# Metal and Pesticide Assessments of Imported and Locally Cultivated Rice (_Oryza sativa_) in Senegal
## 2 Materials and Methods
### Preparation and Measurement of Pesticides  
Prior to the extraction procedure, the rice samples were milled and the dry soil samples were sieved through a 2 mm sieve. An amount of 5 g of milled rice or dried soil sample was weighed and placed into 50 mL centrifuge tubes for extraction. Then, 10 mL of organic solvent consisting of a 1:1 mixture of acetone-dichloromethane (DCM) was added to the samples. The centrifuge tubes with the samples were then placed in an ultrasonic bath and sonicated for one hour. Then, they were centrifuged at 2000 rpm for 15 to 20 min to separate the organic layer from the sample. The extracts were transferred to evaporation tubes by decanting or using a Pasteur pipette. The residue was re-extracted for 30 min with another 10 mL of the acetone-DCM mixture and the second extract was transferred to the same evaporation tube. The residue was re-extracted again for 30 min with another 10 mL of the acetone-DCM mixture and transferred to the same evaporation tube. Then, the whole volume was placed in a water bath at 55 degrees Celsius and evaporated to near dryness. Next, 1 mL of methanol was added and the volume was evaporated to dryness. Finally, the remaining residue was dissolved by cautiously shaking on the vortex with two aliquots of 0.5 mL of methanol that were each transferred to the same 1 mL volumetric tube and made up to volume with methanol. The samples were transferred to 2 mL GC vials and stored in a refrigerator until analysis by GC-MS (7890A-5975C) [26]. The detection limits for deltamethrin, 2,4D, Pyrethrin 1 and 2, Propanil, and Bensulfuron-methyl are 0.01 mg/kg. The recovery of pesticides measured in the range of 70-120%. We analyzed a blank. The analysis was carried out in the Laboratory for Environmental Analysis, Center for Applied Isotope Studies, University of Georgia. Preparation and Measurement of Protein, Lipids, Ash, Lipid, Humidity and Total Carbohydrate  
The main nutrients in rice grains are starch, protein, and lipids. The protein content was performed using a procedure that allows us to account for nitrogen in milling rice grain. A LECO TruSpec model CHN was performed to estimate protein content using the combustion method ([PERSON]'s method). One method consisted of rice-free grain by combustion to measure nitrogen and using a thermal conductivity detector to assess the percent nitrogen content, which will be carried out to calculate the protein content, with 5.95 used as a rice protein conversion factor. The Kjeldahl method has been mostly applied to perform total nitrogen content [27]. Nitrogen content was then multiplied by a factor to arrive at the total protein content. Two assumptions have been taken into consideration based on this approach: dietary carbohydrates and fats do not contain nitrogen, and nearly all the nitrogen in the diet is present as amino acids in proteins. However, all the total nitrogen found in rice samples was multiplied by over 16% for a conversion factor of 6.25. Ash: Typically, samples of 5 g are used in the analysis of ash content. Dry ashing procedures use a high-temperature muffle furnace capable of maintaining temperatures of between 550 \({}^{\circ}\)C for 3 h. Water and other volatile materials are vaporized and organic substances are burned in the presence of oxygen in the air to CO\({}_{2}\), H\({}_{2}\)O, and N\({}_{2}\). The food sample is weighed before and after ashing to determine the concentration of ash present (AOAC Official Method 942.05). Lipids: Accurately weigh 5 g of the sample into the thimble/flask, and dry the sample in an oven at 102 \({}^{\circ}\)C for 5 h. After that, insert the thimble in a Sox let liquid/solid extractor. Accurately weigh a clean, dry 150 mL round bottom flash and put about 90 mL of petroleum ether in the flask. Assemble the extraction unit in an electric heating mantle or a water bath.