Introduction

Chilli (Capsicum annuum L.), member of solanaceae family, is a seasonal crop which is consumed throughout the year. It is native of tropical America and is believed to be introduced by the Portuguese in India in seventeenth century (Parey et al. 2013). Chilli is indispensible to kitchen from day to day curries and pickles to worldwide range of salads and sauces. Being a good source of vitamin A, B, C, and E and minerals like molybdenum, manganese, potassium, and copper, it is becoming very popular (Thamaraikannan et al. 2011). Fresh chilli contains more vitamin C than orange which makes it very effective as immune stimulant and healing agent, especially for cellular damage (Anonymous 2009).

Analogous to every crop, chilli is attacked by various insect pests resulting in yield losses. The crop is attacked by 51 species of insects and 2 species of mites which belong to 27 families under nine orders. Thrips, mites, and pod borer are serious among its pests (Jyot et al. 2013). Moreover, the loss in yield is augmented by bacterial, viral, and fungal attack (Waghulde et al. 2011). To minimize these losses, pesticides are repeatedly applied during the entire period of growth and sometimes even at the fruiting stage. The major problem with the use of pesticides is the persistence of their residues in crops which have malignant effect on humans and animals as well as environment. Moreover, the presence of pesticide residues leads to lowering in quality of produce. Therefore, the use of pesticides is becoming a major constraint in increasing the export of chilli (Thamaraikannan et al. 2011). Insecticide residues have also been found in farmgate samples of chilli. Even with the adoption of Integrated Pest Management System, farmers still prefer chemical control methods due to their immediate results.

Different types of insecticides are used on chilli; the choice depends partly on the time interval between insect attack and crop harvest, and the preharvest interval (PHI) of the insecticides. Frequency of spray mainly depends on the size and nature of insect populations, and partly on climatic and other characterstics. Cypermethrin [(RS)-α-cyano-3-phenoxybenzyl(1RS,3RS;1RS,3SR)-3-(2,2-dichlorovinyl)-2,2-dimethyl-cyclopropane carboxylate] (Fig. 1) is a synthetic pyrethroid having high insecticidal activity and consists of a mixture of four cis- and four trans-isomers. The cis-isomers are considered to be more acutely toxic than the trans-isomers and are used to control many pests, for instance, lepidopterous pests of cotton, fruit, and vegetable crops. This extremely hydrophobic insecticide is both a stomach poison and a contact insecticide (Jin and Webster 1988) that affects the nervous system of insects by affecting voltage-dependent sodium channels and inhibiting ATPase enzyme.

Fig. 1
figure 1

Structure of cypermethrin

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Estimation of pesticide residues in vegetables is imperative to avoid the health hazards. Therefore, the present investigation was carried out to study the persistence and risk assessment of cypermethrin residues in chilli and to suggest the safe waiting period for its consumption.

Materials and methods

Chemicals and reagents

The technical grade analytical standard of cypermethrin (purity 95.1 %) was procured from M/s Sigma Aldrich, India. Super fighter 25 EC used for the application was obtained from M/s Insecticides (India) Ltd., Udhampur, India. Analysis of acetone extract of the formulation showed only cypermethrin and none of its metabolic products. The analysis was found to be accurate with respect to its active ingredient.

Acetonitrile, sodium chloride (NaCl; ASC reagent grade ≥99.9 %), and analytical grade activated anhydrous MgSO4 were obtained from Merck, Darmstadt, Germany. Anhydrous sodium sulfate (Na2SO4; AR grade) was obtained from SD Fine Chemicals, Mumbai. Primary Secondary Amine (PSA) sorbent and activated graphitic carbon black (GCB, 400 mesh) were obtained from Agilent Technologies, Bangalore, India. The solvents were redistilled in all-glass apparatus before use. The suitability of the solvents and other chemicals was ensured by running reagent blanks before actual analysis.

Preparation of standard solution

A standard stock solution of cypermethrin (1 mg mL−1) was prepared in hexane. The standard solutions required for calibration curve (2.00, 1.50, 1.00, 0.50, 0.25, and 0.10 μg mL−1) (Fig. 2) were prepared from stock solution by serial dilutions with hexane. All standard solutions were stored at 4 °C before use.

