Minimization of Imidacloprid Residues in Cucumber and Bell Pepper Through Washing with Citric Acid and Acetic Acid Solutions and Their Dietary Intake Assessment

Abstract

In order to assess the extent and fate of imidacloprid residues, the present research project was designed. Vegetables (cucumber and bell pepper) were collected from a supervised farmer's field. Fresh vegetables were dipped in organic acid solutions such as acetic acid (1.5, 3.0, 6.0, and 9.0%), citric acid (1.5, 3.0, 6.0, and 9.0%), and their combination (50:50 each) for 5 min. Imidacloprid residue level was determined before and after dipping in organic acids through high-performance liquid chromatography. The pesticide residue level was in the range of 0.12 ± 0.04 to 1.92 ± 0.04 mg/kg and 0.52 ± 0.02 to 1.89 ± 0.06 mg/kg in cucumber and bell pepper, respectively. The highest reduction of imidacloprid residue level was found after washing with 9% citric acid solution (93.75%) followed by a mixture of citric acid and acetic acid (1.5% each) solution (91.14%), and the lowest value was found in the mixture of citric acid and acetic acid (0.75% each) solution (48.43%) in cucumber. Likewise, in bell pepper, maximum decrease in imidacloprid content was revealed with 9% citric acid (72.48%), followed by 9% acetic acid (68.78%), and the least was observed with the mixture of citric acid and acetic acid (0.75% each) solution (11.64%). Dietary intake assessment of the pesticide residue was estimated according to the maximum permissible intake of imidacloprid, which is 3.78 mg/person/day. Dipping significantly reduced the amount of maximum permissible intake and ranged from (1.01 to 8.40 and 10.34 to 33.23 mg/kg of body weight/day) in cucumber and bell pepper, respectively. Organic acids dipping significantly reduced the imidacloprid content up to 80% in cucumber and bell pepper.

Keywords:

• Pesticide residue
• Imidacloprid
• Dietary intake assessment
• Food safety
• Vegetables

INTRODUCTION

Vegetables play an imperative role in human health by providing minerals, micronutrients, vitamins, antioxidants, phytosterols, and dietary fiber.[1,2] To cope with insect pest damage in crops, pesticides are commonly used during production of vegetables. Meanwhile, these pesticides are incorporated in the plant tissue when the concentration of applied pesticides exceed the recommended limit and may cause toxic effect. These toxic residues are one of the major reasons to disturb metabolic activity and thereby initiated health problems. In a new epoch, some pesticides and their metabolites are reported to be carcinogenic, mutagenic, and teratogenic in nature.[3] Therefore, there is an increasing social desire to lessen the use of pesticides in horticultural crops,[4] which is also supported by the European Union by a Council Directive.[5]

One of the most applied pesticides in vegetable crops is imidacloprid (Confidor®) in developing countries in farmers’ fields to control or reduce the sucking and chewing insect pest damage. Imidacloprid is a neonicotinoid insecticide, which has a neurotoxic effect through binding protein receptor in the membrane of the nerve synapse. Furthermore, it is classified by the U.S. Environmental Protection Agency in both toxicity class II and class III. It also opens/alters an ion channel that affects on flushes of ions of sodium, potassium, calcium, and chloride.[6] The highest dietary exposure of imidacloprid is approximately 0.06 mg/kg of body weight/day and is associated with the consumption of contaminated vegetation.[7]

Generally, pesticides are absorbed by vegetables and upon consumption, in a minute quantity, may cause serious human health disorders.[8] Persistence and distribution of residues on the edible portions of produce have been reported in major and minor commodities like bell peppers and tomatoes.[9] Fresh vegetables are considered to be the most potential sources of pesticide residues in the diet. In this context, dipping of vegetables in acetic acid solutions (@10% for 20 min) reduced more than 70% of the pesticide residue.[10] Similarly, Kumari[11] also reported that washing curb residues from 20 to 77% by dipping and may increase the reduction up to 32 to 100% through the boiling of vegetables. Moreover, the cooking of tomatoes facilitated to purge most pesticide residues from contaminated vegetables.[12] Earlier, the investigation indicated that washing with water and/or detergent solutions was necessary to decrease the intake of pesticide residues in peppers, cucumbers, and cherry tomatoes after 6 weeks of being sprayed of methamidophos[13] and endosulfan residues.[14] Moreover, Puglies et al.[15] reported that pesticide residue content reduced in nectarines by washing with aqueous solutions of citric acid, H₂O₂, C₂H₅OH, and some other solution as compared with simple tap water. Osman et al.[16] reported that citric acid and acetic acid washing with 1 and 2% solution was more effective than other treatments like H₂O₂, KMnO₄ and tap water for chlorpyrifos residues reduction in vegetables.

