Eicchornia crassipes was collected in the month on March 2018 from three different locations across Pune district, viz, Pashan Lake, Mula river, Mutha river, Pune, India. The samples were authenticated by department of pharmacognosy, Poona College of Pharmacy, India. The leaves, petioles and roots of the plant were segregated and set to dry under shade for 15 days until constant weight of the dry mass was obtained. The dried samples were then ground to fine powder.

The samples were separately weighed, following which they were subjected to microwave digestion (Mars Express Vessel) according to the parameters given in Table 1.

For digestion, 0.5 g of sample was taken into a digestion vessel to which 3 mL of nitric acid (HNO₃ 69% Suprapure grade), 7 mL of hydrochloric acid (HCL 30% Suprapure grade) was added. Blank solutions were prepared similarly. Digested samples were transferred to falcon tubes and diluted with acidified water to a volume of 50 mL. The resulting solutions were subjected to Inductively Coupled Optical Emission Spectroscopy (ICP-OES) and Inductively coupled Plasma Mass Spectrometry (ICP-MS) analysis.

For Iodine determination, 0.5 g of the sample was digested in basic medium by taking 1 mL tetra methyl ammonium hydroxide (TMAH) to which 5 mL of water was added. The solution was subjected to centrifugation, after which it was kept in oven for 2 h at 80℃. The final volume was made up to 50 mL water.

Stock solutions containing 1,000 mg/L of Perkin Elmer Pure was used to prepare different custom-grade multielement standard solutions for Co, I, Se, Ni, Cu, Mn, Zn, Fe by diluting with 50 mL acidified water. Calibration curves for each element were constructed in triplicate using six different concentration. An internal-standard stock solution containing 100 µg/L of rhodium (Rh) was prepared from single-element stock solutions. NIST Standard reference material SRM 1547 (Peach leaves) was used for establishing accuracy of the analytical method. Suprapure grade nitric acid and hydrochloric acid were used for preparing stock solutions of 100 µg/mL and 10 µg/mL for ICP-OES and ICP-MS respectively.

Initially, the sample digests were subjected to a semi-quantitative analysis which allowed the establishment of the range of concentrations of the elements in the various digestion solutions. The error associated with the semiquantitative analysis was calculated to approximately 25%. Based on the results obtained, the elements were divided into two groups depending on the determined concentrations. Group 1 elements (Co, Ni, Cu, I) were determined by ICP-MS (Agilent 7700 series) and group 2 elements (Mn, Zn, Fe) were subjected to ICP-OES (Thermo Fisher Scientific, Bremen Germany, i CAP 6300) analysis. The quantitative determinations were carried out by a calibration curve using multielement standard solutions in which the concentrations of the elements were in an optimal measurement range.

After careful evaluation of all possible isotopes, their abundances, potential interferences (meaningful in the analysis of plants) and the corrections introduced, isotopes selected for quantitative estimation of the respective elements were; ⁵⁹Co, ¹²⁷I, ⁶⁰Ni, ⁶⁶Cu. Pure single element standard solutions of the interfering elements were used to establish the actual interferences on all isotopes under investigation.

The corrections were calculated [12] according to Eq. (1).

Where S_cor is the corrected signal of the analyte; S_meas is the measured signal; S_inter is the signal of the interfering element and A is the % of formation of the respective interfering species determined in the solution of the interfering element at the working conditions specified in Table 2 and Table 3. S_inter is measured in each sample and the isotope abundance is accounted for.

The concentration values of Co, I, Se, Ni, Cu, Mn, Zn, Fe in various parts like roots, leaves and petiole as expressed in mg/Kg are given in Table 4. The total range of concentration of elements was found as low as 0.94 mg/Kg for I and as high as 566 mg/Kg for Mn. The order of overall mean concentration of trace elements in Eichhornia crassipes was found as Mn > Fe > Zn > Cu > Ni > Co > I. Roots of the plant are in direct contact with the surrounding water and as a result they form the principle part of element uptake. Concentration of all measured elements was found to highest in roots. The concentration of Ni, Cu, Mn, was found to be highest in Roots > Petiol > Leaves. Whereas, the concentration of Zn and Fe was found to be highest in roots followed by leaves and petiole. This finding suggests alteration in transport mechanism or storage process involved with Zn and Fe. More detailed studies involving 2D-element mapping are necessary to evaluate the transport and storage mechanism of these elements in Eichhornia crassipes. Concentration of Co was high in roots but in petiole and leaves it was almost similar. An interesting finding was the occurrence of measurable quantities of Iodine in all the parts of the plant despite its non marine source. Occurrence of measureable Iodine in ecosystems far from seashores is not a rare phenomenon. Although high levels of iodine invariably occur in vicinity of costal zones, nevertheless, there is no simple correlation between distance from shoreline and content of iodine in a sample. Among many factors that influence the level of iodine in a sample, supply of iodine, sample’s iodine fixation potential and adsorption of atmospheric iodine on plant’s surface are some important contributors [13]. This data shows that amongst all measured elements, Eichhornia crassipes is exhibited highest bioaccumulation for Mn. This finding is in agreement with another similar study done by Olutona et al. [14].

Recovery value of an analytical method, is a parameter that signifies accuracy of the adopted analytical process. Accuracy is defined as the closeness between the true value of the analyte in the sample and the experimentally obtained value by following the prescribed analytical procedure. A good recovery value ensures that no significant loss or contamination has occurred during the test procedure.

To establish accuracy of the method, certified reference standard SRM 1547 (Peach leaves) was measured and concentration of the desired elements were determined using both ICP-MS and ICP-OES. Good agreement between certified values and measured values was observed (Table 6). This demonstrates the appropriateness of the proposed method for applicability in routine element determination in plant samples.

Recovery values for the desired elements were also calculated in the digested plant samples of leaves, roots and petiole after spiking with 100% of the target limit by standard element solution.

The recovery values obtained for all the measured elements in different parts of Eichhornia crassipes, are shown in Table 7. Recovery values obtained were in between 95% to 101%.

The calibration curves for all the analytes were built on six different concentrations and LOD and LOQ values were determined.

The limits of detection (LOD) were calculated by taking three times the standard deviation of the 10 individually prepared blank solutions.

The limits of quantification (LOQ), were calculated by taking ten times the standard deviation of the 10 individually prepared blank solutions [8]. LOD and LOQ values for the measured elements are given in Table 5.

The correlation coefficients were calculated for the calibration curves for all the seven trace elements on six standard replicates for six different concentrations (Fig. 1). Concentration used for determining linearity were between 10% to 120% of the target limit for each element in the final analysis solution. R² values for the individual elements are shown in Table 5. Good correlation was obtained for all the measured elements. Observed R² values were in the range of 0.9976 to 1.