Determination of Pesticide Residue in Water and Khat (Catha edulis) Leaves Using GC-ECD

In this work the concentration of some selected pesticides such as aldrin, dialdrin, BHC, diazinon, DDT, 4,4-DDE and heptachlor were investigated in water and khat samples that were collected from five different sites. The pesticides were extracted using liquid-liquid and solid phase extraction technique and analyzed by GC-ECD. The recovery test, linearity cure and extraction efficiency were tested by using standard pesticide samples. The highest concentration of pesticide obtained in water samples is diazinon in WS2 (0.0698mg/L), and the lowest concentration is heptachlor in WS3 (0.0006mg/L). The highest and lowest concentrations of pesticides obtained in Khat samples are diazinon in KS1 (0.0323mg/L) and KS3 (0.0001mg/L) respectively. The concentration of diazinon and four isomers of BHC detected in each water sample are higher than the recommended limits set by WHO which shows contamination of the lake water. Diazinon and DDT levels in KS1 (0.0323mg/L), KS2 (0.0293mg/L) and KS1 (0.0134mg/L), KS2 (0.0173mg/L) respectively are above the maximum residue level.


Introduction
The use of chemicals in modern agriculture has significantly increased productivity. But it has also significantly increased the concentration of pesticides in food and in our environment, with associated negative effects on human health. Annually there are dozens of million cases of pesticide poisonings worldwide [1] *E-mail of the corresponding author: dagne.bdagne.bayih@gmail.com JACEP V4 (1) 2019 17 In recent times, the extent of the use of pesticides, and their mode of application including their abuse especially in agriculture have been of much concern to environmental scientists [2] Organochlorine (OCs) and organophosphorus (OPs) pesticides are still being used unofficially in large quantities in many parts of developing countries because of their effectiveness as pesticides and their relatively low cost [3].
Pesticides are mainly sprayed on agricultural fields such as cereal crops and vegetables.
Especially farmers in Eastern Hararge Zone use different types of pesticides for crop and Khat cultivation in different seasons.
Organochlorine pesticides are very persistent, bioaccumulative and toxic, and they can easily find their way into an adjacent water course such as the lakes via soil run-off and leaching.
The aim of this study is to determine the concentration levels of some selected pesticides in water and khat leaves.
The determination of pesticide residues in khat and water can give indication of the extent of aquatic contamination and accumulation characteristics of these compounds in the khat that help in understanding the behavior and fate of these persistent chemicals. This work tries to provide baseline information on levels of pesticide residues including aldrin, dialdrin, BHC, diazinon, DDT, 4,4-DDE and heptachlor in water (Adele lake) and khat leaves (Harmaya Wereda) Eastern Hararge Zone, Ethiopia.

Description of the Study Area
Lake Adele (300 ha) is found in Haramaya Wereda, East Harerghe Zone. The lake is 26 km west of the city of Harar. It is located at latitude of 9° 25' 33" N and longitude of 41° 57' 03" E. The lake is separated by a 15-km-wide strip of cultivated land from Lake Haramaya. The Lake is surrounded by small hills and derives its water directly from rainfall and from several small streams that drain catchments to the west and north; floods from adjacent watersheds also occur [4].

Method Validation
All sampling, extraction and analysis were done in replicates to verify the detected pesticide residue. Recovery tests were carried out using the reference pesticide standards to determine JACEP V4(1) 2019 19 performance of the methodology. Quantification of pesticides residues were carried out using high purity pesticide reference standards. Field blanks and method blanks were also incorporated to check contamination during sampling, transportation and laboratory preparation procedures.

Sampling, Extraction and Cleanup of Pesticides in Water
Water samples were collected on October 2018 from five different locations using plastic bottles, which were previously cleaned with detergent and rinsed with water followed by ethylacetate and then dried for 24 hours. The bottles were also rinsed with sampling water before sampling.  then eluted with 80 mL portion of ethylacetate/dichloromethane at a rate of 5 mL/min into a conical flask as fraction one. The column was eluted again with 50 mL portion of ethylacetate/dichloromethane for the second elution and added to the first extract. All the fractions of each sample was concentrated to dryness using a rotary evaporator at 40 °C. Each residue was then dissolved and collected in 2 mL ethylacetate for GC-ECD analysis.

Collection and Extraction of Pesticides from Khat Leaves
Khat samples were collected from five different farms each in October 2018 from Haramaya Werede (9°24′N42°01′E) Eastern Harerghe where the lake water is used for agriculture. The collected khat samples were packed with polyethylene bags until the next work proceeded. Then, the leaves of khat (consumable part) was separated and washed respectively to clean unwanted materials. The cleaned leaves dried under shade to manage photooxidation.
Pesticide extraction and clean up from khat samples was performed following the procedure described by Huang et al. 2007. The collected leaf samples were dried under shade and powdered. A 1 g of the powdered sample was mixed with 5.0mL water and 1.0 g sodium chloride. The mixture was sonicated for 3 min and kept at room temperature for 30 min. Then the mixture was extracted three times with 4.0 mL each of the extraction solvent [acetone : ethyl acetate : n-hexane (1:2:1 v/v/v)] for 3 min followed by centrifugation at 2,500 rpm for 2 min, and the organic phase from the extractions was combined and dried with 1.0 g of anhydrous sodium sulfate. The solution was then filtered and reduced to about 5mL at 45 0 C. A 5 mL of the concentrated residual extract was introduced into the Solid Phase Extraction (SPE) column, which was preconditioned with 6.0mL of the extraction solvent. The retained pesticides on the column were eluted with 6.0mL of acetone-ethyl acetate mixture (1:2 v/v). The eluent was collected and evaporated to dryness in vacuum. Finally, the residue was re-dissolved in 0.5 mL ethyl acetate and used for GC-ECD analysis.

