Various fruits and vegetables samples were purchased from largest local market for fruits and vegetables in Dhaka named Kawran Bazar. Sampling was done between February, 2021 to May, 2021. The samples were taken to the lab and stored at 4°C until further analysis. The vegetable and fruit samples were extracted and analyzed for the detection of OCPs within 24 hours after collection. Samples were collected on three replicates and a total of 10 types of fruits and 10 types of vegetables were collected randomly. The vegetables analyzed for the present study were cabbage (Brassica oleracea var capitate), cauli flower (Brassica oleracea var botrytis), Radish (Raphanus sativus), Hyacinth bean (Lablab niger), Sweet gourd (Cucurbita maxima), Cucumber (Cucumis sativus), Bitter gourd (Momordica charantia), Brinjal (Solanum melongena), Tomato (Lycopersicon esculentum) and Chilli (Capsicum species). The fruits analyzed for this study were Banana (Musa sapientum), Mango (Mangifera indica), Litchi (Litchi chinensis), Papaya (Carica papaya), Guava (Psidium guajava), Orange (Citrus chrysocarpa), Grape Fruit (Citrus grandis), Pineapple (Ananas comosus), Watermelon (Cucumis melo) and Lemon (Citrus limon). This research was conducted at the Institute of Food Science and Technology (IFST), Bangladesh council for Scientific and Industrial Research (BCSIR), Dhaka, Bangladesh.
2.2 Extraction of sample and clean-up
Sample extraction was done according to the method (Takatori et al. 2011) with slight modification. In short, frozen samples were homogenized and thawed to room temperature. About 10 g of sample was transferred to 50 ml polypropylene tube and 10 ml of acetonitrile was added with it. The samples and the solution were homogenized with vortex mixer at high speed for 1 minute. Then, 4 g anhydrous magnesium sulphate and 1g of sodium chloride was added and shaken for 1 minute. Then, centrifuged for 5 minutes and 4 ml of organic layer supernatant was collected and eluted with two portions of 5 ml acetonitrile in a 50 ml flask (pear- shaped). The elute was then evaporated using a rotary evaporator. The concentrated extract was dissolved in 2 ml ethyl acetate and finally transferred to 2 ml standard vial for GC-MS/MS analysis.
2.3 Chemicals and reagents
Reference standard (with 98% purity) containing a cocktail of 19 different OCPs (Lindane I, Lindane II, Lindane III, Lindane IV, Heptachlor, Aldrin, Heptachlor epoxide, trans-chlordane, α-endosulfan, cis-chlordane, p',p'-DDE, Endrin, β-Endosulfan, Endrin-Ketane, Methoxychlor, Endosulfan sulfate, p',p'-DDT, Phthalic acid, p,p-DDD), Acetonitrile (HPLC grade) and Ethyl acetate were purchased Sigma Aldrich (Germany). Analytical grade anhydrous magnesium sulphate (98% purity) and sodium chloride was purchased from Merck. (Darmstadt, Germany).
2.4 Instrumentation and Gas Chromatography (GC)
The analysis was performed in GC-MS (Model: TRACE 1310, Thermo Fisher Scientific, USA) equipped with Thermo Scientific™ Trace GOLD™ TG-5MS GC Column (0.25 mm X 0.25 µm X 0.25 m) and 5% phenyl phase. Helium was used as the carrier gas in this experiment, with a constant flow rate of 1.2 ml/min. The injection port had a temperature of 230 degrees Celsius. The temperature profile for GC was ranged from 80◦C to 290◦C. The inject volume was 2 µL. Spectral detection was performed by Mass Spectrometer (Model: TSQ DUO, Thermos Scientific, USA).
2.5 Linearity of Calibration curve
Before starting the analysis, the column performance was checked by running only the blank samples. The stock solution of OCPs pesticides containing a cocktail of 19 pesticides was used as standard solution. For the standard calibration curve construction, five different concentrations ranging from 5 ppb to 200 ppb were prepared and injected on the column and all the standards showed a linear range from 5 ppb to 200ppb. The coefficient values (R2) obtained was ranged from 0.94 to 0.99 for all 5 standards.
2.6 Evaluation of Recovery performance
Recovery performance evaluation was used to confirm the method's precision results. For purpose of the recovery performance evaluation, each pesticides was spiked in distill water with two known concentrations (0.5µg/L and 1 µg/L). The spiked samples were extracted and analyzed exactly using the same procedure of sample analysis. The following equation was used to determine the mean percentage recoveries of the OCPs:
Pi= (Si/Ti) × 100Pi= (Si/Ti) × 100
Here, Pi represents the percent recovery, Si represents results from laboratory controls and Ti represents the percentage recovery from the spiked samples of known concentrations. Recovery tests were repeated at least two times for each OCPs and the mean percentage value, standard deviation (SD) was detected and presented in the table 2.
2.7 Determination of limit of detection (LOD) and Limit of quantification (LOQ)
Average blank value method was applied to determine the limit of detection (LOD) and limit of quantification (LOQ). LOD and LOD values were determined by running several blank samples. The LOD value of the OCPs was calculated based on the signal to noise ratio (3:1). LOD is defined as the lowest concentration of OCPs that produced a chromatographic peak that was 3 times larger than the noise background (during the same retention time). The 10 times value of baseline noise in the chromatogram of the blank samples was used to compute the LOQ value.
2.8 Assessment of human health risks
2.8.1 Estimated daily intake (EDI) of OCPs
According to the Codex Alimentarius Commission Procedural Manual (Codex Alimentarius Commission, 2006), the exposure assessment can be defined as “the qualitative and/or quantitative evaluation of the likely intake of biological, chemical, and physical agents via food, as well as exposures from other sources if relevant.” For each pesticide exposure the life time exposure dose (mg/kg/day) can be obtained through multiplication of residual pesticide concentration (mg/kg) in food items with the daily food consumption rate (per capita consumption) (kg/day) and dividing the result by the body weight (kg). The per capita consumption of vegetables and fruits in Bangladesh are 167.30 g/person/day and 35.78 g/person/day respectively (BBS 2017). Dietary exposure was determined by the following formula:
EDI = (FCC ×DFC) /BW
Here, EDI represents Estimated daily intake (EDI) (mg/(kg/day)); FCC indicated food chemical concentration (mg/kg); DFC is stand for daily food consumption (kg/day) and BW is used for Body weight (kg). The hypothetical assumption of U.S. Environmental Protection Agency - USEPA (1986, 1989) about the body weight of adults is 70 kg considered for the dietary exposure calculation.
2.8.2 Hazard Risk Index (HI)
Consumers' health risk assessment from pesticide-contaminated vegetables and fruits was expressed as a Health Risk index (HI). The HI was calculated by multiplying the estimated daily intake (EDI) by the corresponding appropriate daily intake (ADI) values as given by WHO/FAO (Australian Government Department of Health, 2016) and indicated by the equation:
HI = EDI/ADI.
HIv = Estimated daily intake of vegetables (EDIV)/ADIv
Here, EDIv stands for EDI of vegetables and ADIv stands for acceptable daily intake of vegetables.
HIf = Estimated daily intake of fruits (EDIF)/ADIf
Here, EDIf stands for EDI of fruits and ADIf stands for acceptable daily intake of fruits.
If the value of HI is greater than 1, then it can be said that, the population consuming OCPs containing fruits and vegetables are safe from any health hazards.
2.9 Data analysis
The obtained results were summarized, organized, tabulated and analyzed through Microsoft Office Excel 2007 and Statistics 10 (for statistical analysis). Duncan's multiple-range test was used to identify the differences between variables (DMRT).