The present research was designed to evaluate the residual concentration of imidacloprid in cucumber (Cucumis sativus) and bell pepper (Capsicum annuum) along with its reduction through dipping in organic acids solution with different concentrations for 5 min. These vegetables are mostly utilized fresh as salad in homes, restaurants, and in public streets, especially in the summer season of Pakistan. There is a dire need to explore new horizons of the washing/dipping treatments, which can significantly contribute to reduce the pesticide residue levels in food.

MATERIALS AND METHODS

Chemicals and Reagents

Chemicals used for the present study, including citric acid, acetic acid, sodium chloride, sodium sulfate, and ethyl acetate, were purchased from Merck Chemical Co. Standard and high-performance liquid chromatography (HPLC) grade reagents used for imidacloprid residue level determination were purchased from Sigma Aldrich (Tokyo, Japan) and the analytical grade reference insecticide standard of imidacloprid (>98% purity) was purchased from Dr. Ehrenstorfer (GmbH, Germany).

Raw Materials

Vegetables (cucumber and bell pepper) were grown at a supervised farmer's field in Faisalabad District of Pakistan. Both vegetables were grown in separate plots and all cultural practices and imidacloprid applications as per requirement were done at different intervals per normal schedule. The samples were collected at optimum maturity for the analysis. The vegetable samples collected from the supervised farmer's fields were brought to the laboratory of the National Institute of Food Science and Technology, University of Agriculture, Faisalabad. Samples of both vegetables were analyzed after tap water washing and soaking for 5 min in acetic acid, citric acid solutions, as well as the combination of both acids.

Washing with Water

The vegetables were washed in water by rinsing with gentle rotation by hand under normal tap water for 30$ and dried with a disposable paper towel. Grinding of the vegetables was done to prepare the sample for estimation of the pesticide residue.[17]

Dipping in Solution

Both of the vegetables were dipped for 5 min in different concentrations of citric acid (1.5, 3, 6, and 9%), acetic acid (1.5, 3, 6, and 9%), and a combination of both acids (1.5, 3, 6, and 9%).

Extraction of Pesticide Residues

The pesticide residue from vegetables was extracted through a method described by Fernandez-Alba,[18] with some modifications. A well-homogenized vegetable sample (50 g) was mixed with anhydrous sodium sulfate (20 g), sodium chloride (10 g), and ethyl acetate (50 mL). The whole mixture was shaken in a GFL shaker (Germany) at 500 rpm for 2 min and filtered through Whatman filter paper No. 1 (2 μm) to remove any particulate material from the extracted sample. The filtrate was passed through a column containing anhydrous sodium sulfate (5 g) followed by activated charcoal (10 g) for bleaching. The eluent was then evaporated in a rotary evaporator (Eyela, Japan) and dried under a gentle nitrogen stream then re-dissolved in 2 mL of acetonitrile.

High Performance Liquid Chromatography Conditions

Prepared samples (2 mL) were filtered through 0.45-μm filter paper with the help of a syringe filter, and 50 μL of the sample was injected in the isocratic HPLC (Perkin Elmer liquid chromatographic system, series 200) equipped with an auto sampler and UV-VIS detector having a reverse phase C₁₈ column (25 cm × 4.6 mm i.d.), maintained at 30°C. Detection of imidacloprid was done on a UV-Vis detector at 270 nm by using water and acetonitrile (40:60 v/v) mobile phase at a flow rate of 1 mL/min. The mobile phase was pre-filtered through 0.45-μm pore size filter paper by using a vacuum filtration assembly. The retention time of imidacloprid under such conditions was 6.11 min (Fig. 1).

Figure 1 Standard chromatogram of imidacloprid.

The method used for determination of imidacloprid insecticide residues was validated before the analysis of actual samples. For this purpose, 20 μg of imidacloprid was added in the 50 g of blended cucumber and bell pepper and extracted as described above. Clean up and concentration was also performed by following the same set of conditions. Reproducible recoveries of >94% were obtained for the imidacloprid used in this study for both of the vegetables. Linear range of determination for imidacloprid was constructed by using stepwise dilution analysis and run on HPLC. The standard curve showed that linear range was found to be 0.04 to 10.24 μg/mL with correlation coefficients of 0.999 (Fig. 2). The analytical method was found to be highly sensitive to fulfill quality criterion for insecticide residue analysis. The concentration of the unknown sample was quantified by using the standard curve.

Figure 2 Standard curve of imidacloprid with various concentrations. (Color figure available online.)

