1. Introduction

Murraya koenigii L. (curry leaf) belonging to family Rutaceae is a leafy spice characterizing authentic Asian-Indian cuisine and it is used in small quantities for its distinct aroma as well as for preservation purposes., Curry leaf oil an volatile oil produced from the plant has uses in the soap industry (Salikutty and Peter, 2008). Recent studies have shown that carbazole alkaloids have several biological activities such as anti carcinogenic effects in dimethyl hydrazine (DMH) treated rats (Khanum et al., 2000), anti platelet activity and vaso relaxing effects (Wu et al., 1998). Chevalier (1996) also reported that curry leaf has medicinal value as traditionally used in Eastern Asia. Interest in greater use of curry leaf has been stimulated since its high antioxidant potency was reported and this antioxidant activity is attributed due to maha nimbine, murrayanol and mahanine from M. koenigii (Tachibana et al., 2003; Ningappa et al., 2008). Chowdhury et al. (2001) reported that these alkaloids have antimicrobial activity against gram positive and negative bacteria, and fungi. Lee et al. (2002) noted that enrichment of phenolic compounds within the plant extract is correlated with their enhanced antioxidant activity. It is reported to have antioxidant, anti-diabetic, anti carcinogenic, anti dysenteric stimulant, hypo glycaemic and anti microbial activities (Khanum et al., 2000). Biologically active carbazole alkaloids are reported to have anti microbial properties (Ramsewak et al., 1999). Curry leaves have been reported to contain tocopherol, b-carotene, lutein and alkaloids (Khanum et al., 2000). But it is observed that curry leaves have received red alert message from the European Union, the major importers, as the pesticide residue limits were found much beyond the permissible levels.

Maximum Residue Limits (MRLs) are set by Codex Alimentarius Commission (CAC) at international level and as on date, no MRLs are set for any pesticides on curryleaf, 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 at national level based on the Good Agricultural Practices. To control the major insect pest of curry leaf 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 curry leaf are to be recommended based on the scientific evaluation, as the food habits are changing enormously.

2. Materials and Methods

Field trial protocol

For the Decontamination of pesticide residues of commonly detected pesticides a field experiment was conducted utilizing Chlorpyriphos 20% EC @ 300 g a i/ha (1500 ml/ha), cypermethrin 10% EC @ 50 g a i/ha (550 ml/ha), ethion 50% EC @ 500 g a i/ha (1250 ml/ha), profenphos 50% EC @ 500 g a i/ha (1250 ml/ha), Carbendazim 50 WP and triazophos 40% EC @ 500 g ai/ha 1250 ml/ha twice, first at Vegetative stage and second spray after 10 days. Zero ‘0’day samples were collected for estimation of deposits of pesticide within 2 hours of last spray.

Evaluation of decontamination methods for removal of pesticide residues:

The zero day samples which are free from pests and damage were collected from various treatments separately in large quantities and made into thirteen sets, each in four replications. One set of the sample from each treatment (in four replications) was analyzed for deposits of the pesticide. The remaining sets of samples of zero day from each treatment samples were subjected to various decontamination methods separately and the residues were calculated to know the efficiency of the various decontamination methods in the removal of pesticide residues from the curry leaf samples. The decontamination methods used in the study / risk mitigation methods are given (Table 1).

Table 1. Decontamination methods used in the study or risk mitigation methods

Per cent removal of pesticide:

Sample extraction procedure

Curry leaf samples were collected from the fields sprayed with insecticides and brought to the laboratory and analyzed for pesticide residues following the AOAC official method 2007.01 (QuEChERS) after validation of the method in the laboratory. Each sample was homogenized separately with robot coupe blixer and homogenized 15 ± 0.1 g sample was taken in 50 ml centrifuge tube and 30±0.1 ml acetonitrile was added to sample tube. The sample was homogenized at 14000-15000 rpm for 2-3 min using Heidolph silent crusher. 3±0.1 g sodium chloride was added to sample, mixed thor oughly 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±0.1 g anhydrous sodium sulphate to remove the moisture content. 8 ml of extract was taken in to 15 ml tube, containing 0.4±0.01 g PSA sorbent (for dispersive solid phase d-SPE cleanup), 1.2 ±0.01 g anhydrous magnesium sulphate and 0.05 g of GCB (graphatised carbon black).The sample tube was vortexed for 30 sec then followed by centrifugation for 5 min at 2500-3000rpm. The extract of about 1 ml (0.5 g sample) was taken for analysis on LCMS/MS under standard operational conditions (Table 2).

