464 0.461 0.43 0.47 0.554 0.592 0.453 0.536 0.601 0.657 0.574 0.643 0.627 0.595 0.603 0.448
3.79 17.87 0.89 0.57 3.48 3.66 2.62 1.07 3.45 1.11 3.82 2.35 0.62 4.32 4.51 12.75
100 99.5 92.7 101.4 100 106.8 81.9 96.7 100 109.2 95.5 107 100 95 96.3 71.4
DM-H-1
0 30 90 180 0 30 90 180 0 30 90 180 0 30 90 180
FM-H-2
FM-K-1
FM-K-2
1)
Residue (mg kg–1)
DM-H-2
DM-K-1
DM-K-2
Storage time (d) 0 30 90 180 0 30 90 180 0 30 90 180 0 30 90 180
Residue (mg kg–1)
RSD (%)
Recovery (%)
0.413 0.446 0.377 0.397 0.499 0.498 0.449 0.494 0.463 0.417 0.455 0.454 0.538 0.519 0.609 0.586
4.03 0.49 2.36 6.34 2.46 4.43 1.68 3.67 9.85 2.58 2.41 2.24 2.55 8.4 3.64 8.61
100 108.2 91.3 96.3 100 99.9 90 99 100 90.1 98.3 98.1 100 96.5 113.1 108.8
FM-H, fresh maize, homogenized; FM-K, fresh maize, kernal; DM-H, dry maize, homogenized; DM-K, dry maize; 1, repeat 1; 2, repeat 2.
O Cl
H2 C
H2 C
O OH
P
OH
+ KOH
Cl
H2 C
H2 C
OK P
OK
+ H2O
H2C
CH2+KCl+NaH2PO4
Fig. 1 The degradation reaction equation of ethephon.
effect on the derivatization of ethephon in the sample. In this study, the saturated KOH solution was used. To test its fitness for different pH samples, some water samples with different pH were prepared, then the same volume of saturated KOH solution was added (Table 4). Here, KOH played a role of derivatization reagent, also provided an alkaline condition to different pH matrices. The results showed that the peak areas of ethylene produced from three samples were similar, and the RSD was only 1.98%. It predicted that 4 mL saturated KOH solution was enough to derivatize the ethephon in the sample at different pH.
in 2 h. The longer time might cause higher RSD in the actual experiment. Hence 2 h of incubation time was selected. During the procedure of incubation, static standing reduced the recovery due to the difference of phases between solvent and samples (Fig. 2-B). It was more difficult for the ethylene gas to escape from a liquid-solid system than from a liquid system, and this was the biggest difference between solid matrix and liquid matrix. By shaking the vial, the recovery could be increased (Fig. 2-B). The different intervals of 10, 20, 30 min were also compared and showed no significant effects on the results. Then, 30 min of interval was selected.
3.3. Optimization of incubation condition
3.4. Selection of GC-column
According to Hurter et al. (1978), the reaction of ethephon to ethylene could not occur when the temperature was below 60°C. And even at 60°C, the reaction was also time-consuming (overnight was needed). In this research, the incubation temperature was set at 70°C, for higher temperature would lead to higher pressure in the vial, which could result in escape of gas and potential explosion hazard. Higher pressure, furthermore, the competition with water vapor would produce negative influence (Brunetto et al. 2009). Different incubation time of 1, 1.5, 2 and 2.5 h at 70°C was compared in three matrices (Fig. 2-A). It could be observed that the reaction was completed from ethephon to ethylene
For the analysis of most pesticide residues, weak-polarity capillary column, such as HP-5 or DB-1701 was widely used. However, they did not fit to the analysis of ethylene because of its low-boiling, non-polar and small molecular weight. To confirm it, two types of column were investigated including DB-1701 (30 m×0.25 mm I.D., 0.25 μm) and HP-PLOT Q (30 m×0.32 mm I.D., 20 μm). From the chromatograms in Fig. 3, DB-1701 could not separate ethylene from the other interferences well, especially for soil samples, while the capillary column HP-PLOT Q showed good results. The capillary column HP-PLOT Q was applicable to separate and analyse the non-polar hydrocarbon, especially for
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Table 4 Comparison of thylene peak area produced from different pH samples (n=3)1) pH of ethephon standard solution
Amount of saturated KOH solution added (mL)
Ethylene peak area average±SD
4 4 4
2 3495.8±388.3 2 4437.3±426.7 2 3879.3±523.8
pH 1.0 pH 7.0 pH 13.0 1)
The concentration of the ethephon standard solution was 0.5 mg L–1 and the volume was 10 mL.
