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Assessment of Imidacloprid and Acetamiprid Residues in Okra and Eggplant Grown in Peri-Urban Areas and their Dietary Intake in Humans.

Byline: Adnan Amjad, Muhammad Atif Randhawa, Saeed Akhtar, Muhammad Binyameen, Muhammad Riaz and Muhammad Zafarullah

Summary: This research work was designed to estimate the gradually increasing use of insecticides on vegetables to create awareness among the consumers regarding their insecticide residues. The main aim of this study is to highlight the harmful effects of insecticides in comparison with dietary intake assessment of vegetables. In this regard, total 180 (n = 60x3) samples of okra (Abelmoschus esculentus) and eggplant (Solanum melongena) comprising of 30 samples vegetable were collected from the peri-urban farming system of Multan, Faisalabad and Gujranwala to assess insecticide residues along with their dietary intake assessment. The selected samples were quantified for the insecticide residues by using High Performance Liquid Chromatography (HPLC) technique equipped with UV/Visible detector. Results revealed that imidacloprid residues found in eggplant (0.226 mg Kg-1) and okra (0.176 mg Kg-1) from Multan region were greater than the residues reported from Gujranwala and Faisalabad.

Out of all analyzed samples for imidacloprid and acetamaprid residues, 58% and 65% samples contained detectable residues respectively. Whereas 10% and 15% samples exceeded their maximum residue limits (MRLs) established by European Commission. Dietary intake assessment for imidacloprid and acetamiprid was calculated as per their maximum permissible intake (MPI) values i.e. 3.84 and 4.48 mg person-1day-1, respectively, which revealed that although a reasonable proportion of samples exceeded MRLs but their consumption was found within safe limit.

Keywords: Insecticides, Vegetables, Maximum residual limits (MRLs), Maximum permissible intake (MPI).

Introduction

Insecticides boost up agricultural production but their random use also effects human health, pollute environment and effect non-target organisms [1]. Every year about 2.5 million tons of insecticides are applied worldwide. In Pakistan, average number of pesticide application is around 10 or 11 per growing season. [2] Out of total application of insecticides 27% is being utilized on vegetables and fruits. Annual production of vegetables in Pakistan is about 5192 thousand tons [3]. Eggplant (Solanum melongena L.) and okra (Abelmoschus esculentus L.) are widely growing vegetable crops at the countryside. Various agrochemicals are used at different stages of cultivation for their effective role in protecting vegetables from insects-pests. Farmers are widely applying synthetic insecticides like acetamiprid, imidacloprid, organophosphate and pyrethroids for the control of these insect-pests.

Among vegetables; eggplant, tomato, cauliflower and okra are few common vegetables grown throughout the whole country however, severely affected by insect-pest causing various diseases. On eggplant, the average quantity of 4.6L of insecticide including their active ingredients per season per hectare is applied [4].

A new emerging class of insecticides introduced in the market after the commencement of pyrethroids is neonicotinoid insecticides. This class has an annual market share of more than 600 million Euros in which imidacloprid has been ranked as the most selling insecticide all over the world. Neonicotinoids encompass the substitution of organophosphates, carbamates and synthetic pyrethroids that formerly dominate the global market together with a wide spectrum of insecticide action and less severe mammalian toxicity [5]. Neonicotinoids also called neonicotinyls, chloronicotines and chloronicotinyls or new chemistry insecticides having a distinct mode of action. Insecticides present a very significant group of chemical compounds which must be restricted due to their elevated toxicity and extensive use in field as well as for post-harvest protection. In fact, above 1000 compounds might be applied to agricultural crops to control unwanted weeds, insects or molds.

On average, the MRLs are in the range of 0.02-3 mg Kg-1 depending on the composition of mixture or the commodity combination and on the concentration of insecticide [6]. Acetamiprid is a wide-ranging insecticide efficient against several insect groups, including thysanopterans, hemipeterans, coleopterans on fruit trees and vegetables. In addition to stomach and contact, acetamiprid also has systemic and osmotic activity [7]. Imidacloprid accounts for about 11-15% of the total existing market. In 1991, imidacloprid was launched and the products having this insecticide have been registered in 120 countries and it is marketed for about 140 agricultural crops.