Fig. 2
figure 2

Calibration curve of cypermethrin

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Field experiment

Chilli (var. CH-3) was raised during March–August 2013 at Entomological Research Farm, Punjab Agricultural University, Ludhiana, following recommended agronomic practices (Gill and Dhillon 2013). There were three treatments, i.e., control, recommended, and double the recommended dosages, and three replications for each treatment arranged in a randomized block design (RBD), and size of the each plot was 50 m2. The meteorological data comprising temperature, relative humidity, and sunlight hours from the first spray to final sampling is presented in Fig. 3.

Fig. 3
figure 3

Meteorological data comprising temperature, relative humidity, and sunlight hours

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The first application of cypermethrin (Super Fighter 25 EC) at 50 and 100 g a.i. ha−1 was done at 50 % fruiting stage followed by another two applications at 10-day interval. Pesticide was sprayed as foliar application with the help of an ASPEE Knapsack sprayer fitted with hollow cone nozzle.

About 500 g green chilli fruit of marketable size was collected randomly before and 0 (1 h), 1, 3, 5, 7, 10, 15, and 20 days after last application of the insecticide. Red chilli samples were collected at 25th day. The chilli fruit were collected from each plot separately, packed in polyethylene bags, and brought to the laboratory for processing. The samples were processed immediately after sampling.

Extraction and cleanup with QuEChERS method

A sub-sample of 15 g of chilli fruit was weighed into a 50-mL centrifuge tube, and 30 mL acetonitrile was dispensed into it. The samples were homogenized using a high-speed homogenizer (Heidolph Silent Crusher-M®) for 2–3 min at 14,000–15,000 rpm. Sodium chloride (NaCl) 10 ± 0.1 g was added for phase separation. The contents were centrifuged at 2500–3000 rpm for 3 min. An aliquot of 15 mL acetonitrile layer was transferred over 10 ± 0.1 g anhydrous sodium sulfate (Na2SO4) in a test tube. The acetonitrile extract was subjected to cleanup by dispersive solid phase extraction (DSPE). An aliquot of 6 mL acetonitrile was taken in a test tube containing 0.15 ± 0.01 g PSA sorbent, 0.90 ± 0.01 g anhydrous MgSO4, and 0.05 ± 0.01 g graphitic carbon black, and the contents were thoroughly vortexed on a vortex shaker and centrifuged at 2500–3000 rpm for 1 min. Four milliliters of aliquot of this acetonitrile extract was evaporated to dryness using a low-volume rotary evaporator at 35 °C, and final volume was made up to 2 mL with hexane.

Estimation by GLC

Analysis of the cypermethrin residues was carried out using a gas liquid chromatograph (GLC) (Shimadzu Model GC-2010) equipped with electron capture detector (ECD, 63Ni) supplied by M/S Shimadzu, Japan. A capillary column Rxi-5sil MS (30 m × 0.25 mm i.d. × 0.25 μm film thickness of 5 % phenyl and 95 % methyl polysiloxane) was used for estimation of cypermethrin residues. The working conditions of GLC were injector temperature of 290 °C, column temperature of 280 °C, and detector temperature of 300 °C. Carrier gas (N2) flow was maintained at 30 mL min−1. Before use, the column was primed with several injections of standard solution of cypermethrin until a consistent response was obtained. Suitable aliquots of the cleaned up samples were then injected into the ECD mode of the detector. The presence of cypermethrin in the samples was identified and quantified by comparison of the retention time and peak heights of the sample chromatograms with that of standards run under identical operating conditions. Under these operating conditions, the retention time of cypermethrin was found to be 7.647, 7.879, and 7.967 min (Fig. 4).

Fig. 4
figure 4

Gas chromatograms of a standard cypermethrin, b untreated chilli fruit sample, c fortified chilli fruit sample at 0.05 mg kg−1, and d field-treated chilli fruit sample at recommended dosages

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Confirmation by GC-MS

The substrate contamination is found to be more in chilli due to pigmentation which interferes with the baseline during analysis. So the further confirmation of residues of cypermethrin was done by a gas ghromatograph–mass spectrometer GC-MS-QP 2010 plus, Shimadzu (Jyot et al. 2013) equipped with capillary column (30 m × 0.25 mm i.d. × 0.25 um film thickness) in single ion monitoring (SIM) mode. The system software used was GC-MS solution version 2.5. Helium was used as a carrier gas with flow rate of 0.94 mL min−1. The GC-MS operating conditions for cypermethrin were oven (program) initial temperature of 80 °C held for 3 min, ramp of 20 °C min−1 to 180 °C and held for 2 min, then ramp of 2 °C min−1 to 190 °C, held for 2 min, and then again ramp of 5 °C min−1 to 280 °C, held for 10 min; injector temperature was 285 °C. Helium was used as a carrier gas with a flow rate of 0.7 mL min−1. Injection volume was 1 μL in splitless mode. Detector voltage was maintained at 0.9 kV in SIM mode. The mass spectrum of cypermethrin showed ions at m/z 163, 181, and 209. These values were compared with the samples spiked with cypermethrin and samples collected from treated plots for confirmation of cypermethrin residues.