Soil A
Plant
Maize
18 000
Peak area (counts) ×102
16 000 14 000 12 000 10 000 8 000 6 000 4 000 2 000 0
B
0.5
1
120
1.5 2 Incubation time (h) Static
2.5
3
Shaking
Recovery (%)
100 80 60 40 20 0
Plant
Maize
Soil
Fig. 2 Effect of incubation time (A) and shaking (B) on derivatization efficiency.
the C1-C3 isomers. Even if the sample contained water or alcohol, this column could have good reproducibility, chemically inert and long column life (Agilent Technologies 2011). For several matrices like pineapple juice (Chu et al. 2001), mango puree (Li and Zheng 2007), tomato (Zhou and Wang 2008), conventional columns could be used, but from the chromatograms shown in Fig. 3, it was recommended to
hon residues in maize plant, maize kernel and soil was developed by analyzing the head space of ethylene produced from the derivatization of ethephon with saturated potassium hydroxide solution. Using the method, the residue dissipation of ethephon in maize field and stabilities during the sample storage were investigated. Compared with the previous study, the hazardous diazomethane and expensive instruments were avoided, and the LOQ was improved to 0.02 mg kg–1 for maize kernel and 0.05 mg kg–1 for maize plant and soil. This method could fit the monitoring requirements even in England or European Union, which had lower MRL. The low polarity of capillary column HP-PLOT Q used in the study could separate ethylene from other interferences much better, especially for soil samples. The result of the field trial study showed that ethephon degraded rapidly in maize plants and soil. The results of storage stability demonstrated that ethephon could remain stable in both fresh maize and dry maize, homogenized and unhomogenized, in the frozen condition for 6 mon. According to the OECD principles of extrapolation, the residues of ethephon should be stable in high starch content commodities under the frozen condition. To sum up, the method established and the investigation of residue dissipation and storage stability were helpful to monitor the ethephon residue or its dissipation in the maize ecosystem, further to its ecological risk assessment.
5. Materials and methods 5.1. Reagents and chemicals The ethephon reference standard (90.0%) was purchased from J&K Chemical Co., Ltd., (Beijing, China). Methanol was HPLC grade from Honeywell Co., Ltd (Burdick & Jackson). The analytical grade KOH and hydrochloric acid (HCl, 36–38%) were purchased from Beijing Chemical Company (Beijing, China). The water used in standards and analysis procedure was purified using a Milli-Q Water Purification System (Millipore, Bedford, MA, USA).
adopt capillary column HP-PLOT Q as the analysis column.
5.2. Stock and standard solutions of ethephon
4. Conclusion
All the stock and standard solutions of ethephon were prepared in polyethylene graduated volumetric flask or centrifuge tubes, because it was easily absorbed by glassware
A rapid and reliable method for the determination of ethep-
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A UV (x1 000) 4.0 3.0 2.0 Eethylene
1.0 0.0 0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
min
B UV (x1 000) 3.0 2.5 2.0
Ethylene
1.5 1.0 0.5 0.0 0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
min
Fig. 3 GC-FID chromatograms of ethephon standard and controlled soil sample separated on DB-1701 (A) and HP-PLOT Q (B) column.
(Kong et al. 2010). A 1 500 mg L–1 ethephon stock solution was prepared by dissolving the ethephon standard into 0.1 mol L–1 HCl solution and stored in –18°C refrigerator. Then a 100 mg L–1 working solution was diluted with 0.1 mol L–1 HCl and stored in 4°C refrigerator. It was stable for up to 1 mon (Hurter et al. 1978).
5.3. Calibration solutions of the ethephon derivative In the case of plant or soil samples, the calibration solutions were prepared by diluting the 100 mg L–1 solution to 0.05, 0.125, 0.625, 1.25 and 2.5 mg L–1 level with 0.1 mol L–1 HCl. 4 mL of each solution was transferred into 20-mL head space vial, then 6 mL deionized water and 4 mL saturate potassium hydroxide solution were added. In the case of maize kernel samples, the calibration solutions were prepared by diluting the 100 mg L–1 solution to 0.025, 0.0375, 0.0625, 0.125 and 0.25 mg L–1 level, with methanol and 0.1 mol L–1 HCl mixture (6:4, v:v). Four milliliters of each solution were transferred into 20-mL head space vial, then 6 mL methanol and 4 mL saturate potassium hydroxide solution were added. All the vials were closed with a teflon-lined jaw cap and placed in the water bath at 70°C for 2 h for devivatization.
Then the vial was cooled to room temperature prior to GCFID analysis.
5.4. Sample preparation Previously homogenized samples of maize plant (4 g) or soil (10 g) were weighed into a head space vial, 6 mL deionized water and 4 mL saturated potassium hydroxide solution were added. For maize kernel samples, 5 g was weighed, and 6 mL methanol was added instead of deionized water. Then the vial was sealed immediately, and put into water bath at 70°C for 2 h for derivatization. During the derivatization period, the vial was shaken for 30 seconds every half an hour. Before the injection, the vial was treated with ultrasound for 1 min to let ethylene escape from liquid to the head space and then cooled to room temperature.
5.5. Instrumentation The analysis was carried out with a Shimadzu 2010 gas chromatograph equipped with a flame ionization detector (FID). The analytical column used was a capillary column HP-PLOT Q (30 m×0.32 mm I.D., 20 μm), from Agilent. The FID temperature was 250°C and the injector temperature
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was 230°C. The oven temperature of the column was 50°C. The carrier gas was helium, at a flow of 1.0 mL min–1. The analysis was performed in split mode with a ratio of 1:5. Manual sampling was used, and the injection volume was 50 μL.