Table 2. LCMS/MS standard operating parameters

Results of fortification and recovery studies in curryleaf:

The control curryleaf samples were fortified at 0.50 mg/kg, 0.25 and 0.05 mg/kg levels adding required quantity of Profenofos, Triazophos, Ethion, Chlorpyriphos, Carbendazim and Cypermethrin standards and replicated thrice. The following are the recoveries of Profenofos, Triazophos, Ethion, Chlorpyriphos, carbendazim and Cypermethrin at three different fortification levels (Table-3).

Table 3. Recoveries of Profenofos, carbendazim, Triazophos, Chlorpyriphos, Cypermethrin and Ethion at various fortification levels in curryleaf samples

3. Results and Discussion

The residues of, Triazophos, Chlorpyriphos, Ethion, Carbendazim, Cypermethrin and Profenofos in curryleaf samples have got substantial reduction by different house hold processing methods. The reduction percentage and residue levels are presented in Table 4 and depicted in Fig 1.

Table 4. Effectiveness of various decontamination methods

Figure 1. Per cent removal of pesticide residues from curry leaf by various decontamination methods

Triazophos

Triazophos is a non-systemic, broad spectrum, organophosphorus insecticide with contact and stomach action, acaricide with some nematicidal properties. Frying curry leaf for 10 min was found to be most effective with 27.53 per cent decontamination when compared to other treatments. The percentage removal of triazophos residues due to various decontamination treatments in descending order are tamarind water (27.20%) > biowash (25.43%) > butter milk (24.44%) > formula 1 (24.40%) > tap water (18.43%) > acetic acid (17.74%) > drying (17.33%) > lemon water (14.06%) > salt solution (13.84%) > sodium bicarbonate (12.66%) > cooking (9.07%). Based on the percentage removal of residues, it is statistically proved that there is significant difference in the efficiency of decontaminating methods in removing triazophos residues. Percentage removal of triazophos residues from various decontamination methods is depicted in Fig 2.

Figure 2. Per cent removal of triazophos residues from curry leaf by various decontamination methods

Chlorpyriphos

Various decontamination methods were evaluated in order to know their efficiency in removing chlorpyriphos residues from curry leaf. Results revealed that all the treatments significantly differed among each other in their efficiency in removing chlorpyriphos residues. Dipping in formula 1 for 10 min was found to be most effective with 58.76 per cent decontamination when compared to other treatments. Next promising treatment was dipping in Butter milk (46.55%). Other treatments like tamarind water (44.28%), tap water (43.37%), cooking (40.27%), biowash (39.13%), sodium bicarbonate (35.28%), frying (32.09%), lemon water (27.81%), acetic acid (22.26%), salt solution (6.97%) and drying (2.23%) were also found to remove significant amount of residues from curry leaf. Percentage removal of chlorpyriphos residues from various decontamination methods is depicted in Fig 3.

Figure 3. Per cent removal of chlorpyriphos residues from curry leaf by various decontamination methods

Ethion

Curry leaf sprayed with recommended dose of ethion were collected two hours after spraying and subjected to various decontamination methods. Among the different treatments employed, dipping leaves in Formula 1 solution for 10 min was found to be more effective with 19.44 per cent decontamination than other treatments. Biowash and sodium bicarbonate were found to be next promising treatments with 17.54 per cent decontamination followed by salt solution (15.15%), tamarind water (14.39%), cooking (12.99%), butter milk (12.03%), acetic acid (10.45%), drying (6.67%), tap water (4.14%), frying (2.84%) and lemon water (2.19%).Percentage removal of ethion residues from various decontamination methods is depicted in Fig 4.

Figure 4. Per cent removal of ethion residues from curry leaf by various decontamination methods

Carbendazim

Carbendazim is a systemic fungicide with protective and curative action. Cooking of curry leaf for 10 min in pressure cooker was found to be most effective treatment with 69.11 per cent reduction in residues of carbendazim than other treatments. The percentage removal of carbendazim residues due to various decontamination treatments in descending order are tamarind water (69.05%) > biowash (59.26%) > butter milk (54.51%) > tap water (52.77%) > formula 1 (52.11%) > lemon water (49.68%) > acetic acid (41.27%) > salt solution (40.88%) > sodium bicarbonate (37.25%) > frying (18.19%) > drying (0.90%). Based on the percentage removal of residues, it was statistically proved that there is significant difference in the efficiency of decontaminating solutions in removing carbendazim residues. Percentage removal of carbendazim residues from various decontamination methods is depicted in Fig 5.

Figure 5. Per cent removal of carbendazim residues from curry leaf by various decontamination methods

Cypermethrin

Cypermethrin residues in curry leaf were removed significantly when subjected to different decontamination solutions after two hours of spraying. Results revealed that dipping in tamarind water for 10 min was found to be most effective than other treatments. In this treatment residues were reduced up to 61.35 per cent. Next promising treatment was formula 1 (59.33%) followed by tap water (58.36%), bio wash (55.22%), butter milk (54.45%), lemon water (52.19%), acetic acid (44.53%), salt solution (37.93%), sodium bicarbonate (19.21%), frying (16.79%), cooking (16.67%) and drying (2.07%).Percentage removal of cypermethrin residues from various decontamination methods is depicted in Fig 6.