Dietary Intake Assessment

Dietary intake assessment of imidacloprid was calculated by following the procedure as described by Aziz et al.[19] and compared with different washing treatments effects on cucumbers and bell pepper. The prescribed acceptable daily intake (ADI) of imidacloprid is 0.06 mg/kg body weight/day for humans (Department of Health and Ageing, Government of Australia, 2011).[20] Multiplying the upper limits of ADI with the body weight of an average Pakistani of 63 Kg, the maximum permissible intake (MPI) was calculated to be 3.78 mg/person/day. The values of dietary exposure were then determined by multiplying the imidacloprid concentration in the individual vegetable with average per capita daily consumption of cucumber (8.49 g) and bell pepper (19.9 g).[21,22]

Statistical Analysis

The data obtained from all the parameters was subjected to statistical analysis though one way ANOVA at 5% probability level by using Statistix 8.1 software (Tallahassee, FL, USA). Differences in means were carried out by using the least significant difference technique.[23]

Table 1  Pesticide residue level and its reduction of cucumber and bell pepper through washing with organic acid solutions

Treatments	Cucumber (mg / kg)	Reduction (%)	Bell pepper (mg/kg)	Reduction (%)
Control (tap water)	1.92 ± 0.04a	—	1.89 ± 0.061a	—
Citric acid (1.5%)	0.48 ± 0.02e	75.00	1.02 ± 0.031e	46.03
Citric acid (3%)	0.31 ± 0.01g	83.85	0.87 ± 0.040g	53.96
Citric acid (6%)	0.24 ± 0.09h	87.50	0.78 ± 0.028g	58.73
Citric acid (9%)	0.12 ± 0.04j	93.75	0.52 ± 0.019i	72.48
Acetic acid (1.5%)	0.71 ± 0.013c	63.02	1.60 ± 0.058b	15.34
Acetic acid (3%)	0.48 ± 0.026e	75.00	0.95 ± 0.034ef	49.73
Acetic acid (6%)	0.37 ± 0.018f	80.72	0.68 ± 0.024h	64.02
Acetic acid (9%)	0.34 ± 0.018fg	82.29	0.59 ± 0.021i	68.78
Citric acid (0.75%) + Acetic acid (0.75%)	0.99 ± 0.036b	48.43	1.67 ± 0.067a	11.64
Citric acid (1.5%) + Acetic acid (1.5%)	0.53 ± 0.019d	72.39	1.49 ± 0.053c	21.16
Citric acid (3.0%) + Acetic acid (3.0%)	0.32 ± 0.011g	83.33	1.12 ± 0.040d	40.74
Citric acid (4.5%) + Acetic acid (4.5%)	0.17 ± 0.06i	91.14	0.82 ± 0.029g	56.61
Values are triplicates and expressed as mean ± SD. The same letters in a column showed non-significant differences among treatments.

RESULTS AND DISCUSSION

Imidacloprid Residues in Cucumber and Bell Pepper

Imidacloprid residue contents estimated in cucumber and bell pepper after washing with tap water and citric acid and acetic acid and their combinations are depicted in Table 1. The imidacloprid level in the cucumber was found to be in the range of 0.12 ± 0.04 to 1.92 ± 0.04 mg/kg. The highest imidacloprid content was found in the control (1.92 ± 0.04 mg/kg) followed by a mixture of citric acid and acetic acid (0.75% each) solutions (0.99 ± 0.03 mg/kg). The least residue concentration of 0.12 ± 0.04 mg/kg was observed when the cucumber was washed with citric acid (9%) solution. Dipping of vegetables in organic acids may significantly decline the pesticide residue level of cucumber. On the other hand, bell pepper showed more concentration of selected pesticide as compared with cucumber, which illustrated that it may absorb more pesticide residue. The results shown in Table 1 depicted that imidacloprid residue ranges from 0.52 ± 0.02 to 1.89 ± 0.06 mg/kg. The highest concentration was found in the control (1.89 ± 0.06 mg/kg) followed by a mixture of citric acid and acetic acid (0.75%) solutions (1.67 ± 0.06 mg/kg), whereas the lowest content was observed in 9% citric acid (0.52 ± 0.02 mg/kg). Imidacloprid reduction percentage in bell pepper was found to be in the range of 11.64 to 72.48% with citric acid, acetic acid, and their combined solutions washing.

The results obtained in this study showed a comprehensive picture of the different organic solutions used in an instant study with varied concentrations and their combinations. One of the best washing solutions among the present studied solution for the significant reduction in imidacloprid level in cucumber was found with 9% solution of citric acid and the least effective was observed by a combined solution of citric acid (0.75%) and acetic acid (0.75%) in all the treatments. Conclusively, for the cucumber and bell pepper, 9% citric acid solution washing was more effective than acetic acid and a combination of citric acid and acetic acid.