From mid 1990s, neonicotinoids are commonly used in Pakistan to manage and control sucking insect-pest on vegetables, fruits, ornamental flora and cotton. Various studies conducted in Pakistan on insecticide residues showed that most of the samples were contaminated by neonicotinoid [8], pyrethroid [9], [10] and chlorinated pesticide [11] a quantity of them were over and above the MRLs, which could be a threat for consumers. The household vegetable processing techniques like washing, peeling and cooking can significantly reduce the pesticide residues below MRL values [12], [13].

For ensuring that insecticide residues are not exceeding their MRL, dietary intake assessment of vegetables is an integral part. Comparison between the acceptable daily intake (ADI) and MPI values of insecticide residues, is a tool to evaluate the present scenario. Dietary intake assessment is required to assure the safe consumption limits of vegetables with respect to exposure of insecticide residues. Such studies are feasible to predict the safety level of insecticide residues [14]. The objective of this research was to determine residual levels of imidacloprid and acetamiprid residues in okra and eggplant with respect to their dietary intake assessment in the proposed studied areas (Multan, Faisalabad and Gujranwala) of Punjab, Pakistan.

Experimental

Sample procurement

Different growers of okra and eggplant were selected before collection of samples. Farmers data including their total area of vegetable production, insecticide dosages and number of irrigation was maintained accordingly. Samples of eggplant (Solanum melongena L.) and okra (Abelmoschus esculentus L.) were collected from peri-urban farming system of Faisalabad, Gujranwala and Multan. Thirty growers of these vegetables were selected in each district. One kg sample of each vegetable was collected from each grower keeping in mind the maturity stage of these vegetables. In this way total 30 samples of each vegetable from each district were collected (total samples n = 180). Samples were immediately brought to Pesticide Residue and Food Safety Laboratory of National Institute of Food Science and Technology, in University of Agriculture, Faisalabad with minimum delay after harvesting and extracted for targeted insecticide residues as explained below.

Extraction, clean up and concentration

One kg of the representative samples was chopped and blended by National household blender. In this way, homogenous slurry was obtained by blending. After blending the homogenous samples were stored at -40AdegC. For extraction, 50 g of the sample was taken out in quick fit bottles from slurry, 20 g of anhydrous sodium sulfate and 10 mL saturated solution of sodium chloride was added to the flask and mixed thoroughly. Ethyl acetate 70-80 ml was added in homogenous slurry of samples. Shaking of the samples was done by using mechanical shaker at a speed of 240 rpm for the time of 1 hr. The supernatant obtained after extraction of samples was filtered by filter-paper. The sample residues were rinsed by adding 50 ml of ethyl acetate twice and filtered. The clean liquid after filtration was collected separately and stored at -40AdegC. For clean-up, the filtered extract was passed through the column containing activated charcoal, silica gel and layers of anhydrous sodium sulfate.

Ring of glass wool was placed in the neck of the column. The extracted samples were passed through the clean-up column and rinsed by 15-20 ml of ethyl acetate. The filtrate was taken to the rotary evaporator for the evaporation of samples by using 45-50AdegC water bath. After evaporation from rotary evaporator 1-2 ml of the sample was taken in glass vials and further dried up by using gentle stream of nitrogen gas till a concentrated spot of sample was left.

The recovery rates for imidacloprid and acetamiprid were determined by fortification of known amount of active ingredients in concentration range of 0.1-1.0 mg kg-1 taken from the mix insecticide solution (0.5 mL L-1). The fortified samples were passed through the entire process of extraction then cleaned up and analyzed for recovery tests of each insecticides. The recovery values were calculated as well as the results obtained from the residue determinations; were corrected according to recovery percentages. The percent mean recovery values (triplicate) with relative standard deviation were ranged from 83.7+-2.43 to 91.6+-4.39; and 88.7+-3.14 to 96.2+-3.62 for imidacloprid and acetamiprid, respectively.

Determination of imidacloprid and acetamiprid residues

The analytical technique employed for the estimation of insecticides in vegetables is important for evaluation and interpretation of the data. It is necessary to authenticate the analytical methods for the reliability of data. Determination of imidacloprid and acetamiprid residues was done by using HPLC technique equipped with UV/VIS detector (Perkin Elmer series 200) as described by [15]. For preparing mobile phase, methanol and water were mixed together in ratio of (45: 55). After mixing the methanol and water, the mobile phase was subjected to fume hood for filtration by using specially designed filtration assembly having pore size of 0.2 um. The mobile phase was subjected to the sonicator to remove the air bubbles. Injection volume of 20 uL was used with C18 column (25 cm x 4.6 mm i.d.) bonded silica based reverse phase having particle size of 5um.

