Pesticide Residues in Eggplant Fruit from Khartoum State, Sudan

Background. Eggplant is a popular food item in Sudan, however pesticides are heavily used. Objective. To investigate the presence of pesticide residues in fresh eggplants in Khartoum State, Sudan. Methods. Eggplant fruit samples from three different regions in Khartoum State (central vegetable market, east Nile farms, and west Nile farms) were analyzed for residues of commonly used pesticides. Pesticide residues were analyzed by gas chromatography coupled with mass spectrometry and results were expressed in μg/kg fruit. Results. Out of the 11 active ingredients analyzed, residues were identified for four pesticides (imidacloprid, dimethoate, endosulfan (α and β isomers) and 2, 4-D). Levels of omethoate, diazinon, malathion, chlorpyrifos, atrazine, and pendimethalin were below the detection limits. Conclusions. Residues of four insecticides out of the 11 analyzed (imidacloprid, dimethoate, endosulfan (α, β isomers), and 2, 4-D) were detected in the current study. The health implications of these violative levels should be regularly observed along with strict enforcement of laws and regulations coupled with agricultural extension interventions. Competing Interests. The authors declare no competing financial interests.


Introduction
Vegetables are an important part of the human diet due to their high nutritional value and as important sources of vitamins (C, A, B6, thiamine, niacin, E), minerals, and dietary fiber. 1,2 Production of vegetables in Sudan is rapidly increasing to meet the needs of the growing population. A wide range of vegetables and fruits are grown in Sudan due to the high availability of arable land, supply of irrigation water and suitable climatic conditions. According to the Food and Agriculture Organization of the United Nations (FAO), vegetable production in Sudan has increased annually in cultivated area and quantity. 3 The percentage increases in cultivated area (hectares) and production (tons) between 2012-2017 were 31.67% and 86.78% for eggplant, 73.48% and 81.41% for tomatoes, 10.32% and 11.75 % for cucumbers, 78.48 and 88.44% for okra, 16.37% and 31.86 % for carrots, 61.74 % and 58.33% for onions, 26.10% and 28.53% for green chilies and peppers, and 68.92% and 71.38% for dry chilies and peppers, respectively. The main vegetables grown in the Sudan include tomato (Lycopersicom esculentum Miller), onion (Allium cepa), eggplant (Solanum melongena L.), and cucurbits. 4 Eggplant, S. melongena L., is a typical and very profitable vegetable crop for farmers. It is locally known as bedingan or aswad and has several common names: aubergine, garden egg and brinjial. 5,6 Eggplant come in different types, shapes, sizes, and colors. 7 The popularity of eggplant in Sudan is mainly attributed to its affordability, the diversity of ways in which it can be cooked and its successful growth under warm weather conditions such as those in Sudan. 8, 9 The history of eggplant in Sudan is unknown, although its entry into Sudan was probably through Egypt. 10 In 2017, the total area grown with eggplant in Sudan was about 13 000 hectares with a total production of 90 336 tons. 3 Worldwide, eggplant production has been steadily increasing, with the largest production in China,India,Egypt,Turkey and Japan. 11 However, eggplant production negatively affects the environment due to its heavy requirement for pesticides to combat insect pests and diseases. Insecticides are repeatedly applied Research during the entire period of growth and sometimes even at the fruiting stage. Indiscriminate use of pesticides during its farming season, particularly at the fruiting stage, and non-adoption of a pre-harvest interval may lead to accumulation of pesticide residues in the fruits, consumers and the environment. [12][13][14][15] Previous studies indicate limited knowledge of vegetable farmers in Khartoum and Gezira states on the safe use of pesticides and their potential risks to human health and the environment. [16][17][18] Extension services available to farmers were sub-standard and require more focus in current and future development projects. Furthermore, studies in Sudan indicate the presence of high levels of malathion, fenitrothion, chlorpyrifos, profenofos, dimethoate, heptachlor, diazinon, ethephon, oxyfluorfen and omethoate in fresh fruits. [17][18][19][20][21][22][23] Prewashing fruits reduces the levels of some pesticides. 21,22,24 Due to limited knowledge of pesticide safety, farmers indiscriminately and heavily use pesticides on eggplants. This study was initiated to investigate the presence of pesticide residues in fresh eggplant fruits in Khartoum State.

