Dissipation Pattern of Five Pesticides in Vegetable Samples

Pesticides are frequently used in agricultural elds in Bangladesh. Residual pesticides in vegetable samples above maximum residue limits (MRL) are illegal use of pesticides and absence of Good Agricultural Practice (GAP). The Government made Safe Food Law to ensure safe food for all citizens of the country. To assess the dissipation of pesticides in vegetable samples for consumer safety, ve locally banded pesticides i.e., Vitaban (chlorpyrifos), Double (mixture of imidacloprid and cypermethrin), Nitro (mixture of chlorpyrifos and cypermethrin), Acephate and Reeva (lambda cyhalothrin) were applied to eight different vegetables at the dose which farmers apply in their eld in a large vegetable cultivation area. Samples were harvested from the farmer’s elds at 2h (0 day) after application of pesticides and analyzed at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 days keeping them at ambient temperature. Samples were extracted following Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) method and cleaned up using primary secondary amine (PSA), and nally analysed by Gas Chromatograph Electron Captured Detector (GC-ECD. Limit of detection (LOD) and limit of quantication (LOQ) of chlorpyrifos, cypermethrin, imidacloprid, acephate and lambda cyhalothrin were 0.019 and 0.057, 0.019 and 0.057, 0.009 and 0.027, 0.019 and 0.057, and 0.009 and 0.057 µg/mL, respectively. Recoveries of these pesticides in these vegetables samples were within acceptable range of 74–110%. Level below MRL value of cypermethrin was found to be 2–5 days while chlorpyrifos, imidacloprid, acephate and lambda cyhalothrin treated vegetables were varied 3–8 days and considered to consume after these days, respectively.


Introduction
Bangladesh is an agricultural country with a population of 160 million in 56,000 square miles. Although the economy of the country mainly depends on the agriculture small industries for example garments, leather etc are increasing and contributing to the national GDP. The country is also facing natural disaster like cyclone, tornado, ood, thunderstorms, monsoon and heavy rain, and is vulnerable for climate change. Rice, wheat, jute, cotton, tobacco, potato, sugarcane, pulse, lentils, tea and vegetables are the major crops grown in the country , Chowdhury et al. 2013]. In order to meet the demand of food supply for a huge number of populations, a large number of fertilizer and pesticides are being used in agricultural crops. Due to climatic condition of Bangladesh the widely cultivated and highyielding variety plants are highly vulnerable to pest and other diseases and therefore, the use of pesticides is an integral part for pest control [Chowdhury et al. 2013].
The vegetables are low in fat, high in dietary bers; contain minerals and vitamins, possessing a very high nutritional density [Nahar et al. 2016]. However, vegetables are usually susceptible to pest attack and different kinds of pesticides are being used in cultivation around the world including Bangladesh. Use of pesticide is necessary to increase crop production. However, indiscriminate use of insecticides and without maintaining the proper pre-harvest interval can make the food unsafe, especially when tamotoes are used as salad [Nahar et al. 2012]. Pesticide residues in the agricultural products depend on the dose of application, climatic condition of each country and also on seasons like winter and summer [Nahar et al. 2014]. We earlier reported dissipation pattern of fenvalerate in tea, and cypermethrin and quinalphos in tomato, bean, cauli ower in experimental elds [Nahar et al. 2012[Nahar et al. , 2014

Reagents and materials
The standards of chlorpyrifos, cypermethrin, imidacloprid, acephate, lambda-cyhalothrin (99% pure) were purchased from Dr. Ehrenstorfer, Germany. Primary secondary amine (PSA) from Supelco USA, was used in clean-up steps. Analytical grade magnesium sulphate, sodium chloride (anhydrous), ethyl acetate and ultra-pure GC-grade n-hexane were obtained from Merck KGaA, Germany.