5.6. Field trial The field trial was conducted in Beijing City (BJ), Shandong Province (SD), and Anhui Province (AH), according to ‘‘Guidelines on Pesticide Residue Trials” issued by the Ministry of Agriculture, People’s Republic of China (NY/T 788, 2004). Two formulations were applied in the field. The first was a product containing 3% DA-6 and 27% ethephon aqueous solution and it was applied in BJ and SD. The second contained 40% ethephon and it was applied in BJ and AH. Water consumption was 2.5 L for each plot, which was 30 m2. There were three replicate plots for each treatment. The formulation was sprayed at several different dosages (Table 2) when the maize plant was at the growth stage of 10 leaves unfolded. The plant and soil was sampled 2, 6 and 12 h, then 1, 3, 5, 7 , 10 and 14 d after the application. All the samples were stored at –18°C prior to analysis.
5.7. Storage stability of ethephon in maize No ethephon residue was detected in any of the maize kernel samples. Maize spiked with 0.5 mg kg–1 ethephon was used for quality control. In many countries and regions, fresh maize was edible or used as fodder, so both fresh and dry maize were researched. In consideration of the fact, two statuses might be adopted for storage, which were homogenized and unhomogenized. There were four treatments with two replicates: whole fresh maize kernel, whole dry maize kernel, homogenized fresh maize and homogenized dry maize. The samples were packaged separately and stored in –18°C.
5.8. Calculation The ethephon residue concentration in plant, soil and maize (both fresh and dry) were calculated following the equations (eqs.) 1 and 2:
C (plant or soil)= C (maize)=
CstdA×Ssam×4 SstdA×m
CstdB×Ssam×4 SstdB×m
(1) (2)
Where, C was the concentration of ethephon residues, CstdA was the concentrations of 4 mL standard solution diluted by 0.1 mol L–1 HCl, SstdA was the area of peak of the corresponding standard solution, CstdB was the concentrations of
4 mL standard solution diluted by methanol and 0.1 mol L–1 HCl mixture, SstdB was the area of peak of the corresponding standard solution, Ssam was the area of peak of actual samples, and m was mass of actual samples. The dissipation concentration and half-life of ethephon residue were calculated by the first-order kinetics eqs. 3 and 4, respectively. C=C0e–KT (3) ln2 (4) K Where, T was the time (in days) after pesticide application, C was the residue concentration at time T, C0 was the initial concentration after application (at T=0), K was the rate constant, and T1/2 was the half-life of residue dissipation. The recovery in the storage stability was calculated by the concentration after storage divided by the initial concentration. T1/2=
Acknowledgements This study was partly supported by the National Natural Science Foundation of China (21177155).
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26, 287–289. (in Chinese) Marín J M, Pozo Ó J, Beltrán J, Hernádez F. 2006. An ionpairing liquid chromatography / tandem mass spectrometric method for the determination of ethephon residues in vegetables. Rapid Communications in Mass Spectrometry, 20, 419–426. NY/T1016. 2006. Determination of ethephon residue in fruits and vegetables gas chromatogram method. Ministry of Agriculture of the People’s Republic of China. (in Chinese) NY/T 788. 2004. Guideline on pesticide residue trials. Ministry of Agriculture of the People’s Republic of China. (in Chinese) OECD (Organization for Economic Co-operation and Development). 2007. OECD guideline for the testing of chemicals. Test No 506: stability of pesticide residues in stored commodities. [2007-10-16]. http://www.oecd-ilibrary. org/content/book/9789264061927-en Ripollés C, Marín J M, Sancho J V, López F J, Hernández F. 2011. Analytical study on ethephon residue determination in water by ion-pairing liquid chromatographytandem mass spectrometry. International Journal of Environmental Analytical Chemistry, 91, 1380–1391. Yang S F. 1969. Ethylene evolution from 2-chloroethylphosphonic acid. Plant Physiology, 44, 1203–1204. Yao W Q, Li F G, Shang D J, Wang Z N. 2008. Determination of residue of ethephon in tomato paste by capillary gas chromatography with Auto HS headspace sampler. Chinese Journal of Health Laboratory Technology, 18, 1537–1692. (in Chinese) Yu W H, Gao Y Q, Zhao W, Qi X J, Yang J. 2006. Study on mutation of ethephon in mice. Chinese Journal of Pesticide Science, 8, 184–186. (in Chinese) Zhou Y M, Wang X. 2008. Analysis of uncertaintity of ethephon in tomato detected by gas chromatography. Science and Technology of Food Industry, 29, 278–280. (in Chinese) (Managing editor SUN Lu-juan)
ScienceDirect
RESEARCH ARTICLE
Dissipation and residue of ethephon in maize field DONG Jian-nan1, MA Yong-qiang1, LIU Feng-mao1, JIANG Nai-wen1, JIAN Qiu2 1 2
Department of Applied Chemistry, College of Science, China Agricultural University, Beijing 100193, P.R.China Institute for the Control of Agrochemicals, Ministry of Agriculture, Beijing 100125, P.R.China
