Insecticide Residues in Cotton, Sorghum and Fallow Soil from the Nuba Mountains Cotton Corporation of South Kordofan State, Sudan

Background. Soil is the final depot of most environmental contaminants, including pesticides. Soil may be contaminated by pesticides as a result of direct application or drift during spray activities. Soil contamination with pesticide residues may affect the quality of food crops, animal products, and soil micro-organisms which may in turn negatively affect human health and the environment. Objectives. The main objective of the current study was to determine the soil residues of commonly used pesticides in rain-fed crops grown by the Nuba Mountains Cotton Corporation (NMCC) in South Kordofan state of Western Sudan. Methods. Four locations (representing the four directions around the state capital Kadugli) were chosen for sample collection: Alefain (East Kadugli), Elmashaish (West Kadugli), Ed Dalling uncultivated area (North Kadugli) and Lagawa (Southwest Kadugli). Nine soil samples were randomly taken from each location representing areas under cotton, sorghum, and uncultivated land covered with natural vegetation. Soil samples were analyzed by gas chromatography (GC) equipped with electron capture detector (ECD) and GC-mass spectrometry (MS). Results. The results generally indicated that organophosphate levels were greater than organochlorine and pyrethroids with heptachlor, malathion, and dimethoate present in all samples analyzed, while the level of p,p-dichlorodiphenyltrichloroethane (DDT) was below the detection limit. Endosulfan α and β isomers were detected in some samples. Dimethoate had the highest level (22.02 mg/kg), while β endosulfan was found at the lowest level (0.015 mg/kg). Generally, samples collected from cotton soils showed higher residue levels compared to sorghum soil with average concentrations of 307.25 mg/kg versus 58.63 mg/kg, respectively. Almashaish showed the highest residues levels followed by Alefain, Lagawa, and Ed Dalling with total residues of 57.56 mg/kg, 26.34 mg/kg, 22.63 mg/kg, and 17.07 mg/kg, respectively. Conclusions. The current study sheds light on the residue levels of some of the commonly used pesticides in the cotton rain-fed scheme in South Kordofan State, western Sudan. The study calls for regular residue monitoring in various environmental components in the area and suggests possible management measures. Competing Interests. The authors declare no competing financial interests.


Introduction
Sudan has one the highest rates of pesticide use in the Middle East and Africa. 1 The annual import of pesticides reached about 5000 tons for crop protection and control of vector borne diseases in 1990. 1 However, this amount decreased to a range of 2000-3000 tons after the 1990's 2-3 due to adoption of integrated pest management (IPM) programs, changes in agricultural policy, and reduction in areas allotted to cotton. Irrigated cotton growing areas were known for intensive use of pesticides. 2,4-5 On the other hand, rain-fed cotton was known to receive relatively lower levels of pesticides (less than 10%) and attract less investigation. 2 Among the most important rain-fed cotton growing areas in Sudan is the Nuba Mountains Cotton Corporation Research (NMCC) of South Kordofan State, Western Sudan. South Kordofan State is rich in crop diversity and famous for production of cereals, oil seeds, vegetables, fruits, and cotton. 6 From 1986-2003 this area experienced intensive and extensive spraying of different groups of insecticides due to an increase in the population density of many agricultural and public health pests. 6 Cotton and sorghum were the dominant crops grown with a three year crop rotation (cotton, sorghum, and fallow). Cotton is the main cash crop and is repeatedly sprayed with insecticides during the growing season, while sorghum and fallow soils do not receive authorized pesticide application. However, they may become contaminated from previous season applications, unauthorized use, or from nearby control operations. Earlier studies reported a measurable level of organochlorine insecticides in soil and blood samples from the surrounding areas in western Sudan. 7 Monitoring the level of contamination with pesticide residues in soil is important for a safe environment and healthy production of food crops. Soil is the final depot of most applied pesticides. Studies have shown that only 0.1% of the applied pesticides reach the target pest 8-9 and the remaining 99.99% of the applied dose goes to the environment and ultimately ends up in soil, 8,10 thus presenting a potential source of contamination to crops, surface and underground water, living biota and the associated food chain. 8 Although most pesticides degrade fairly quickly on crops and in soil, some may persist in one form or another for a longer time. Pesticide active ingredients can be inactivated in soil by adsorption to clay and organic matter or degraded by microorganisms that digest and decompose organic matter in soil. The ability of pesticides to be degraded by micro-organisms (biodegradability) or by chemical action varies according to their chemical structure, degree of exposure, types of microorganisms, and environmental factors such as soil temperature, moisture content and climatic conditions. 11-17 Considering the past and current intensive use of pesticides in the NMCC area and the expected acute and/or chronic adverse effects on humans, grazing animals, and other non-target organisms together with the expected negative impact on the quality of food crops grown in the area, this study aimed to examine residue levels of some of the most commonly used pesticides in soil grown with cotton, sorghum and fallow soil in the NMCC. 6 As the primary cash crop, cotton receives repeated spraying with many insecticides during the growing season, while sorghum and fallow soils do not receive authorized pesticide application, however they may become contaminated from previous season applications. They were included in the investigation because they may reflect residues from previous season use and/or unauthorized use or from nearby control operations. The absence of previous studies in this area further strengthens these goals. The specific objectives of the study were to determine the residue concentrations of some of the commonly used pesticides in soil grown with cotton, sorghum, and fallow land in the NMCC area, to determine the level of pesticide residues in the soil of various production areas in the state (east, west, north, and south), and to provide policy makers and officials with base line data needed for designing suitable mitigation measures and formulating future environmental management plans.