Sample collection
Eight commonly consumed vegetable i.e., cabbage, cucumber, bottle gourd, sweet gourd, sponce gourd, green chili, cauli ower and tomato were treated with ve locally used pesticides namely, Vitaban 48 EC (chlorpyrifos), Double 50 EC (mix formulation of imidacloprid and cypermethrin), Nitro 505 EC (mix formulation of chlorpyrifos and cypermethrin), Asataf 75 SP (acephate) and Reeva 2.5 EC (lambdacyhalothrin) at farmer's eld in Nuritola, Comilla, Bangladesh. Vegetable were harvested 2 hours after application of pesticides and a total of 40 samples were collected to analyze the dissipation pattern and the safe level from consumer perception. Fresh and untreated (with pesticides) vegetables of all eight types were rst collected in zip-locked bag before application of these pesticides, brought to the laboratory. Only control samples were kept in freezer at -20 0 C and the rest of the samples were kept at ambient temperature, so that they could be in a contact with air, wind, light and heat. From these samples, working samples were chopped, homogenized by kitchen blender and then extracted and cleaned-up on consecutive 1st, 3rd, 5th, 8th and 10th day from the application day.

Application of pesticides
Nitro was sprayed at a dose of 1 mL L − 1 of water/ha; Vitaban 48 EC was sprayed at a dose of 3 mL L − 1 of water/ ha, Double 50 EC was sprayed at a dose of 800 mL/ ha, Reeva 2.5 EC was sprayed at a dose of 0.5 mL L − 1 of water/ ha; Asataf 75 SP was used at a dose of 1gm L − 1 of water/ ha.

Extraction
The extraction process followed the Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) method [Anastassiades et al. 2003]. To homogenized vegetable sample, weighed of about 10g was taken in a te on tube and 20 mL of ethyl acetate was added to it. The tube was then vortexed for about 1 minute. Then 6 g of MgSO4 and 1.5 g of NaCl were added and again vortexed for 1 minute. The mixture was then centrifuged for 5 minutes at 5,000 rpm maintaining temperature 20 0 C. About 10 mL of supernatant was taken in a round bottom ask and dried to dryness. 5 mL n-hexane was added to the dried mass and from there; 2 mL solution was taken in a test tube.
For clean-up, 150 mg primary secondary amine (PSA) and 750 mg MgSO 4 were added and the test tube vortexed for about 1 minute. It was then centrifuged for another 5 minutes and then the extract was passed through pipette with cotton plugged inside which was washed successively with methanol, acetone and nally with n-hexane.
Pesticide residues were identi ed and quanti ed using a Shimadzu 2010 gas chromatograph (GC) equipped with a 63 Ni electron capture detector (ECD). Separation was on an ultra-performance quartz capillary column (HP 5MS, 30 m, 250 µm internal diameter, 0.25 µm lm thickness, Agilent, USA). The injector and detector temperatures were 250°C and 260°C, respectively. For determination of chlorpyrifos, lambda-cyhalothrin and acephate, the oven temperature was programmed as: initial temperature of 140°C hold for 1 minute; increased at 15°C min − 1 to 260°C; hold for 2 minutes but for determination of cypermethrin and imidacloprid, the oven temperature was programmed as: initial temperature of 120°C held for 1 minute; increased at 14°C min − 1 to 260°C; held for 3 minutes. Nitrogen gas was used as carrier and make up gas, and the ow rate was 1.92 mL min − 1 . Samples were injected manually in the splitless (1 min)/split mode.
The recovery experiments of pesticides in spiked control vegetable samples were carried out in two different concentrations levels and were extracted and cleaned up following the same procedure as described above.

Storage ability
The storage stability was determined by spiking the control vegetable samples with the ve pesticides and then storing them in a freezer at -20 0 C. The control vegetables were spiked with chlorpyrifos, cypermethrin and imidacloprid at a concentration level of 1.0 µg mL − 1 and with lambda-cyhalothrin and acephate at a concentration level of 0.5 and 2.0 µg mL − 1 , respectively. Then these spiked samples were stored in freezer for 35 days and extracted, cleaned-up and analyzed following the same procedure to nd out the stability of the pesticides at freezing condition during storage.