Abstract A rapid and reliable method was developed for analysis of ethephon residues in maize, in combination with the investigation of its dissipation in field condition and stabilities during the sample storage. The residue analytical method in maize plant, maize kernel and soil was developed based on the quantification of ethylene produced from the derivatization of ethephon residue by adding the saturated potassium hydroxide solution to the sample. The determination was carried out by using the head space gas chromatography with flame ionization detector (HS-GC-FID). The limit of quantification (LOQ) of the method for maize plant was 0.05, 0.02 mg kg–1 for maize kernel and 0.05 mg kg–1 for soil, respectively. The fortified recoveries of the method were from 84.6–102.6%, with relative standard deviations of 7.9–3.8%. Using the methods, the dissipation of ephethon in maize plant or soil was investigated. The half life of ethephon degradation was from 0.6 to 3.3 d for plant and 0.7 to 5.7 d for soil, respectively. The storage stabilities of ethephon residues were determined in fresh and dry kernels with homogenization and without homogenization process. And the result showed that ethephon residues in maize kernels were stable under –18°C for 6 mon. The results were helpful to monitor the residue dissipation of ethephon in the maize ecosystem for further ecological risk assessment. Keywords: ethephon, residue, maize, degradation, storage stability
1 Introduction Ethephon (2-chlorethylphosphonic acid) was a plant growth regulator commonly used on fruit and vegetable crops (Gao et al. 2009). Its excellent biological action made it to be one of the most important plant growth regulators in agriculture since its discovery in the 1970s. Harm to the environment and non-target organism was reported and acute toxicity,
Received 20 November, 2013 Accepted 27 March, 2014 DONG Jian-nan, E-mail: [email protected]; Correspondence LIU Feng-mao, Tel: +86-10-62731978, Fax: +86-10-62733620, E-mail: [email protected] © 2015, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(14)60768-1
sub-chronic toxicity and mutation (Yu et al. 2006) were demonstrated. The ethephon was also registered on maize crop, and the maximum residue limit (MRL) of ethephon on maize was set at 0.5 mg kg–1 in Japan and China, and 0.05 mg kg–1 in England and European Union. For the analysis of ethephon residue, there were some difficulties to analyze the ethephon residue directly. The transfer rate of ethephon residue to organic solvent from the sample was very low because of its higher water solubility and lower n-octanol/water partition coefficient. Ultraviolet (UV) detector in liquid chromatography (LC) did not fit to the analysis due to its low sensitivity to UV irradiation. GC did not fit to its analysis due to its thermal instability (Marín et al. 2006). From the literatures, the analytical methods of ethephon residue were as follows. Method I: Methylation with diazomethane of ethephon residue in methanol and GC determination with phosphorous-selective detection. This
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was a classical and full-fledged method for detection of trace ethephon (Bache and Assoc 1970; Cochrane et al. 1976), and was adopted as the standard method to analyze the ethephon residue in China (NY/T1016, 2006). Its limit of quantification (LOQ) was 0.01 mg kg–1 in fruits and vegetables. However, the most serious problem was its complexity, and the derivatization reagent (diazomethane) was carcinogenic and explosive. Method II: Analysis with ion chromatography coupled plasma mass spectrometry. The ion chromatography was fit to the analysis of ethephon residue in water directly or in organic extractant (Marín et al. 2006; Guo et al. 2007; Ripollés et al. 2011). And the plasma mass spectrometry had excellent selectivity and sensitivity provided by selected reaction monitoring (SRM) mode (Kuster et al. 2009). Its limit of detection (LOD) was 0.1 μg L–1 in water and 0.02 mg kg–1 in vegetables. The method avoided laborious and time-consuming procedures. However, the equipment itself was expensive and not popular in common laboratories on pesticide residue analysis. Method III: Analysis of ethylene instead of ethephon residues by derivatization in alkali condition, which was first published in 1978 (Hurter et al. 1978). It was a rapid method suitable for routine analysis of ethephon residues in several fruits (Li and Zheng 2007), vegetables (Zhou and Wang 2008), juices (Chu et al. 2001), jams (Yao et al. 2008), meat (Kong et al. 2010) and water (Efer et al. 1993). The method was based upon the quantity of ethylene released from ethephon at high pH. Its advantage was using head space of ethylene, which made the clean-up step easier. Most of the matrices studied were water samples or samples with high water content. For solid samples and matrices with low water content, such as soil or plants, seldom researches were reported (Krautz and Hanika 1990). However, the dissipation of ethephon in maize plant and soil has not been reported. Although the storage stability of ethephon in several fruits, vegetables and wheat, cotton was researched (FAO 1994), the stability of ethephon in maize was not reported. In this research, based on method III, a rapid and simple method was developed to determine the residue of ethephon in maize plant, maize kernel and soil
by head space gas chromatography with flame ionization detector (HS-GC-FID) after derivatization with potassium hydroxide. Using the method, the dissipations of ethephon in plant or soil samples, and the storage stability of ethephon in maize kernels were studied.