Study area
The study area is located in South Kordofan State, between longitude 29º-32º east and latitude 9º-12º north. The area is characterized by semiarid conditions and summer rains. The average maximum temperature ranges between 36 to 40ºC, while the minimum temperature ranges between 17-20ºC. Rainfall ranges from 600 to 850 mm per year. Half of the area is covered by dark cracking clay soil and grown with sorghum, cotton, and sesame as well as grazing plants. A considerable area of the northern part of the state is covered by sandy soil cultivated with millet. 18

Interview
Agricultural inspectors were interviewed based on their knowledge of pests and pesticides used in the area. Short questions were asked to ten plant protection inspectors in South Kordufan state. Questions asked included years of previous experience, types of crops grown, major pest, types of pesticides applied, and number of spraying per growing season (Supplemental Material Table 1 -5).

Extraction and cleanup of samples
The Association of Official Analytical Chemists (AOAC) 20 method No. 5 was followed for extraction and cleanup of samples. Fifty (50) grams of dried soil of each sample was placed into a 250-ml glass bottle and wetted with 15 ml of distilled water, and then 50 ml of re-distilled acetone and 50 ml of n-hexane were added. The bottle was tightly closed and placed firmly in an end-over-end shaker for three hours. The samples were then left to stand for 5 minutes to allow soil particles to settle down and then filtered through 18-mm filter paper. The filtrate was collected in a round bottle flask. This was placed in a rotary evaporator (Büchi Rotavapor R-200) at 40°C to reduce the filtrate volume to about 80 ml. The content was then transferred to a separatory funnel (capacity 1000 ml) then 600 ml distilled water and 10 ml of saturated sodium chloride solution (23% w/v) were added and shaken for one minute (the cock was opened several times to release pressure). The organic phase was transferred to a 500-ml separatory funnel, while the aqueous phase was re-extracted twice with 30 ml n-hexane. The combined n-hexane extracts were washed twice with 100 ml of distilled water and 5 ml of saturated sodium chloride solution. The organic phase was dried in a rotary evaporator to reduce the volume to 10 ml then filtered through filter paper containing 10 g of anhydrous sodium sulphate to absorb the moisture. The dried filtrate was then collected in tubes (10 ml) with a Teflon-lined screw cap, tightly closed, sealed with Teflon tape, and stored in a refrigerator at 4 o C for GC analysis.

Recovery test
Spiked samples were prepared and analyzed under similar conditions to check for method recovery. Sixteen grams of soil were weighted, divided into four groups of 4 g each. Each soil was placed in a glass bottle as previously mentioned, and then treated with analytical standards of candidate insecticides: heptachlor, 0.0134 mg/kg; p,p-DDT, 0.045 mg/ kg; deltamethrin, 0.0135 mg/kg; α-β endosulfan, 0.0124 mg/kg; and endosulfan sulfate, 0.0156 mg/kg. Fortified samples were subject to the same extraction and clean up procedures. The recoveries of the method are greater than 91% as given in Supplemental Material Table 6.  Table 7.

Gas chromatography with mass spectroscopy
Three representative samples were reanalyzed using a Shimadzu GC-MS Qp2010 system (Japan) with an AOC-5000 auto sampler.

Research
The Wiley7.LIB, NIST147.LIB, and NIST27.LIB libraries were searched for reconfirmation of identity of compounds detected by the GC-ECD. Libraries were searched for similarity of molecular weight and fragmentation pattern.