Methods
Eggplant fruit samples (2-3 kg per sample) were collected randomly from three different areas in Khartoum State, Sudan: two central vegetable market locations (15.6726 N, 32.5376 E, 15.5304 N, 32.5576 E) and three east Nile farms (15.5133 N, 32.6534 E) and three west Nile farms (15.2578 N, 32.5015 E) (Figure 1). Twentyseven (27) samples were collected in total, nine from each of the three study areas. The FAO Codex method was followed in sample collection at all locations. 25 The collected samples were placed in paper bags, labeled and transferred immediately for extraction to the pesticide analytical laboratory, Faculty of Agriculture, University of Khartoum.

Extraction and partitioning
The collected samples (unpeeled eggplant fruits) were sliced into small pieces, mixed thoroughly and about 50 g of each were weighed using a sensitive balance and blended with 10 g of anhydrous sodium sulphate at high speed (22000 rpm) for two minutes using a chemical resistent blender (National Analytical Corporation, Mumbai, India). The sample was then extracted with 100 ml Abdelbagi

Clean-up
Clean-up was carried out using a solid phase extraction column using readypacked columns with silica gel and activated charcoal (5:1 wt/wt) topped with anhydrous sodium sulphate. The columns were pre-washed with 50 ml of acetone and hexane, respectively. Finally, the extract was passed through the columns and eluted with a 125 ml mixture of acetone:hexane (3:7 vol/vol). The cleaned extract was dried by rotary evaporator at 40°C until complete dryness and then reconstituted in 10 ml hexane and stored 4°C for analysis by gas chromatography coupled with mass spectrometry.

Chromatographic analysis
The pesticide residue analysis was determined using gas chromatography coupled with mass spectrometry. Samples were analyzed using a Shimadzu GC-MSQP 2010 (Tokyo, Japan) with an AOC-5000 auto sampler. The gas chromatograph was fitted with an Rtx5-MS capillary column 30 m x 0.25 mm internal diameter x 0.25 µm film thicknesses from Restek (UK). Helium (purity ≥ 99.999%) was used as a carrier gas at a flow rate of 1.69 ml/min. The splitless injection temperature was 230°C. The oven temperature was programmed from initial temperature of 50°C for 3 minutes, raised at 10°C per minute to 200°C and held for 5 minutes, then increased by 3°C per minute to 230°C and held for 2 minutes. The mass spectrometer was operated with an electron impact source in scan mode. The electron energy was 70 eV, and the interface temperature was maintained at 200°C. The solvent delay was set to 10 minutes. The retention time of standard pesticides are shown in Figures 2, 3 and 4. Detection limits are given in Table 1. The recovery of the method ranged from 70-120%. No pesticide residues were detected in the calibration blanks.
The pesticide residues present in the eggplant samples were identified by matching their retention times and mass-spectrum to those of analytical Table 1

-Minimum Detectable Levels and Retention Times of Studied Pesticides
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Pesticide Residues in Eggplant Fruit from Khartoum State, Sudan standards. About 1.0 µl of various concentrations of each analytical standard was injected in the gas chromatography and their peak areas were used for the construction of the standard curves. Duplicate samples of 1 µl from each extract were injected and concentration was determined using Equation 1.

