2.1 Study Area and Population
A Cross-sectional study on a rural area, namely Sonargaon, Narayanganj, Dhaka, was performed because this area is abundant with industrial and agricultural activities (Banglapedia). Hence these two effects in tandem lead to large-scale contamination of the soil, groundwater, crops and affect the general population. Moreover, this area is at a communicable distance from Dhaka city, and there have been many research activities in this area. As a result, people are aware and are more inclined to participate in research. In Sonargaon, this study is the first of its kind.
The sampling population selected were adults who were full-time or part-time involved in agricultural activities. A voter list of the 47 villages of Sonargaon was collected, and certain people were requested to appear at the Research sites as volunteers for this study. The selected people were invited to the research center and briefed about the cause of the invitation and were assigned IDs. Researchers along with physicians filled up datasheets, which recorded the healthy and CKD, affected people of the selected area age, profession, habits, occupation, education, monthly income, food habits, etc. The healthy and CKD affected people of the selected area were told to come early in the morning on a different day when their blood and urine were collected. The biological samples (Blood and Urine) of 50 representative people from 16 of the 47 villages were collected in metal-free containers. The controls and CKD affected people of the selected area have been divided into two groups, namely 'exposed' and 'controls'. The patients of CKD were confirmed by examining parameters like eGFR, serum creatinine, and ACR over 3 months. Only the confirmed cases of CKD, who did not have High Blood Pressure or Diabetes but had abnormal levels of eGFR and ACR were considered for our study. The controls are the people who have not been diagnosed with CKD.
Fig. 1. Schematic diagram of our research
This study was approved by the ethics committee of the collaborating hospital and the Institutional Review Board (IRB), Atomic Energy Centre, Bangladesh Atomic Energy Commission, Dhaka, and written informed consent was obtained from all of the volunteers, including patients and healthy controls.
2.2 Chemicals and Reagents
In our study, all chemical reagents were of analytical grade or of supra pure quality (E. Merck, Germany). High purity deionized water (Milli-Q System, Millipore, Thermo scientific, MA USA) with the resistivity 18.2MΩ cm−1 was used for the preparation of all solutions. Nitric acid (HNO3) (70%, purified by redistillation, ≥99.999% trace metals basis) was obtained from Sigma Aldrich (St. Louis, MO, USA). Single element standard reference metal solution of Pb, Cd, Cr, Cu, Zn (1000 μg/L; Spectropure, USA) from Agilent Technologies (Santa Clara, CA, USA) was procured. Quality control standard was prepared from multielement standard solution (500 μg/L; Agiland, Santa Clara, USA).
As the tubes and pipette tips (Eppendorf type; Brinkmann Instruments, Inc., Westbury, NY 11590) are unused, they were directly soaked in 5% nitric acid for 24 hours, followed by washing in deionized water for another 24 hours, and then dried and kept in cleaned capped plastic containers.
2.3 Sample Preparation and Analysis
The urine was transported from the sample collection site in iceboxes and was finally preserved at -80 0C. Blood was collected in Polypropylene tubes and stored in an icebox. Later it was subjected to centrifugation at 3000 rpm for 15 minutes at room temperature. Thus, serum was obtained, and the serum was preserved at -80 0C in metal-free polypropylene tubes.
After receiving the serum and urine samples in the Analytical Chemistry Laboratory, dilution of samples was rendered to 10 mL using ultrapure deionized water if required. Analytical calibration standards, internal quality control, and spiked samples with matrix from known standards were prepared from the standard stock solution. A calibration curve for each metal of 0.1–10 μg/L was constructed. The Pb, Cr, and Cd in urine and Cu, Zn in serum were determined by Atomic Absorption Spectrophotometer (Varian AA240 FS) equipped with hollow cathode lamp. The wavelength and lamp current of Pb, Cd, Cr, Cu, Zn are 217.0 nm and 10mA, 228.8 nm and 4mA, 357.9 nm and 7mA, 324.8 nm and 4mA, 213.9 nm and 3mA, respectively. The type of flame was Air/Acetylene, the flow rate of Air and Acetylene was 13.50 L/min and 2.90 L/min, respectively. Moreover, the purity of Acetylene gas was 99.99% pure used for the flame. Replicate samples/standards, quality control standard, spike recovery, and method blanks were used to monitor the performance of the instrument and the quality of the data. The recoveries of spiked samples were from 88 to 99%.
2.4 Statistical Analysis
For statistical analysis SPSS Statistics 22.0 (IBM Corp, Armonk, NY, USA) was used, including analysis of variance, correlation analysis, and principal component analysis. Origin 2019 (OriginLab Corp, Northampton, MA, USA) was used for distribution tests and charting. The Monto Carlo simulation was performed using Crystal Ball Software (11.1 Oracle Inc., Oracle, CA, USA).
2.5 Monte Carlo Simulation
The probabilistic risk assessment of heavy metals was performed using a simulation of Monte Carlo. Weak correlation or independence between the input variables was assumed in this simulation. Input variables (Pb, Cd, Cr, Cu) were modeled as specific probability distribution functions (Table 4). To ensure the reliability of the results, 20,000 random iteractions of each input variable were carried out in each simulation. The input variables were randomly extracted from the defined probability distributions (Table 4). In this study, the 5th, 25th, 50th, 75th, and 95th percentiles for each heavy metal were extracted from the probability distribution (Fig.4).