Statistical analysis
Collected data were tabulated and statistically analyzed using the MINITAP software statistical package. Descriptive statistics was used in terms of means and standard deviation, in addition to median and range.

Results
Interviews with agricultural inspectors indicated that they relied on different types of insecticides for crop protection purposes, mostly organophosphates (OPs), some pyrethroids, and occasionally organochlorines (OCs) (when available). Chemical control decisions were based on regular surveys of pests on cotton, and based on farmers' observations for sorghum, although the latter is rarely sprayed. The most commonly used insecticides reported by the respondents are presented in Supplemental Material Tables 3-7.

Levels of pesticide residues detected in soils under cotton
The results revealed the presence of detectable levels of at least four pesticides in each location (  Figure 1). Generally, cotton soils showed higher levels compared to sorghum soils and uncultivated land in various locations ( Table 1 and Figure  1). Gas chromatography analysis revealed the presence of detectable levels of heptachlor, malathion, and dimethoate, and the absence of detectable levels of p,p-DDT in all analyzed samples.

Figure 1 -Total soil residue levels (mg/kg) of the main insecticide groups detected across locations
Journal of Health & Pollution Vol. 11, No. 30 -June 2021 6 Research Table 1

-Total Soil Residue Levels (mg/kg) of the Main Insecticide Groups Detected Across Locations
Mohamed et al

Insecticide Residues in Cotton, Sorghum and Soil in Sudan
Research (0.021 mg/kg).

Levels detected in soil under sorghum
Sampling of sorghum soil was possible in two locations Alefain, East Kadugli and Almashaish, West Kadugli, in addition to uncultivated soil. There was no sorghum grown in the south of the state during that season. The soil of West Kadugli showed the highest level, followed by East Kadugli, and lastly the uncultivated soils. Generally, organophosphorus compounds were found at higher levels compared to organochlorines, while pyrethroids were below the detection limit ( Table  2 and Figure 1).

Table 2 -Concentration (mg/kg) of Insecticide Residues Detected in Soil under Sorghum in South Kordofan State
Research and dimethoate in all samples analyzed, while the levels of p,p DDT, endosulfan sulfate, and deltamethrin were below the detection limit ( Table  2). The highest levels detected at this site corresponded to dimethoate (average 22.02 mg/kg and range of 21.25-22.68 mg/kg), while the lowest level detected corresponded to β endosulfan (average 0.129 mg/kg and range of 0.099-164 mg/kg).
The analysis ( Table 2) of soil samples from uncultivated soil indicated the presence of detectable levels of heptachlor, α endosulfan, β endosulfan, malathion, and dimethoate in all samples analyzed, while the levels of p,p-DDT, endosulfan sulfate, and deltamethrin were below the detection limit ( Table 2). The highest levels detected correspond to dimethoate (average 16.17 and ranges of 1.57-16.54 mg/ kg), while the lowest levels detected belonged to β endosulfan (average 0.02 and range 0.018-0.024) mg/kg).

Discussion
The results generally indicated the presence of detectable residues of some insecticides in all soil samples analyzed. Heptachlor, malathion and dimethoate were detected in all samples, while the level of p,p-DDT was below the detection limit. On the other hand, endosulfan β, endosulfan sulfate, and deltamethrin were found in cotton soil at one location only (Almashaish

Study limitations
The present study focused on the soil residues of some of the most commonly used pesticides. Residue levels in food sources and other environmental compartments were not assessed. Since the current study reported measurable levels of some pesticides and the study was performed with a limited number of samples, this indicates the need for a regular monitoring program of residues of all pesticides in the crops grown in the area and the surrounding environment. The study did not address the level of awareness of pesticide safety among farmers, which should be covered in future work. Proposals of suitable management plans and possible mitigation measures are also needed.

Conclusions
Heptachlor, malathion and dimethoate were detected in all samples analyzed in the present study. Dimethoate was found at the highest level (22.02 mg/ kg), while malathion and heptachlor were found at lower levels (8.91 mg/ kg and 1.56 mg/kg, respectively). Cotton soil showed higher residue levels compared to sorghum soil with average concentrations of 43.69 mg/ kg versus 41.46 mg/kg, respectively. Almashaish in west Kadugli had the highest residues levels, followed by Alefain, Lagawa, and Ed Dalling with total residues of 57.56 mg/kg, 27.95 mg/kg, 20.58 mg/kg, and 17.07 mg/kg, respectively.
The current study sheds light on the residue levels of some of the commonly used pesticides in the cotton rain-fed scheme in South Kordofan State, western Sudan. The study calls for regular residue monitoring in various environmental components in the area and suggests possible management measures.