These results are in harmony with the finding of Abou-Arab et al.[12] that washing with organic acids significantly reduced the pesticides residues concentration in the raw and processed vegetables. In general, washing with water and/or detergent to remove most of the surface residues is worthwhile prior to utilizing vegetables. Furthermore, some of the pesticides penetrate from the surface into the deep layers of the plant tissue through dissolving in the top wax-like layers and progress to the inside.^[12]

Earlier, it was reported that acetic acid washing is comparatively better than sodium chlorine solution washing with the same time and concentration. On the other hand, there was a gradual increase in the percent residue reduction through increasing the concentration of washing solution for the same-time treatment. The present results are in corroboration with the findings of Ismail et al.[24] and Soliman.[25] Likewise, Zhang et al.[10] also reported that partial removal of pesticide residue was done by the washing operation in cabbages. Osman et al.[16] reported that citric acid and acetic acid with 1 and 2% solution washing was more effective than other treatments like H₂O₂, KMnO₄, and tap water for chlorpyrifos residues reduction in horticultural crops. Similar results were also observed by Puglies et al.[15] that pesticide residue level was significantly reduced in nectarines by washing with aqueous solutions of citric acid, C₂H₅OH, glycerol, H₂O₂, KMnO₄, sodium metabisulfite, sodium laurylsulfate, sodium hypochlorite, and urea when compared with tap water washing. Acetic acid solution washing provided up to 91.5, 86.0, and 93.7% loss in the case of dimethoate, profenofos, and pirimiphos-methyl contamination in tomato, respectively. Moreover, ascorbic acid (10%) and citric acid (10%) solutions reduced aldrin content by approximately 85–90% in vegetables.[26]

Dietary Intake Assessment

Data of the dietary intake assessment revealed that all the treatments of the washing with organic acids, i.e., citric acid, acetic acid, and their mixture with various concentrations, significantly decreased imidacloprid contents in cucumbers and bell pepper. Maximum permissible intake of cucumber for imidacloprid was observed in dipping solution treatments varying from 1.01 to 8.40 mg/kg of the body weight (Table 2). Similarly, imidacloprid residue concentration in bell pepper was higher than that of cucumber and ranged from 10.34 to 33.23 mg/kg of the body weight.

Table 2  Dietary intake assessment of imidacloprid residue level in cucumber and bell pepper after dipping in citric acid and acetic acid solutions

Treatments	Cucumber (mg/kg)	Bell pepper (mg/kg)
Control (tap water)	16.30a	37.61a
Citric acid (1.5%)	4.07e	20.29e
Citric acid (3%)	2.63g	17.3g
Citric acid (6%)	2.03h	15.52g
Citric acid (9%)	1.01j	10.34i
Acetic acid (1.5%)	6.02c	31.84b
Acetic acid (3%)	4.07e	18.90ef
Acetic acid (6%)	3.14f	13.53h
Acetic acid (9%)	3.02fg	11.74i
Citric acid (0.75%) + Acetic acid (0.75%)	8.40b	33.23a
Citric acid (1.5%) + Acetic acid (1.5%)	4.49d	29.65c
Citric acid (3%) + Acetic acid (3%)	2.71g	22.28d
Citric acid (4.5%) + Acetic acid (4.5%)	1.44i	16.31g
Values are triplicates and expressed as mean ± SD. The same letters in a column showed non-significant differences among treatments.

Dikshit et al.[27] reported the dietary intake assessment of imidacloprid residue level in tomato and concluded that the judicious application of pesticide reduced the dietary exposure up to safety limits as recommended by ADI of pesticide in a supervised field trial. Audenhaegea et al.[28] compared the pesticide residue and dietary intake of the French general population and vegetarian population. Higher fruit- and vegetable-consuming segments of the population appear to be preferentially exposed to pesticide residue threats. Recently, pesticide residue levels and risk assessment were assessed by Jang et al.,29 and it was reported that the deterministic risk indexes of diethofencarb, indoxacarb, diniconazole, and chlorothalonil were 7.33, 5.13, 3.92, and 3.09% in the population of Seoul, respectively.

CONCLUSIONS

The organic acids solution washing treatments significantly reduced the pesticide residue level in cucumber and bell pepper. Among studied organic acids solutions, citric acid (9%) solution showed better reduction in pesticide residue in cucumber and bell pepper. Generally, concentration of washing solution increased; momentous reduction was observed in imidacloprid residue level. Similarly, dietary assessment revealed that the organic acids solution washing significantly reduced the intake level of imidacloprid residue and reduced the food-related disorders associated with intake of pesticides residue. Convincingly, citric acid solutions could be recommended for the removal of pesticide residue level in fresh vegetables consumed as fresh salad.

ACKNOWLEDGMENT

The authors are highly thankful to the Higher Education Commission of Pakistan for the financial assistance.