2. Results 2.1. Recovery and detection limits For the fortified recovery study, the working standard solution of ethephon was spiked to the control samples of maize plant, maize kernel and soil at three levels with five replicates. The results are listed in Table 1. The average recoveries ranged from 84.6 to 99.1% with relative standard deviation (RSD) from 3.8 to 7.9% for maize plant, 86.7 to 93.4% with RSD from 5.0 to 5.5% for maize kernel, and 93.1 to 102.6% with RSD from 5.2 to 7.6% for soil, respectively. The LOQ was set at the lowest spiking level and the LOD was considered to be the concentration that produced a signal-to-noise of 3. From Table 1, this method was more sensitive than the previous report, in which the lowest spiked concentration was 0.1 mg kg–1 in pineapple juices (Chu et al. 2001), 0.5 mg kg–1 in tomato jams (Yao et al. 2008) and 0.1 mg kg–1 in maize (Huang et al. 2012). In the study, it was found that higher injection volume could increase the sensitivity of method, and 50 μL of injection volume was selected.
2.2. Residue dissipation of ethephon in maize plant and soil The dissipation of ethephon in maize plant and soil fit to the first order kinetics, and the results are shown in Table 2. From the result, it could be concluded that the dissipation of ethephon in plant or soil was rapid. The highest initial concentration was 315.4 mg kg–1 in plant and 22.2 mg kg–1 in soil, and the half life was not longer than 3.3 d for plant and 5.4 d for soil. It demonstrated that ethephon was degraded rapidly. The rate of degradation in plants was faster than in soil for the growing diluting factors in plant. No obvious
Table 1 The average recovery, limit of detection (LOD) and limit of quantification (LOQ) of ethephon in three matrices (n=5) Sample Maize plant
Maize kernel
Soil
Spiked level (mg kg–1)
Average recovery (%)
0.05 0.5 1 0.02 0.05 0.5 0.05 0.5 1
91.4 84.6 99.1 93.4 89.5 86.7 93.1 102.6 100.0
Relative standard deviation (RSD, %) LOD (mg kg–1) LOQ (mg kg–1) 7.9 4.5 3.8 5.5 5.1 5.0 7.6 5.4 5.2
0.01
0.05
0.005
0.02
0.003
0.05
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Table 2 The residue dissipation of ephethon in maize plant and soil Sample Plant
Soil
1) 2)
Date 2010.08.01 2010.07.07 2011.06.19 2011.07.20 2011.06.19 2011.07.26 2012.06.03 2012.07.30 2010.08.01 2010.07.07 2011.06.19 2011.08.01 2011.07.20 2011.06.19 2011.07.26 2012.06.03 2012.07.14
Region1) Formulation2) Dosage (g.a.i ha–1) Initial concentration (mg kg–1) BJ A 152 8.82 SD A 152 2.57 315.4 BJ A 3 037 50.5 SD A 1 498 BJ B 135 10.4 AH B 135 11.9 BJ B 135 1.73 AH B 135 21.19 BJ A 759 7.81 SD A 15 2.33 3.29 BJ A 3 037 22.2 BJ A 16 200 0.62 SD A 1 498 BJ B 135 0.32 AH B 135 0.43 BJ B 135 0.26 AH B 135 0.58
Kinetic equation C=15e–0.52T C=4.2e–0.97T C=350e–0.55T C=56e–0.24T C=20e–0.69T C=15.5e–0.61T C=1.889e–0.71T C=24e–1.32T C=9.795e–0.23T C=3e–0.96T C=4e–1.32T C=24.5e–0.23T C=0.65e–0.66T C=0.32e–0.23T C=0.43e–0.21T C=0.26e–0.08T C=0.6e–0.22T
R2 Half life (d) 0.943 2.3 0.733 1.2 0.940 1.5 0.982 3.3 0.960 2.0 0.936 1.5 0.980 1.1 0.730 0.6 0.988 3.9 0.894 1.7 0.942 0.7 0.984 3.4 0.319 1.1 0.840 3.0 0.902 3.3 0.727 5.7 0.935 3.2
BJ, Beijing City; SD, Shandong Province; AH, Anhui Province. A, 30% DA-6·ethephon aqueous solution; B, 40% ethephon aqueous solution.
correlation was found between the rate of degradation and dosage applied in plants or soil.
residue of ethephon was also stable in the commodities in high starch content category.