Results
The results of residues analysis of samples are summarized in Tables  2 and 3  Generally, endosulfan α showed the lowest levels, except in east Nile farm samples where its average was 1040 µgkg -1 with a range of ND-3050 µgkg -1 . The highest insecticide loads per kilogram fruit were found in the samples collected from east Nile farms (average total 4175.3 µgkg -1 ), followed by central vegetable market (1742.7 µgkg -1 ) and west Nile farms (1711.1 µgkg -1 ). In addition, the highest frequency of detection (+ ve samples %) and the highest frequency of violative levels (violation %) followed the same order (Tables  2 and 3 and Supplemental Material). Levels of omethoate, diazinon, malathion, chlorpyrifos, atrazine, and pendimethalin were below the detection limit (Tables 2 and 4).
The herbicide 2,4-D was the only herbicide detected and only in samples from east Nile and west Nile farms. Its level was relatively higher in east Nile farms (38.7 µgkg -1 ) versus west Nile farm samples (37.7 µgkg -1 ). Its frequency of detection followed the same order (66% in east Nile farms versus 33% in west Nile farms). Herbicide 2,4-D levels detected were below FAO Codex MRLs. Atrazine and pendimethalin levels were below the detection limit (Table 4 and Supplemental Material).
In summary, the average residue load of insecticides and herbicides per kg fruit was about 2443.1 µgkg -1 with a load range of ND-3892.7 µgkg -1 , while the total frequency of detection of pesticide residues was 66% ( Table  5). Out of the total samples, about 55% were violative. Generally, the highest levels were detected in the east Nile farm samples (3837.8 µgkg -1 ) followed by west Nile farm samples (1748.8 µgkg -1 ) and central vegetable market samples (1742.7 µgkg -1 ). In addition, the level of violations and the frequency of detection followed the same order (Table 3,5 and Supplemental Material).

Discussion
The current study investigated the level of 11 commonly used pesticides in eggplant samples collected from

-Levels of Herbicides Residues (µgkg -1 ) Detected in Eggplant Samples Collected from Central Vegetable Market and Vegetable Farms in Khartoum State
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Furthermore, endosulfan was reported as the main causative agent of human poisoning in Sudan, 38-40 although most of the reported cases were due to consumption of endosulfancontaminated food.
The herbicide 2,4-D was detected in samples from east Nile and west Nile farms, with a relatively higher level in the east Nile farms. The presence of 2,4-D in the samples may be due to its recent use in the area (although not claimed by the farmers interviewed), or from drift from nearby farms or from contaminated equipment. 30 Fantke et al. reported that 2,4-D and parathion were the most damaging pesticides across the various crops studied. 41 Levels of omethoate, diazinon, malathion, chlorpyrifos, atrazine and pendimethalin were below the detection limit. The absence of their residues may be explained by the absence or limited use in the area. Previous studies in Sudan indicated the presence of high levels of malathion, fenitrothion, chlorpyrifos, profenofos, dimethoate, heptachlor, diazinon, ethephon, oxyfluorfen, dimethoate and omethoate in fresh vegetables. [16][17][18][19][20][21] Variation across studies may be explained by differences in location and use patterns in different vegetable crops.
Violations (greater than FAO-Codex MRLs) ranged from 11% (endosulfan α) to 55% (imidacloprid Limitations of the present study included a lack of detailed information on pesticide use patterns and safety aspects as well as lack of policy enforcement in the study areas. In addition, the effect that processing eggplant fruits has on the level of pesticide residues was not considered. Further studies examining pesticide residues in other commonly consumed vegetables in the Sudan are needed as there is no regular monitoring program for pesticide residues in vegetables in this area and few studies have examined this issue.

Conclusions
Residues of four insecticides out of the 11 analyzed (imidacloprid, dimethoate, endosulfan (α, β isomers), and 2, 4-D) were detected in the current study. Violative levels were found for imidacloprid and endosulfan. The highest levels and frequency of detection and violation were found for imidacloprid. The highest levels and frequency of detection and frequency of violation were associated with east Nile farms, while the lowest were associated with central vegetable market. Levels of omethoate, diazinon, malathion, chlorpyrifos, atrazine, pendimethalin were below detection limits. The health implications of these violative levels should be regularly observed along with strict enforcement of laws and regulations coupled with agricultural extension interventions.

Codex Pesticides Residues in Food Online
Database [Internet]. Rome: Codex Alimentarius.