Results And Discussion
The residual level of ve pesticides namely chlorpyrifos, cypermethrin, imidacloprid, acephate and lambda-cyhalothrin were analyzed in eight vegetable samples i.e., tomato, cabbage, cauli ower, bottle gourd, sweet gourd, sponge gourd, green chili and cucumber by GC-ECD using external standard calibration method. The percentage recoveries of chlorpyrifos at 0. 5  The degradation of a component is described by the rst order function (C t = C 0 x e kt ). The half-lives of the pesticides were obtained by the equation t ½ = ln2/k, where C t is the concentration (µg L 1 ) at time t (days) after application, C 0 is the initial concentration (µg L 1 ) and k is the rst order rate constant (day 1 (Fig. 1). Ravi and Verma (1997) reported that initial chlorpyriphos was degraded below MRL (0.2 mg kg − 1 ) within 20 days from its initial residues of were 5.41 mg kg − 1 . Subhash (2014) reported that chlorpyrifos dissipated below MRL in okra value after 15 days. Our results are also in consistent that the persistence of chlorpyrifos and is lengthier than other pesticides and dissipates slowly. It was reported that dissipation rate of chlorpyrifos is slower than other modern pesticides.
Nitro 505 EC is a mix formulation of chlorpyrifos (50%) and cypermethrin (5%) and was sprayed on the eight kind of vegetables at a dose of 1 mL per Liter of water/ha.  ]. The initial amounts of chlorpyrifos and cypermethrin in all vegetables on the rst day were much higher than the MRL values of both pesticides. Cypermethrin in tomato, cauli ower, sweet gourd, sponce gourd, green chili and cucumber went below MRL value on 1st day after application of pesticide but within 3rd day of application in cabbage and sponce gourd. 100% dissipation of cypermethrin occurs within 5 days after application of pesticides. The dissipation of chlorpyrifos and cypermethrin followed pseudo rst-order kinetic patterns (Fig. 2-3). The half-lives of cypermethrin for tomato, cabbage, bottle gourd, cauli ower, were 0.6, 0.5, 0.4, 0.5 days, respectively.
From the dissipation curves, the average half-lives of chlorpyrifos and cypermethrin in nitro were found to be 0.92 and 0.58 days, respectively. The average half -life value in chlorpyrifos is higher than that of cypermethrin. Similar results were reported by Awasthi (1994) whereas he determined persistence and degradation of different pyrethroids on green chilli fruits [Awasthi 1994]. The half-life of permethrin was 0.42-0.68 days as that of cypermethrin in the current study was 0.46-0.81 days.
The MRL value of acephate in tomato, cucumber and green chili is 1 µg g − 1 and cabbage, cauli ower and sweet gourd is 2 µg g − 1 .The present study revealed that residual acephate in all vegetable samples were less than MRL value on 3rd day except cabbage and cauli ower which went down to MRL on 5th day. Acephate might have more persistency in vegetables like cabbage and cauli ower (brassica vegetables).
This can be explained that they grow in upper direction and their surface are large and nonhomogeneous, and pesticide has much space to accommodate. As a results pesticide reaches to the target point properly.
The active ingredient of Reeva 2.5 EC is lambda-cyhalothrin. The percentage recoveries of lambdacyhalothrin at 0.03 and 0.20 µg mL − 1 forti cation levels in tomato, green chili, cucumber and at 0.03 and 0.50 µg mL −  respectively. The LOD and LOQ of lambda-cyhalothrinin in these vegetables were 0.009 and 0.027 µg mL − 1 .
To analyze the storage stability of the pesticides at freezing condition, control vegetable samples were forti edwith higher concentrations of the ve pesticides with two replicates each and the samples were then stored in a freezer. The vegetable samples forti ed with chlorpyrifos, cypermethrin and imidacloprid, were freezed on 12th January, 2016 and kept at freezer for about 35 days. Control vegetable samples spiked with lambda-cyhalothrin and acephate were freezed for about 39 days, from 18th February to 27th March, 2016. Then these spiked samples were extracted, cleaned-up and analyzed following the same procedure to nd out the stability of the pesticide residues at freezing condition.
The average recoveries of the pesticides were found in the range of 84-93% with RSD value ranging from 1.1 to 4.2 which indicated that pesticide residues were quite stable at -20°C storage condition because the recovery results were consistent with the range listed in the Codex guidelines (Codex 2010). The storage stability results showed that the pesticides were quiet persistent at freezing condition and not degraded during the period of this test.

Conclusions
All pesticides are suitable to use in different vegetables under agro climatic condition of Bangladesh. However, farmers should apply pesticide as recommended dose and harvest after 2-3 days depending on pesticides. The present study recommends to keep vegetable at ambient temperature rather keeping in refrigerator after buying from marker for consumer safety. Figure 1 Dissipation curve of chlorpyrifos in tomato, cauli ower and sponce gourd Figure 2 Dissipation curve of chlorpyrifos in cauli ower, cabbage and sweet gourd as the constituent of Nitro 505 EC