Results and discussion

Quality control and quality assurance of analytical method

Green chilli and red chilli samples were spiked with cypermethrin at three concentration levels (0.05, 0.25, and 0.50 mg kg−1) and analyzed as per the methodology described in the “Materials and methods” section. The recoveries of cypermethrin in green chilli and red chilli ranged between 82.52–87.40 and 81.85–86.79 %, respectively (Table 1). The percent recoveries of cypermethrin were found to be consistent and more than 80 %. Therefore, the results have been presented as such without applying any correction factor. The precision of the method was determined by repeatability studies and expressed by relative standard deviation (RSD). The RSD of cypermethrin were found to be 2.50–3.60 and 2.75–3.38 %, on green and red chilli, respectively (Table 1). Quantification was accomplished by standard calibration curve prepared by diluting the stock solution. Good linearity was achieved with a correlation coefficient of 0.998 for cypermethrin (Fig. 2).

Table 1 Recovery for cypermethrin in spiked green and red chilli fruit samples at different concentration levels
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Limit of detection and quantification

Cypermethrin residues were determined by comparison of peak areas of the reference standards with that of the unknown or spiked samples run under identical working conditions of the instruments employed. For cypermethrin, half-scale deflection was obtained for 0.5 ng which could be easily identified from the baseline, and 0.1 ng of the compound produced 10 % deflection. The processed sample when injected (2 μL equivalent to 2 mg) did not produce any background interference. Thus, limit of quantification (LOQ) was found to be 0.05 mg kg−1 and limit of detection (LOD) being 0.017 mg kg−1.

Persistence of cypermethrin residues on green chilli samples

The average initial deposits of cypermethrin on green chilli were found to be 1.46 and 3.11 mg kg−1, respectively, following two applications at 10-day interval of cypermethrin 25 EC at 50 and 100 g a.i. ha−1. The initial deposits dissipated to 0.90 and 1.74 mg kg−1 after 3 days at recommended and double the recommended dosages, respectively, thereby showing a loss of about 38.36 and 44.10 %. About 80 % of residues of cypermethrin dissipated after 10 days of last spray at both the dosages. At 20th day, cypermethrin residues were found to be 0.06 and 0.14 mg kg−1 for recommended and double recommended dosages, respectively (Table 2).

Table 2 Residues of cypermethrin (mg kg−1) on chilli fruits at different times after the application of cypermethrin 25 EC at 50 and 100 g a.i. ha−1
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These results are in agreement with those of Singh et al. (1990) who studied the residues of cypermethrin on cauliflower. Following the application of cypermethrin on cauliflower crop at 50 g a.i. ha−1, the maximum initial deposits of these insecticides on heads and leaves observed were 1.10 and 0.75 mg kg−1, respectively. In a study on persistence of cypermethrin on chickpea following its application at 60 and 90 g a.i ha−1, the initial deposits were found to be 0.42 and 0.60 mg kg−1 at low and high doses, respectively. The rate of dissipation was rapid up to the fifth day following gradual decrease at subsequent intervals. Cypermethrin residues persisted beyond 15 days at both the doses (Kumar et al. 1998). Singh et al. (2004) studied the persistence of cypermethrin on okra following applications of cypermethrin at 50 and 100 g a.i. ha−1. The initial deposits were found to be 0.27 and 0.38 mg kg−1 at recommended and double the recommended dosages, respectively. The residues dissipated to more than 80 % at 10 days. But, the residues persisted even after 15 days. Duara et al. (2003) studied the cypermethrin residues in top portion of brinjal plant (15–20 cm) comprising of stem, leaves, flowers, and fruits. The initial deposits of cypermethrin on plant parts were found to be 0.31, 0.58, and 0.93 mg kg−1 following application at 22.5, 45, and 75 g a.i. ha−1, respectively. These residues were dissipated to more than 70 % at all the dosages. These residues were found to below determination limit of 0.001 mg kg−1 at lower dosages at 10 days. But, cypermethrin residues were found to be present (0.07 mg kg−1) following application at 75 g a.i. ha−1 at 10 days.