The flow rate was set to 1 ml min-1, with column oven temperature maintained at 30AdegC, pressure 170 kg fcm-2 and detector wavelength was set at 254 nm with sample run time of 20 min. Retention times were observed with respect to the peak values for both insecticides (Fig. 1). The limit of detection (LOD) and limit of quantification (LOQ) for imidacloprid in the present study were obtained in the range of 0.003 to 0.01 mg kg-1 and 0.013 to 0.06 mg kg-1, respectively. For acetamiprid the LOD and LOQ were found to be 0.01 and 0.002 mg kg-1 respectively.

Dietary Intake Assessment

Dietary intake assessment was calculated with some modifications by following the method adopted by Randhawa [8]. The dietary intake assessment of imidacloprid and acetamiprid residues estimated by computing the MPI of imidacloprid and acetamiprid. The recommended ADI of imidacloprid is 0.06 mg kg-1 body weight day-1 and for acetamiprid 0.07 mg kg-1 body weight day-1 in humans, whereas an average weight of a Pakistani adult is 64 kg. By multiplying the average weight of a Pakistani adult with ADI, the MPI values found for imidacloprid and acetamiprid were (3.84 mg person-1day-1) and (4.48 mg person-1day-1), respectively. Afterwards, the dietary exposure was determined by multiplying average per capita daily consumption of eggplant (58 g) and okra (57.77 g) with their respective imidacloprid and acetamiprid residue levels [16]. The samples were analyzed on HPLC for quantification of insecticide residues. The results obtained are summarized as below:

Results and Discussion

Residual values of imidacloprid in eggplant and okra collected from peri-urban areas of Punjab

The residual value of imidacloprid in eggplant and okra are given in Table-1. The recommended maximum residue limit for imidacloprid in okra and eggplant by European Commission is 0.5 mg kg-1. Mean values for residues of imidacloprid found in okra from Multan (0.176 mg kg-1) region were slightly greater than the residues of imidacloprid collected from both Gujranwala (0.140 mg kg-1) and Faisalabad (0.138 mg kg-1). There were total 30 samples of each vegetable (okra, eggplant) collected from each of three regions. The positive samples found in okra collected from Multan were 17 (56%) with 2 (6%) samples above MRLs. The contamination rate found in Gujranwala and Faisalabad region was 18 (60%) and 14 (46%) samples, and the samples exceeding MRLs were 1(3%) and 4 (13%), respectively.

As vegetables are short interval crops hence, to resist the prevailing subtropical conditions as well as against the insect attacks; frequent use of insecticides during the whole period of plant growth is common in these regions. Imidacloprid is not stable class of insecticides due to their volatile nature and cause less toxicity as compared to carbamate and organophosphate insecticides. The samples exceeding MRLs values showed that farmers are not adopting proper withholding period for harvesting of vegetables after spray. Mansoor [17], had reported residues of imidacloprid (0.030-0.015 ppm), acephate (0.067-0.040 ppm) and chlorpyriphos (0.075-0.039 ppm), respectively in eggplant. The residues were investigated after the spraying period of few hours to 3 and 7 days. The quantification of residues was negligible after 7 days and it was suggested that consumption of eggplant after 2-3 days of spraying is safe for human beings.

Results revealed that eggplant samples collected from Multan showed 24 positive samples (80%) 5 samples exceeding the MRLs (16%). Mean values of imidacloprid residues found in eggplant from Multan region (0.226 mg kg-1) were greater than the residues reported from Gujranwala (0.146 mg kg-1) and Faisalabad (0.216 mg kg-1). Similarly, 30 samples of eggplant were collected from Gujranwala showing total 12 positive samples (40%) with 3 samples exceeding the MRLs (10%). While the contamination rate of eggplant found in Faisalabad region showed 19 positive samples (63%) with 3 samples exceeding MRLs (10%). However, imidacloprid residues in eggplant fruit collected from Faisalabad were greater than mean residues estimated from Gujranwala but lower than Multan (Table-1).

Table-1: Imidacloprid residues in eggplant and okra grown in peri-urban environment.