2.3. Storage stability of ethephon residue under laboratory condition
3. Discussion
Results of stability of ethephon in maize, homogenized and unhomogenized in –18°C refrigerator are presented in Table 3. There was almost no variation of ethephon residue level during the storage. The residue of ethephon was stable, in fresh or dry maize, whether it was homogenized or unhomogenized, under the condition of –18°C for 6 mon. The JMPR report (FAO 1994) summarized the stability of ethephon in apples, blackberries, cherries, grapes, pineapples, cantaloupes, peppers, tomatoes, walnuts, wheat grain and wheat straw, cotton seed, but maize was not included. In most matrixes the residue was stable for more than six months in frozen conditions (except walnuts). It showed that although ethephon was not stable from the perspective of physicochemical property, it could remain stable in many agricultural products under the frozen condition for six months in the laboratory. According to the principles of extrapolation in OECD guidelines (OECD 2007), the stability of ethephon in more than three diverse commodities in high water content category (apples, cherries, cantaloupes, peppers, tomatoes) and more than two diverse commodities in high acid content category (blackberries, grapes, pineapples) has been confirmed, so the residue of ethephon of other crops that belong to both categories should be stable. For the high starch content category in OECD, only wheat was mentioned. In this research, the result of maize was a makeup to OECD results. It was concluded that the
3.1. Derivatization The reaction path on the degradation of ethephon at pH>4.0 was expounded particularly by Yang (1969), as shown in Fig.1. This enabled the indirect determination of residue of ethephon by testing the ethylene produced. The chromatogram peaks for ethylene coincide perfectly with the concentration range expected for ethephon, which demonstrated that the percent conversion was stable. This made it feasible to determine the amount of ethephon conveniently. Then the head space volume was decreased to 6 mL, which was less than 10 mL, half of the inner volume of the head space vial, to improve the sensitivity of the method. Deionized water was added to soil or plant sample to increase the volume of liquid-solid phase, and methanol was added to maize sample to prevent the starch from becoming a gelatin, which would make it too viscous to stir. The concentration of ethephon in matrices was so low that the ethylene released by the degradation reaction had no effect on the pressure in head space violently. It was feasible for the manual sampling for small change of the pressure in the sample vial.
3.2. Influence of pH From the previous researches (Efer et al. 1993; Chu et al. 2001; Li and Zheng 2007; Yao et al. 2008), higher concentrations of potassium hydroxide (KOH) solution showed better
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Table 3 The ethephon residue recovery in fresh and dry maize with different status during the storage Sample type1)
Storage time (d)
FM-H-1
RSD (%)
Recovery (%)
Sample type1)
0.464 0.461 0.43 0.47 0.554 0.592 0.453 0.536 0.601 0.657 0.574 0.643 0.627 0.595 0.603 0.448
3.79 17.87 0.89 0.57 3.48 3.66 2.62 1.07 3.45 1.11 3.82 2.35 0.62 4.32 4.51 12.75
100 99.5 92.7 101.4 100 106.8 81.9 96.7 100 109.2 95.5 107 100 95 96.3 71.4
DM-H-1
0 30 90 180 0 30 90 180 0 30 90 180 0 30 90 180
FM-H-2
FM-K-1
FM-K-2
1)
Residue (mg kg–1)
DM-H-2
DM-K-1
DM-K-2
Storage time (d) 0 30 90 180 0 30 90 180 0 30 90 180 0 30 90 180
Residue (mg kg–1)
RSD (%)
Recovery (%)
0.413 0.446 0.377 0.397 0.499 0.498 0.449 0.494 0.463 0.417 0.455 0.454 0.538 0.519 0.609 0.586
4.03 0.49 2.36 6.34 2.46 4.43 1.68 3.67 9.85 2.58 2.41 2.24 2.55 8.4 3.64 8.61
100 108.2 91.3 96.3 100 99.9 90 99 100 90.1 98.3 98.1 100 96.5 113.1 108.8
FM-H, fresh maize, homogenized; FM-K, fresh maize, kernal; DM-H, dry maize, homogenized; DM-K, dry maize; 1, repeat 1; 2, repeat 2.
O Cl
H2 C
H2 C
O OH
P
OH
+ KOH
Cl
H2 C
H2 C
OK P
OK
+ H2O
H2C
CH2+KCl+NaH2PO4
Fig. 1 The degradation reaction equation of ethephon.
effect on the derivatization of ethephon in the sample. In this study, the saturated KOH solution was used. To test its fitness for different pH samples, some water samples with different pH were prepared, then the same volume of saturated KOH solution was added (Table 4). Here, KOH played a role of derivatization reagent, also provided an alkaline condition to different pH matrices. The results showed that the peak areas of ethylene produced from three samples were similar, and the RSD was only 1.98%. It predicted that 4 mL saturated KOH solution was enough to derivatize the ethephon in the sample at different pH.
in 2 h. The longer time might cause higher RSD in the actual experiment. Hence 2 h of incubation time was selected. During the procedure of incubation, static standing reduced the recovery due to the difference of phases between solvent and samples (Fig. 2-B). It was more difficult for the ethylene gas to escape from a liquid-solid system than from a liquid system, and this was the biggest difference between solid matrix and liquid matrix. By shaking the vial, the recovery could be increased (Fig. 2-B). The different intervals of 10, 20, 30 min were also compared and showed no significant effects on the results. Then, 30 min of interval was selected.