Figure 6. Per cent removal of cypermethrin residues from curry leaf by various decontamination methods

Profenophos

The percentage removal of profenophos residues in curry leaf when subjected to different decontamination solutions after two hours of spraying showed that dipping in butter milk for ten min was found to be most effective removing 64.53 per cent residues, than other treatments. The next promising treatment was washing with tamarind water (63.62%), followed by bio wash (59.34%), tap water (57.64%), acetic acid (50.08%), formula 1 (47.74%), lemon water (45.36%), drying (43.46%), salt solution (42.98%), sodium bicarbonate (17.63%), cooking (15.85%) and frying (0.84%). Fig -7. Based on the percentage removal of residues, it was statistically proved that there is significant difference in the efficiency of decontaminating methods in removing residues of above mentioned pesticides. Per cent removal of pesticide residues from curry leaf by various decontamination methods is depicted in fig 1.

Figure 7. Per cent removal of profenophos residues from curry leaf by various decontamination methods

Scientists and food processors have long been interested in the effect of processing on pesticide residues in food commodities. The extent to which pesticide residues are removed by processing depends on a variety of factors, such as chemical properties of the pesticides, the nature of food commodity, the processing step and the length of time the compound has been in contact with the food (Farris et al., 1992., Holland et al., 1994 and Kumar et al., 2010). In a developing country like India, dissipation techniques at the household level can serve as an effective tool in reducing risk related to dietary exposure to residues and henceforth controlling pesticide related adversities. Washing is the most common form of processing which is a preliminary step in both household and commercial preparation. Loosely held residues of several pesticides are removed with reasonable efficiency by varied types of washing processes (Street, 1969). Several studies have examined the effects of washing solutions on removing pesticide residues from various food commodities as follows. To minimize dietary exposure to pesticides, it is pertinent to explore strategies that effectively help in reducing the residue content at individual level. Twelve simple, labour-less and cost effective unit operations were imparted to curry leaf samples for reducing dietary consumption of pesticide residues which can be even followed in poor populace. Out of all treatments imparted each pesticide has its own treatment of reduction. There is no common treatment observed which can remove all pesticides effectively. Triazophos was effectively removed by frying (27.53%). In case of other pesticides scenario is like chlorpyriphos (formula 1 - 58.76%), ethion (formula 1 – 19.44%), carbendazim (cooking – 69.11%), cypermethrin (tamarind water – 61.35%) and profenophos (butter milk – 64.53%). The results obtained in the present studies are in line with the work done by Pallavi et al. (2014) who observed a loss of 62.4, 54.4, 56.2, 68.2 and 45.6 per cent of malathion, chlorpyriphos, quinalphos, profenophos and cypermethrin residues in curry leaf by treating with 2 per cent common salt for 15 min and reduction of 41.8, 47.3, 51.48, 52.77 and 50.25 by treating with 2 per cent vinegar for 15 min. she also observed reduction upto 66.7, 11.4, 41.9, 48.3 and 22.7 per cent of malathion, chlorpyriphos, quinalphos, profenophos and cypermethrin by treating with 2 per cent buttermilk for 15 min. whereas by by treating with 2 per cent vinegar for 15 min the loss was 41.8, 47.3, 51.48, 52.77 and 50.25 per cent respectively. Randhawa et al (2008) also reported that washing reduced cypermethrin residues by 33.42-35 per cent in brinjal. Chlorpyriphos residues were reduced by 33 per cent in spinach by washing (Randhawa et al. 2007). Wasim et al. (2010) reported that washing with 4 per cent acetic acid solution, biowash and formula 1 play a role in the reduction of pesticide residues in cabbage. Direct cooking process has shown less effect in the removal of residues when compared with 2 per cent salt water washing because of all the processed pesticides are having the property of high water solubility. Similar results were observed by Shashi et al. (2014) in tomato. The results of earlier workers like Raghu et al. (2015) were similar to our findings as they found that profenophos residues reduced to 47.64 per cent, 78.48 per cent, 65.48 per cent and 28.95 per cent by 2 per cent by salt solution, bio wash, 4 per cent acetic acid solution and 0.1 per cent baking soda solution in chilli. Geetha (2015) got reduction of chlorpyriphos residues by boiling (54.43%) in spinach which is in line with present work. Shashi et al. (2015) observed similar results of reducing the residues of profenophos in brinjal by 47.07 per cent by running tap water washing which proved that washing is an effective and easy tool to dislodge surface residues to greater extent in a simple way.