###Range###Mean +- SD

###Location/cities###Vegetables###Samples Exceeded MRLs (%)

###(mg kg-1)###(mg kg-1)###Positive samples

###Eggplant###0.003-0.561###0.226a +- 0.147###24###5

###Multan

###Okra###0.006-0.604###0.176d +- 0.080###17###2

###Eggplant###0.001-0.650###0.216b +- 0.200###19###3

###Faisalabad

###Okra###0.001-0.721###0.138f +- 0.058###14###4

###Eggplant###0.002-0.522###0.146e +- 0.112###12###3

###Gujranwala

###Okra###0.001-0.521###0.200c +- 0.140###18###1

Table-2: Acetamiprid residues in eggplant and okra grown in peri-urban environment.

###Vegetables###Range###Means +- SD###Positive samples

###Location/cities###Samples Exceeded MRLs (%)

###(n=30)###(mg kg-1)###(mg kg-1)###(%)

###b

###Eggplant###0.002-0.150###0.032 +- 0.024###16###3

###Multan

###Okra###0.003-0.038###0.011d +- 0.009###19###5

###Eggplant###0.001-0.125###0.020c +- 0.014###15###4

###Faisalabad

###Okra###0.001-0.014###0.005f +- 0.004###23###5

###Eggplant###0.003-0.310###0.059a +- 0.024###24###6

###Gujranwala

###Okra###0.001-0.017###0.008e +- 0.005###20###4

Similar results of imidacloprid in eggplant collected from Noshera were reported by Iqbal [18]. Different insecticides like, chlorpyriphos (0.060 mg kg-1), carbofuran (0.034 mg kg-1) and imidacloprid (0.028 mg kg-1) were found in year of 2006. Recently, Latif [19], also assessed six different pesticide (carbofuran, chlorpyrifos, [alpha]-endosulfan, [beta]-endosulfan, malathion, fenvalerate) residues in Hyderabad region of Pakistan from different samples of eight vegetables i.e. cauliflower, eggplant, green chili, peas, tomato, bitter gourd, apple gourd and spinach. It concluded that 39% samples out of total 200, contained pesticide residues below the MRLs and 61% vegetable samples contained the residues of pesticide above the MRLs. This shows the excessive use of insecticides which can be attributed to the illiteracy of farmers about the judicious and safe use of insecticides. Furthermore, the farmers also tend to over-dose the insecticides to secure the high value crop.

Residual values of acetamiprid in eggplant and okra collected from peri-urban areas of Punjab

The residual value of acetamiprid in eggplant and okra are given in Table-2. The recommended MRLs for acetamiprid in okra and eggplant established by European Union is 0.01 mg kg-1 and 0.1 mg kg-1, respectively. For the assessment of acetamiprid residues, 30 samples of each vegetable (okra, eggplant) were collected from peri-urban farming system of each district. The observed data for eggplant revealed that there were 16 positive samples (53%) with 3 samples exceeding MRLs (10%) in Multan, 24 positive samples (80%) with 6 samples exceeding MRLs (20%) in Gujranwala and 15 contaminated samples (50%) with 4 samples exceeding MRLs (13%) in Faisalabad. Whereas the data calculated for acetamiprid residues in okra showed 19 positive samples (64%) with 5 samples above MRLs (16%) in Multan, 20 positive samples (67%) with 4 samples above MRLs (13%) in Gujranwala and 23 positive samples (76%) with 5 samples above MRLs (16%) in Faisalabad, respectively.

Significant difference was investigated between the residues of acetamiprid in okra in all three districts (Table-2). The data indicated an excessive application of acetamiprid in okra in peri urban areas of Multan as compared to Faisalabad and Gujranwala. However, the acetamiprid residues in eggplant fruit collected from Gujranwala (0.059 mg kg-1) were greater than the residues reported from Multan (0.032 mg kg-1) and Faisalabad (0.02 mg kg-1). The acetamiprid residues in okra collected from Multan (0.011 mg kg-1) were greater than the mean residues reported from Gujranwala (0.008 mg kg-1) and Faisalabad (0.005 mg kg-1).

The results depicted that acetamiprid is excessively used against several insect groups, including thysanopterans, hemipterans, coleopterans on fruit trees and vegetables. As Multan is a cotton belt area and the attack of insects like white fly, jassids and thrips are much more in this region as compared to other areas under study. Therefore, residues of acetamiprid were higher in Multan region. Acetamiprid residues in okra fruit were also analyzed by Singh and Kulshrestha [20], after 5 and 7 days of its application. Initially deposits of thiamethoxam (0.335 ug g-1) and acetamiprid (0.475 ug g-1) were found with gradual dissipation in both residues. More than 50 % dissipation was observed at the end of 3rd day of application. About 80 % dissipation was observed by the 5th day and finally by the end of 7th day of thiamethoxam and acetamiprid application, the residues were almost completely dissipated.