3.3. Optimization of incubation condition
3.4. Selection of GC-column
According to Hurter et al. (1978), the reaction of ethephon to ethylene could not occur when the temperature was below 60°C. And even at 60°C, the reaction was also time-consuming (overnight was needed). In this research, the incubation temperature was set at 70°C, for higher temperature would lead to higher pressure in the vial, which could result in escape of gas and potential explosion hazard. Higher pressure, furthermore, the competition with water vapor would produce negative influence (Brunetto et al. 2009). Different incubation time of 1, 1.5, 2 and 2.5 h at 70°C was compared in three matrices (Fig. 2-A). It could be observed that the reaction was completed from ethephon to ethylene
For the analysis of most pesticide residues, weak-polarity capillary column, such as HP-5 or DB-1701 was widely used. However, they did not fit to the analysis of ethylene because of its low-boiling, non-polar and small molecular weight. To confirm it, two types of column were investigated including DB-1701 (30 m×0.25 mm I.D., 0.25 μm) and HP-PLOT Q (30 m×0.32 mm I.D., 20 μm). From the chromatograms in Fig. 3, DB-1701 could not separate ethylene from the other interferences well, especially for soil samples, while the capillary column HP-PLOT Q showed good results. The capillary column HP-PLOT Q was applicable to separate and analyse the non-polar hydrocarbon, especially for
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Table 4 Comparison of thylene peak area produced from different pH samples (n=3)1) pH of ethephon standard solution
Amount of saturated KOH solution added (mL)
Ethylene peak area average±SD
4 4 4
2 3495.8±388.3 2 4437.3±426.7 2 3879.3±523.8
pH 1.0 pH 7.0 pH 13.0 1)
The concentration of the ethephon standard solution was 0.5 mg L–1 and the volume was 10 mL.
Soil A
Plant
Maize
18 000
Peak area (counts) ×102
16 000 14 000 12 000 10 000 8 000 6 000 4 000 2 000 0
B
0.5
1
120
1.5 2 Incubation time (h) Static
2.5
3
Shaking
Recovery (%)
100 80 60 40 20 0
Plant
Maize
Soil
Fig. 2 Effect of incubation time (A) and shaking (B) on derivatization efficiency.
the C1-C3 isomers. Even if the sample contained water or alcohol, this column could have good reproducibility, chemically inert and long column life (Agilent Technologies 2011). For several matrices like pineapple juice (Chu et al. 2001), mango puree (Li and Zheng 2007), tomato (Zhou and Wang 2008), conventional columns could be used, but from the chromatograms shown in Fig. 3, it was recommended to
hon residues in maize plant, maize kernel and soil was developed by analyzing the head space of ethylene produced from the derivatization of ethephon with saturated potassium hydroxide solution. Using the method, the residue dissipation of ethephon in maize field and stabilities during the sample storage were investigated. Compared with the previous study, the hazardous diazomethane and expensive instruments were avoided, and the LOQ was improved to 0.02 mg kg–1 for maize kernel and 0.05 mg kg–1 for maize plant and soil. This method could fit the monitoring requirements even in England or European Union, which had lower MRL. The low polarity of capillary column HP-PLOT Q used in the study could separate ethylene from other interferences much better, especially for soil samples. The result of the field trial study showed that ethephon degraded rapidly in maize plants and soil. The results of storage stability demonstrated that ethephon could remain stable in both fresh maize and dry maize, homogenized and unhomogenized, in the frozen condition for 6 mon. According to the OECD principles of extrapolation, the residues of ethephon should be stable in high starch content commodities under the frozen condition. To sum up, the method established and the investigation of residue dissipation and storage stability were helpful to monitor the ethephon residue or its dissipation in the maize ecosystem, further to its ecological risk assessment.
5. Materials and methods 5.1. Reagents and chemicals The ethephon reference standard (90.0%) was purchased from J&K Chemical Co., Ltd., (Beijing, China). Methanol was HPLC grade from Honeywell Co., Ltd (Burdick & Jackson). The analytical grade KOH and hydrochloric acid (HCl, 36–38%) were purchased from Beijing Chemical Company (Beijing, China). The water used in standards and analysis procedure was purified using a Milli-Q Water Purification System (Millipore, Bedford, MA, USA).
adopt capillary column HP-PLOT Q as the analysis column.
5.2. Stock and standard solutions of ethephon
4. Conclusion
All the stock and standard solutions of ethephon were prepared in polyethylene graduated volumetric flask or centrifuge tubes, because it was easily absorbed by glassware
A rapid and reliable method for the determination of ethep-
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A UV (x1 000) 4.0 3.0 2.0 Eethylene
1.0 0.0 0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
min
B UV (x1 000) 3.0 2.5 2.0
Ethylene
1.5 1.0 0.5 0.0 0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
min
Fig. 3 GC-FID chromatograms of ethephon standard and controlled soil sample separated on DB-1701 (A) and HP-PLOT Q (B) column.
