Risk Mitigation Methods for Removal of Pesticide Residues in Brinjal for Food Safety

The commercial production of highly cultivated and consumed brinjal is highly dependent on regular usage of insecticides to protect the crop from insect pests. The increased consumer awareness and legal issues on food safety, with special reference to insecticide residues in foods, led us to attempt for cheap and effective methods for removal of pesticide residues to address the issues of consumer and food safety, as the farmers are not following the Good Agricultural Practices i.e pre-harvest intervals. The most commonly used pesticides such as profenophos, chlorpyriphos, dimethoate, malathion, phosalone, quinalphos, triazophos and -cyhalothrin were sprayed at recommended doses at brinjal formation stage, samples were collected at 2 hours after treatment to quantify the deposits. The samples were subjected to various household treatments (tap water wash, lemon water wash, dipping in 2% salt water for 15 min, dipping in 2% tamarind water for 10 min, washing with 0.1% sodium bicarbonate solution, washing with 4% acetic acid solution, biowash, cooking), each in three replications, and analysed for residues using validated quick, easy cheap rugged and simple method (Quechers method and GC-ECD, ( GAS Chromatography Electron capture detector) FPD( Flame photometric detector) and GC-MS( Gas Chromatography Mass spectrometry) so as to estimate the % removal and their effectiveness. Out of all treatments, dipping in 2% salt solution for 10 minutes is very effective in removing 45%, 43%, 52%, 50%, 54%, 48% and 76% of dimethoate, chlorpyriphos, quinalphos, profenophos, phosalone, -cyhalothrin and malathion, respectively, and cooking removed insecticides in the range 55-80%. Dipping fruits and vegetables in 2% salt solution for 15 minutes is the best household method for removal of pesticide residues, and also the method is effective in reducing the residues below MRL (Maximum Residue Limits).


Introduction
Brinjal is the most popular vegetable in India, and state of Andhra Pradesh is third most important growing Brinjal producing 1.615 M mt with a share of 12% (NHB, 2013) during 2012-13. In India, about 13-14% of the total pesticides used in agriculture are used for fruits and vegetables covering only 3% of the cropped area. Repeated application of pesticides on vegetables often results in the buildup of their residues. Surveys carried out in the country indicated that 50-70% of vegetables are contaminated with insecticide residues).Studies on farm gate monitoring of vegetables carried out in different places revealed contamination mostly with organophosphorous and synthetic pyrethroids insecticides, indicating clearly the changes in the usage pattern from organochlorine to other groups of pesticides.
Maximum Residue Limits (MRLs) are set by Codex Alimentarius Commission (CAC) at international level and as on date, MRLs are set for 17 pesticides on brinjal, and by Food Safety and Standards Authority of India (FSSAI) of Ministry of Health and Family Welfare, Government of India, as per Food Safety and Standards Act, 2006 (Food Safety and Standards Regulation, 2011) at national level based on the Good Agricultural Practices. The major insect pest of brinjal is fruit and shoot borer ( Leucinoders orbonalis)for which farmers apply insecticides at almost weekly interval, and hence the risk of pesticide residues in foods need to be addressed as per FSSAI (Food Safety and Standards Authority of India) for the protection of consumer health and interests. In this context, household risk mitigation methods for removal of pesticide residues in brinjal are to be recommended based on the scientific evaluation, as the food habits are changing enormously.

Field Trial Protocol
A supervised field trial was conducted during Rabi 280 Risk Mitigation Methods for Removal of Pesticide Residues in Brinjal for Food Safety 2012-13 in order to study the effect of house hold processing methods in the removal of certain pesticides in brinjal resulting from spray application of most commonly used insecticides viz., Profenophos 50EC@ 2ml/lit,Chlorpyrifos 20EC@ 2ml/lit, Dimethoate 30EC @ 4ml/lit, Malathion 50EC@3ml/lit, Phosalone 35EC@3ml/lit, Quinalphos 25EC@ 2ml/lit, Triazophos 40EC @ 2.5ml/lit, Lamda cyhalothrin 5EC@ 0.6ml/lit. Single spray was given at fruiting stage and brinjal fruit samples were collected after 2 hours and brought to the laboratory for further analysis. The field trail was conducted in randomized block design, and all the treatments were replicated thrice .

Residue Analysis Method Validation
Prior to sample collection, AOAC official method 2007.01 (QuEChERS quick,easy cheap rugged and simple method) for residue analysis of dimethoate, profenophos, chlorpyrifos, malathion, phosalone, quinalphos, triazophos, -cyhalothrin was validated by fortifying control samples at 0.50 mg/kg level, and the results indicated that the method was good as the recovery per cent was 97, 119, 96, 103, 114, 93, 99 and 97, respectively, and hence the method is used for analysis. The details of the method are as follows


Brinjal samples were homogenized with robot coupe blixer (high volume homogenizer). 15±0.1g sample was taken in 50ml centrifuge tube, and 30±0.1 ml acetonitrile was added.  The sample was homogenized (low volume homogenizer) at 14000-15000 rpm for 2-3 min using Heidolph silent crusher, then added with 3±0.1g sodium chloride, mixed by shaking gently followed by centrifugation for 3 min at 2500-3000 rpm to separate the organic layer.


The top organic layer of about 16 ml was taken into the 50 ml centrifuge tube and added with 9±0.1g anhydrous sodium sulphate to remove the moisture content.  8 ml of extract was taken in to 15 ml tube, containing 0.4±0.01g PSA sorbent (for dispersive solid phase d-SPE cleanup) and 1.2±0.01gr anhydrous magnesium sulphate. The sample tube was vortexed for 30sec then followed by centrifugation for 5min at 2500-3000rpm.