Cypermethrin residues on red chilli samples

Residues of cypermethrin in red chilli collected after the 25th day of last spray were found to be below its determination limit of 0.05 mg kg−1 at both the dosages (Table 3). The residues of fipronil and profenofos in dried red chillies (collected at harvest) were also found to below detectable limit by Reddy et al. (2007a). Similarly, the residues of endosulfan, dicofol, dimethoate, and λ-cyhalothrin in harvested red chillies were found to be below detectable levels in all the treatments (Reddy et al. 2007b).

Table 3 Maximum permissible intake (MPI) and theoretical maximum residue contribution (TMRC) of cypermethrin in chilli fruits
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Half-life values of cypermethrin on green chilli samples

The correlation coefficients were found to be 0.993 and 0.9792 at recommended and double the recommended dosages, respectively (Fig. 5). The dissipation of residues of cypermethrin from chilli fruits followed first-order kinetics at recommended dosages. Half-life value (T1/2) is usually defined as the time required for half of the given quantity of material to dissipate (Gunther and Blinn 1955). The T1/2 of cypermethrin was calculated using Hoskins (1961) formula. Half-life (T1/2) of cypermethrin on chilli was observed to be 4.43 and 4.70 days, respectively, following application of cypermethrin at 50 and 100 g a.i. ha−1 (Fig. 5). Half-life values of cypermethrin on chickpea green pods were 8.36 and 9.40 days following application of cypermethrin at 60 and 90 g a.i. ha−1, respectively (Kumar et al. 1998). Similarly, half-life value of cypermethrin on okra fruits was 2.25 days following application of cypermethrin at 0.012 % at 10-day interval initiating from 30 days (Khan et al. 1999). The half-life values of cypermethrin on brinjal plant parts were found to be 3.30, 3.39, and 3.16 days following application at 22.5, 45, and 75 g a.i. ha−1, respectively (Duara et al. 2003). The half-life values of cypermethrin on okra fruits were found to be 3.4 and 3.5 days at recommended and double the recommended dosages (Singh et al. 2004).

Fig. 5
figure 5

Semi-logarithm graph showing dissipation kinetics of cypermethrin residues on chilli

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Risk assessment of cypermethrin on green chilli fruits

Human health risk situation is a function of hazard and exposure to that hazard. If the hazard is small and fixed, then the risk will be proportional to exposure, which can be reduced to low and occasional (Bates, 2002). The use of pesticides on food crops leads to unwanted residues, which may constitute barriers to exporters and domestic consumptions when they exceed maximum residue limit (MRL). In the present study, theoretical maximum residue contribution (TMRC) values were calculated by considering recommended consumption of chilli as 2.5 g in Indian context (Anonymous 2002) and compared with maximum permissible intake (MPI) to evaluate the risk to the consumer. The prescribed acceptable daily intake (ADI) of cypermethrin is 0.05 mg kg−1 body weight−1 day−1 (Sharma 2007). Maximum permissible intake (MPI) was obtained by multiplying the ADI with the weight of average Indian person (55 kg) (Mukherjee and Gopal 2000). MPI was calculated to be 2750 μg person−1 day−1. The TMRC values on 0 day were found to be 4.100 and 8.800 μg−1 person−1 day−1 (Table 3) and were found to be below the MPI on chilli fruit even on 0 day. Hence, the insecticide would not cause any adverse effect after consumption of chilli fruits. Therefore, on the basis of TMRC and MPI values, it is suggested that a waiting period of 1 day should be observed before consumption of chilli fruit sprayed with cypermethrin at the recommended dosages.

Conclusions

Half-life values for cypermethrin following two applications at recommended and double the recommended dosages on chilli fruits were observed to be 4.43 and 4.70 days, respectively. The Theoretical maximum residues contribution (TMRC) values were found to be quite low as compared to maximum permissible intake (MPI); hence, it was concluded that the insecticide will not cause any adverse effects after consumption of green chilli fruits. A waiting period of 1 day is suggested to reduce the risk before consumption of chilli fruits. Therefore, application of cypermethrin at the recommended dose on chilli is quite safe from the point of view of crop protection, environmental contamination, and use by the consumer.