(Kong et al. 2010). A 1 500 mg L–1 ethephon stock solution was prepared by dissolving the ethephon standard into 0.1 mol L–1 HCl solution and stored in –18°C refrigerator. Then a 100 mg L–1 working solution was diluted with 0.1 mol L–1 HCl and stored in 4°C refrigerator. It was stable for up to 1 mon (Hurter et al. 1978).
5.3. Calibration solutions of the ethephon derivative In the case of plant or soil samples, the calibration solutions were prepared by diluting the 100 mg L–1 solution to 0.05, 0.125, 0.625, 1.25 and 2.5 mg L–1 level with 0.1 mol L–1 HCl. 4 mL of each solution was transferred into 20-mL head space vial, then 6 mL deionized water and 4 mL saturate potassium hydroxide solution were added. In the case of maize kernel samples, the calibration solutions were prepared by diluting the 100 mg L–1 solution to 0.025, 0.0375, 0.0625, 0.125 and 0.25 mg L–1 level, with methanol and 0.1 mol L–1 HCl mixture (6:4, v:v). Four milliliters of each solution were transferred into 20-mL head space vial, then 6 mL methanol and 4 mL saturate potassium hydroxide solution were added. All the vials were closed with a teflon-lined jaw cap and placed in the water bath at 70°C for 2 h for devivatization.
Then the vial was cooled to room temperature prior to GCFID analysis.
5.4. Sample preparation Previously homogenized samples of maize plant (4 g) or soil (10 g) were weighed into a head space vial, 6 mL deionized water and 4 mL saturated potassium hydroxide solution were added. For maize kernel samples, 5 g was weighed, and 6 mL methanol was added instead of deionized water. Then the vial was sealed immediately, and put into water bath at 70°C for 2 h for derivatization. During the derivatization period, the vial was shaken for 30 seconds every half an hour. Before the injection, the vial was treated with ultrasound for 1 min to let ethylene escape from liquid to the head space and then cooled to room temperature.
5.5. Instrumentation The analysis was carried out with a Shimadzu 2010 gas chromatograph equipped with a flame ionization detector (FID). The analytical column used was a capillary column HP-PLOT Q (30 m×0.32 mm I.D., 20 μm), from Agilent. The FID temperature was 250°C and the injector temperature
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was 230°C. The oven temperature of the column was 50°C. The carrier gas was helium, at a flow of 1.0 mL min–1. The analysis was performed in split mode with a ratio of 1:5. Manual sampling was used, and the injection volume was 50 μL.
5.6. Field trial The field trial was conducted in Beijing City (BJ), Shandong Province (SD), and Anhui Province (AH), according to ‘‘Guidelines on Pesticide Residue Trials” issued by the Ministry of Agriculture, People’s Republic of China (NY/T 788, 2004). Two formulations were applied in the field. The first was a product containing 3% DA-6 and 27% ethephon aqueous solution and it was applied in BJ and SD. The second contained 40% ethephon and it was applied in BJ and AH. Water consumption was 2.5 L for each plot, which was 30 m2. There were three replicate plots for each treatment. The formulation was sprayed at several different dosages (Table 2) when the maize plant was at the growth stage of 10 leaves unfolded. The plant and soil was sampled 2, 6 and 12 h, then 1, 3, 5, 7 , 10 and 14 d after the application. All the samples were stored at –18°C prior to analysis.
5.7. Storage stability of ethephon in maize No ethephon residue was detected in any of the maize kernel samples. Maize spiked with 0.5 mg kg–1 ethephon was used for quality control. In many countries and regions, fresh maize was edible or used as fodder, so both fresh and dry maize were researched. In consideration of the fact, two statuses might be adopted for storage, which were homogenized and unhomogenized. There were four treatments with two replicates: whole fresh maize kernel, whole dry maize kernel, homogenized fresh maize and homogenized dry maize. The samples were packaged separately and stored in –18°C.
5.8. Calculation The ethephon residue concentration in plant, soil and maize (both fresh and dry) were calculated following the equations (eqs.) 1 and 2:
C (plant or soil)= C (maize)=
CstdA×Ssam×4 SstdA×m
CstdB×Ssam×4 SstdB×m
(1) (2)
Where, C was the concentration of ethephon residues, CstdA was the concentrations of 4 mL standard solution diluted by 0.1 mol L–1 HCl, SstdA was the area of peak of the corresponding standard solution, CstdB was the concentrations of
4 mL standard solution diluted by methanol and 0.1 mol L–1 HCl mixture, SstdB was the area of peak of the corresponding standard solution, Ssam was the area of peak of actual samples, and m was mass of actual samples. The dissipation concentration and half-life of ethephon residue were calculated by the first-order kinetics eqs. 3 and 4, respectively. C=C0e–KT (3) ln2 (4) K Where, T was the time (in days) after pesticide application, C was the residue concentration at time T, C0 was the initial concentration after application (at T=0), K was the rate constant, and T1/2 was the half-life of residue dissipation. The recovery in the storage stability was calculated by the concentration after storage divided by the initial concentration. T1/2=
Acknowledgements This study was partly supported by the National Natural Science Foundation of China (21177155).
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