JACEP V4(1) 2019 21
Five surface-water and khat as well as quality control samples were analyzed using GC-ECD for the determination of pesticide concentration. Performance of the GC-ECD method was assessed by examining data for chemical surrogates and spiked samples.
Mean recoveries of each analyte, including surrogates, should ideally be 100 percent; mean recoveries substantially different from 100 percent may indicate inaccurate spike (or surrogate) solution concentrations, degradation, extraction efficiencies less than 100 percent, sample contamination, or analytical interference [8].   Diazinon is one of the organophosphorous insecticides used to control insects. It is the highest concentration of all pesticides detected in water sample. Its recommended concentration limit set by WHO is 0.001mg/L. Therefore, the concentration of diazinon detected in each sample is higher than the recommended limit. This indicates that the farmers around the lake use diazinon in large amount.
Heptachlor is applied as a soil treatment, as a seed treatment (maize, small grains and sorghum) or directly to foliage. It is used to control ants, cutworms, maggots, termites, thrips, weevils, wireworms and many other insect pests in both cultivated and uncultivated soils. Heptachlor also controls household insects and pests of humans and domestic animals [9]. The value obtained in the water sample is less than the value set by WHO (0.004mg/L).
Aldrin and dieldrin are highly effective insecticides for soil-dwelling pests and for the protection of wooden structures against termites and wood borers. Dieldrin has also been used against insects of public health importance [10]. Aldrin and dieldrin are the common names of two

Concentration of Pesticide Residue on Khat Leaves
A summary of the pesticide residues found in khat samples from five different farms of Haramaya werede Eastern Harerege is shown in 2. Some level of a-BHC was detected in KS 1 , KS 2 and KS 3 . In KS 1 and KS 2 its level are higher which implies its excess use in this farms than the rest. The highest concentration of α-BHC is detected in water sample compared with that of khat leaves.
Diazinon was detected almost in all the khat samples except KS 5 even if it is below the quantification level in some samples (the maximum residue level (MRL) under EU-regulation set for vegetables and fruits is 10g/kg). Diazinon level in KS 1 and KS 2 is higher or above MRL which indicates it is frequently used in these farms than the others. Diazinon in Ethiopia is registered for the control of pests that attack vegetables and fruits and some concentrations of residue observed in khat samples with higher amount indicate its excessive use in that farms. The concentration of diazinon is slightly greater in water sample than is khat leaves.
Currently the use of aldrin in Ethiopia is restricted. However, its presence in samples (KS 1 , KS 2 and KS 4 below MRL) could be from historical application and could be attributed to the persistence of organochlorine pesticides in the environment. The concentration of aldrin in khat leaves are less than that of water sample obtained in this work. However, because of its easy accessible to the farmers from the depots meant for malarial control, it is illegally used on khat farms.
Several reviews on the adverse health effects of chewing khat documented negative health impact of the active compounds in khat and in some cases in association with smoking (commonly used in khat chewing sessions) but did not account the role that may be contributed by pesticides. Organochlorines have been shown to cause abnormalities in the reproduction and immune systems of birds and marine mammals and several abnormalities caused by organophosphorous have been reported in many organisms [14]. The incidence of oral and oesophageal cancer among khat chewers may also be in part attributed to the carcinogenic effects of organochlorine pesticides [15].

Conclusion
The result of the recovery test indicates that most of them are in the range of good recovery (70-120%). Alpha-BHC, diazinon, DDT and 4,4-DDE were detected in all the water samples.
However, the other pesticides were undetected at list in one water sample. The highest concentration of pesticide obtained in water sample is diazinon in WS 2 (0.069845), and the lowest concentration is heptachlor in WS 3 (0.000594). The concentration of diazinon, adrin, JACEP V4 (1) 2019 26 dialdrin, DDE and α-BHC were higher in water samples than is khat leaves. However, the concentration of heptachlor and DDT were higher in khat leaves.
The concentration of all the pesticides detected, except heptachlor, in each water, samples were higher than the recommended limit set by WHO. This shows that even if some of the pesticides including DDT are banned to use in the country, farmers are still using them for different purpose and the lake water is highly contaminated by these pesticides. The value of DDT and DDE obtained in this work are in agreement with the values that were investigated by other researchers in Ghana but slightly greater than the values obtained at Iran. The value of diazinon obtained is higher than the value that was obtained in Nigeria.