Study area
Geographically, Uddanam is located at 19.0167 degrees north latitude and 84.6833 degrees east longitude, with an average elevation of 41 meters above sea level. It consists of 7 revenue Mandals (Ichapuram, Kanchili, Kaviti, Mandasa, Palasa, Sompeta, and Vajrapukotturu). We have chosen Kaviti revenue Mandal because of high prevalence of CKD. We selected three revenue villages out of 21 revenue Villages in Kaviti Mandal based on the patients number and data obtained from the Community Hospital Sompeta. These three villages are Kusmapuram (L1), Baraka (L2) and Bolivanka (L3) (Figure 1-A, B, C, D).
Sample collection and Preparation
We identified six locally cultivated vegetables for Trace Elemental and Heavy Metal analysis (Table 1), which are grown in their Coconut orchards as inter crops and are frequently consumed. In which 2 are belong to the family Solanaceae, and one each from Caricaceae, Cucurbitaceae, Moringaceae and Musaceae (Table 1).
Table 1. Frequently consumed local Vegetables used in the study
Sample No.
|
Botanical Name
|
Family
|
English name
|
Vernacular name
|
S1
S2
S3
S4
S5
S6
|
Capsicum frutescence L.
Carica papaya L.
Momordica charantia L.
Moringa oliefera Lam.
Musa sapientum L.
Solanum melongena L.
|
Solanaceae
Caricaceae
Cucurbitaceae
Moringaceae
Musaceae
Solanaceae
|
Chilli
Papaya
Bitter gourd
Drumsticks
Plantain
Brinjal
|
Pachchi mirapa kaya
Boppayi kaya
Kakara kaya
Munaga Kada
Arati kaya
Vanga kaya
|
The fresh samples of the selected vegetables collected from the fields directly, from the three Villages are kept in zip locked bags and are brought to the laboratory. The samples are washed twice with distilled water and then subjected to Acid Digestion process, i.e. fast and accurate Wet Digestion method [19] is followed for the digestion of samples to digest all organic matter in the samples and placed in ICP-MS to obtain the Trace Elemental levels.
Procedure
We used the ICP-MS instrument (Agilent Technologies- Model 7700) available in Centre for Studies on Bay of Bengal, Andhra University, Visakhapatnam. The Sample preparation for ICP-MS analysis followed here is based on the oxidation and solubility of organic matter in plants when H2O2 (33%) is added directly to the concentrated HNO3 and heated for a short time. The instrument was switched on and is first run with the Standard Reference Materials of 0 PPB, 0.5 PPB, 1 PPB, 5 PPB, 10 PPB, 25 PPB, 50 PPB, and 100 PPB then ran the samples and obtained the levels of 24 Elements (Li, Be, Al, Sc, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, As, Se, Rb, Sr, Ag, Cd, Cs, Ba, TI, Rb, and U).
ICP-MS instrument (Agilent Technologies - Model 7700) available at the Center for studies on Bay of Bengal, Andhra University, Visakhapatnam was used for analysis. The following sample preparation for ICP-MS analysis is based on the oxidation and solubility of organic matter in plants when H2O2 (33%) is added directly to concentrated HNO3 and heated for short time. First we turn on the instrument and run with standard reference materials of 0 PPB, 0.5 PPB, 1 PPB, 5 PPB, 10 PPB, 25 PPB, 50 PPB, and 100 PPB, then followed by samples to find 24 Elements. (Li, Be, Al, Sc, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, As, Se, Rb, Sr, Ag, Cd, Cs, Ba, TI, Rb and U).
Wet Digestion
Modified HNO3/H2O2 wet digestion method (Pequerul. et al. 1993) [19] was followed. 2 gr. sample is taken, kept in a Teflon beaker, mixed with 8 ml HNO3 and left overnight. Heat the solution on a hot plate at 120-125 °C for 1 hour and add 4 mL H2O2 (33%) once or twice until the organic matter completely digested. Then we added H2O2 until the solution is clear or colorless (indicating complete digestion of organic matter). Place the residue in a dry place at 80° C., shake well, cool to room temperature and filter through Whattman filter paper. The solution is then diluted to a known volume (50 mL) with Milli-Q water. These digested solutions are stored in high-density polyethylene bottles (100 mL) at room temperature for further use.