The results obtained from this study indicated that withholding or pre-harvest interval time of fresh produce before transferring into market; is an important factor in lowering the concentration of residues in vegetables. It also indicated that the effect of insecticides dissipates with the passage of time. On the other hand, excessive and uncontrolled application of insecticides is an alarming situation which needs immediate attention of concerned authorities.

Dietary intake assessment of imidacloprid and acetamiprid residues with their safety status in eggplant and okra

The results of imidacloprid and acetamiprid residues with respect to their dietary intake assessment have been elaborated in Fig. 2 and 3, respectively. The MPI calculated for Pakistani adult (average weight of 64 Kg) for imidacloprid and acetamiprid was 3.84 mg person-1day-1 and 4.48 mg person-1day-1, respectively. In Pakistan, per capita eggplant and okra consumption is 58 and 57.77g. The dietary intake assessment based on per capita consumption of eggplant and okra revealed the maximum quantity of imidacloprid residues taken by a person in Multan by consuming eggplant (0.035 mg person-1day-1) and okra (0.032 mg person-1day-1). Similarly, in Faisalabad the dietary intake of imidacloprid in eggplant was (0.038 mg person-1day-1) and okra (0.0302 mg person-1day-1), respectively. On the other hand, in Gujranwala the dietary intake was (0.030 mg person-1day-1) for both eggplant and okra.

The dietary intake assessment of imidacloprid (0.127 mg person-1day-1) was relatively high as compared to dietary intake assessment of acetamiprid (0.002 mg person-1day-1) in Multan region. Whereas acetamiprid was found to be high in Multan for eggplant and okra as 0.009 and 0.002 mg person-1day-1 as compared to Faisalabad region (eggplant 0.0011, okra 0.0003 mg person-1day-1) and Gujranwala region (eggplant 0.003 and okra 0.003 mg person-1day-1) as illustrated in Fig. 3.

In Pakistan, per capita daily vegetable consumption has been reported to be 134 g. Considering the okra and eggplant as sole contributor in the vegetables intake, we must determine the safety status of okra and eggplant in such a case. Therefore, dietary intake assessment was also calculated based on 134 g vegetable intake per person per day (Figs 2-3). The maximum dietary intakes found for imidacloprid residues in Multan region were, eggplant 0.0302 and okra 0.0235 mg person-1day-1, respectively.

By comparing the dietary intake assessment of the individual vegetables (okra = 57.77 g, eggplant = 58 g) with the overall vegetable consumption, it was concluded that there was no health risk even if we consider the per capita consumption of okra and eggplant average per capita vegetable consumption i.e. 134 g. Although some of the samples in the present study exceeded their MRLs values but their dietary intake results showed that they were far below than MPI limits. Hence, after calculating the dietary assessment of the current vegetable samples it can be suggested that okra and eggplant can be consumed without any hesitation. There was very low risk factor in consumption of these vegetables even when the residues of imidacloprid and acetamiprid were exceeding the MRLs.

These results are correlated with the findings of Randhawa [13], who reported the endosulfan residues along with their dietary intakes in fresh and processed vegetables including okra, eggplant, spinach, potato and tomato. Maximum residues were found in fresh vegetables as compared to the processed form, (eggplant 1.50 mg kg-1), (spinach 1.16 mg kg-1), (okra 1.71 mg kg-1) and potato (0.130 mg kg-1). Okra in fresh stage exceeded the dietary intake values due to highest residues of endosulfan as compared to others. However, dietary intake assessment results revealed that these were well below the MPI values and falling under safe limits. The results are also correlated with findings of Dikshit [14], who determined the imidacloprid and beta-cyfluthrin residues in tomato. It was suggested that controlled application of insecticides could be helpful in reducing the dietary intake of insecticides.

Conclusion

It is concluded that benefits of using insecticides in farming to produce healthier crops must be considered while keeping in mind the probable health hazards due to poisonous insecticide residues in food. Although higher residue of imidacloprid and acetamiprid residues were detected in some samples of okra and eggplant collected from three regions but their dietary intake assessment levels were found within the safe limits. Good Agricultural Practices (GAPs) following the controlled and adequate application of insecticides must be adopted.

Acknowledgement

The authors acknowledge Punjab Agriculture Research Board (PARB) for providing the opportunity to conduct this project.

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