The extract of about 2ml was transferred into test tubes and evaporated to dryness using turbovap with nitrogen gas and reconstituted with 1ml n-Hexane for GC (GAS Chromatography) and GC-MS( Gas Chromatography Mass spectrometry) analysis with ECD( Electron capture detector) FPD( Flame photometric detector). The GC column end at detector was fitted with Universal "Y" splitter for simultaneous analysis of insecticides on both detectors for confirmatory analysis. All pesticides could be detected and quantified on both ECD and FPD, except for triazophos and -cyhalothrin which could be detected only on ECD and FPD, respectively. The samples were also analysed on GC-MS/MS ( Gas chromatography Mass spectrometry-Mass spectrometry)(triple quadrupole) for confirmatory analysis.

Decontamination Methods
After spray of pesticide, about 15 kgs of brinjal were collected randomly in polythene bags from each plot to avoid cross contamination. Each lot from treatment plot was divided in to 8 sub-lots, where one lot was analysed for initial deposits, and remaining lots were subjected to various rick mitigation methods prior to analysis. All samples were replicated thrice. The decontamination methods used in the study are presented in Table 1.

T3
Dipping in 2% tamarind Solution for 10 min: 80 grams of tamarind is added to 4 lts of water, and 1 kg brinjal sample dipped in salt water for 10 min.

T4
Dipping in Lemon water (1Lemon/1lit) for 10min: Juice of 4 lemons is added to 4 lts of water, and 1 kg brinjal samples is dipped in lemon water for 10 min.

T5
Dipping in 0.1% Sodium Bicarbonate solution for 10min: 4 grams of sodium bicarbonate is added to 4 lts of water; 1 kg brinjal sample is dipped in solution for 10 min.

T6
Dipping in 4% Acetic acid solution for 1min: 160 ml of acetic acid is added to 4 lts of water; 1 kg brinjal samples dipped in the solution for 10 min.

T8
Cooking in Pressure cooker: 1 kg brinjal sample is cooked in pressure cooker for 5min.

T9
Washing with Bio wash keep it for 10min: 8 ml of commercial formula Biowash is added to 4 lts of water and 1 kg brinjal samples is dipped in solution for 10 min.

Results of fortification and recovery studies in Brinjal
The control / Brinjal samples were fortified at 0.50 mg/kg levels adding required quantity of Profenophos, Chlorpyrifos, Dimethoate, Malathion, Phosalone, Quinalphos, Triazophos, Lamda cyhalothrin standards and replicated thrice.The following are the recoveries of Profenophos, Chlorpyrifos, Dimethoate, Malathion, Phosalone, Quinalphos, Triazophos, Lamda cyhalothrin at three different fortification levels. The recovery of Triazophos was 99% from the Brinjal samples fortified at 0.50 mg/kg. and the recovery of Lambda cyhalothrin was 97%

Results and Discussion
The residues of dimethoate, profenophos, chlorpyrifos, malathion, phosalone, quinalphos, triazophos -cyhalothrin in brinjal samples have got substantial reduction by different house hold processing methods. The reduction percentage and residue levels have been presented in Table 3.

Results
In the process of washing under running tap water malathion residues were reduced up to 70.30%, whereas phosalone 44.10%, Quinolphos 45.60%, ^cyhalothrin 40.90% , Profenophos 42%., Chlorpyriphos 35.30%, and dimethoate were reduced to 30.70% .By washing the brinjal samples under running tap water the residue levels of dimethoate were not degraded much. With the method of direct cooking malathion residues were reduced up to 81.40%, dimethoate 64%, Profenophos 52%.^cyhalothrin 48.70%, and least reduction was seen in quinolphos 39.40%.
The direct cooking method has shown better effect when compared with Tap water washing. By washing with 2% s alt water malathion residues reduced by 76.50%, Dimethoate 45.30%, phosalone 54%, quinalphos 52.10%, and the lowest reduction was seen in chlorpyriphos residues with a reduction of 43%.
Among all the treatments dipping in 2% tamarind solution, washing with 0.1% sodium bicarbonate solution ,4% acetic acid solution, washing with formula -I were less effective in reducing the pesticide residues compared to washing with tap water, lemon water, washing methods utilized. Among all the methods utilized Direct cooking and washing with 2% salt solution were most effective.

Discussion
Pesticides are used indiscriminately and excessively throughout the globe, and these residues remain in the food materials, water, fruits, vegetables (Baptista et al.,2008, Lazic et al., 2009) and in total diet. Excessive use of pesticides, their toxic residues has been reported in various environmental commodities (Lazic et al.,2009). These pesticide residues enter in to the human body by consumption of the pesticide contaminated food which leads to the chronic disorders. Thus the removal of these residues from food commodities utilizing different processing methods is very essential)The different house hold preparations such as washing with tap water, washing with lemon water, dipping in 2% tamarind solution, cooking, dipping in 2% salt solution. washing with 4% acetic acid solution, biowash and washing with formula-I play a role in the reduction of pesticide residues (Wasim Aktar et al.,2010).
Thus, based on the results obtained in this study it can be concluded that by processing the brinjal with the traditional processing methods if it helps in the removal of pesticide residues below MRL levels, then it is safe for human consumption. The results of earlier workers ( Dhiman et al., 2006, Kumari.2008