2.1 Chemicals and reference materials
All reagents were analytical-reagent grade. Nitric acid (65%), hydrochloric acid (36%), and hydrogen peroxide (30%) were purchased from Merck (Darmstadt, Germany). For all dilutions, deionized water was used.
iTEVA software from Thermo Scientific (Cambridge, UK) was used to collect and analyze the data (Pavlović et al., 2020). Multi-element standard solution IV of the microelements Al, As, Ba, Be, B, Cd, Cr, Co, Cu, Fe, Pb, Mn, Ni, Se, Tl, V and Zn, standard solution III of the macroelements Ca, K, Mg and Na, as well as individual standard solutions of Si, P and Hg (Trace CERT, Fluka Analytical, Switzerland) were used for calibration.
The accuracy of our analytical method was determined using the Certified reference material (CRM LGC7162): K, Ca, Mg, P, Cr, Mn, Fe, Ni and Zn. The found value is reported as value ± standard deviation (SD) (Table S1). Accuracy was expressed as percentage differences between the measured concentration and the certified value to CRM (%). Method precision was evaluated as repeatability and is expressed through the relative standard deviation as a percentage (%). Differences between certified values and quantified concentrations were below 10%. The recovery values were in range of 93.6 and 106.2%. All the results are presented in Table S1.
2.2 Sample collection
The aerial parts of Satureja kitaibelii Wierzb. ex Heuff. family Lamiaceae were collected during 2020 from a natural population at the Kravlje village, southeastern Serbia at three different stages of development: vegetative stage (June, M1); flowering stage (July, M2 and August, M3); after flowering stage (September, M4; October, M5 and November, M6). The plants were collected on the fifteenth in the months mentioned. Dr. Marija Marković did identification of plant material, and the voucher specimen (accession number 13220) is deposited at the Herbarium of the Department of Biology and Ecology, Faculty of Science and Mathematics, University of Niš (Herbarium Moesiacum Niš – HMN). Basic characteristics of the locality are given in Table S2.
Seven sample locations were selected, and seven trace element contents were studied: B, Si, Cr, Mn, Ni, Cu, and Zn. The topsoil (0–20 cm) of the sample mixture, consisting of three small samples, was collected 10 m apart at each sampling point. Seven examples of S. kitaibelii in the same growth phase were taken at each sample site within 20 × 20 × 20 cm soil blocks, cut using a stainless steel spade. The soil dust and other materials in the savory samples were removed with a plastic brush, washed repeatedly with distilled water, and then stored in pre-cleaned polythene bags. The collected samples were then brought to the laboratory for further processing.
2.3. Soil sampling and preparation
Each soil sample was carefully mixed and external materials such as stones and pebbles, were extracted. The sample was then heated in an electric oven at 60°C until a constant weight was obtained. Dried soil samples are ground into fine powder. Weighted soil sample mass (1,00 g) was placed into an Erlenmeyer and treated with 16 mL mixture of conc. HCl and conc. HNO3 (3:1) (v/v). The mixture was heated to 190 oC for about an hour, then 5 mL of H2O2 (30%) were added and evaporated to a small volume. Then, it was cooled, filtered (grade 589/3 blue ribbon) and diluted with 0.5% HNO3 (in ultra-pure deionized water, 0.05 µS/cm) up to the volume of 25 mL. A blank sample was also prepared using a similar experimental procedure (Addis and Abebaw, 2017).
2.4 Plant sampling and preparation
Savory samples were dried in an electric oven at 60°C until a constant weight was obtained and then powdered. Powdered soil and savory samples were sieved through a 63 µm sieve shaker.
Digestion of plant samples was realized according to slightly modified procedure of Mosetlha et al. (2007). 1,00 g of each sample was mineralized in an Erlenmeyer flask with 15 mL of conc. HNO3, covered with a watch glass and left over-night. After that, the mixture was heated up to 150 oC and H2O2 (30%) was added. Digestion procedure was applied to obtained mixtures to reduce the volume and improve decomposition. Another portion of H2O2 was added and evaporation continued. After cooling, the mixture was filtered (grade 589/3 blue ribbon) and diluted with 0.5% HNO3 up to 25 mL. A blank sample was prepared in the same way.
All analysis was carried out on iCAP 6000 inductively coupled plasma optical emission spectrometer (Thermo Scientific, Cambridge, UK) that uses an Echelle optical design and a change injection device solid-state detector. The operating conditions for the ICP-OES instrument were: flush pump rate 100 rpm, analysis pump rate 50 rpm, RF power 1150 W, nebulizer gas flow rate 0.7 L min− 1, coolant gas flow rate 12 L min− 1, auxiliary gas flow rate 0.5 L min− 1, dual (axial/radial) viewed plasma mode and sample uptake delay 30 s.
All measurements were performed in triplicate. Parameters of conducted ICP-OES analysis based on a calibration curve: wavelength of selected emission lines, correlation coefficient (r), limit of detection (LOD) and limit of quantification (LOQ) of the calibration for each element determination are given in Table S3. The LOD and LOQ values were calculated using the 3σ and 10σ criterion (Uhrovčík 2014).
2.6 Statistical Analysis
Statistical analyses were performed with Statistica 8 (StatSoft, Tulsa) software packages. All chemical analyses were carried out in triplicate and the results were expressed as mean ± SD. To determine the statistical significance of variation of accumulation elements in plant and soil during different stages of development, student’s t-test was used. It determines whether any observed differences between the content of elements in plant and soil during different stages of development statistically significant or not. The significance of differences was defined at p < 0.05. The same software carried out hierarchical cluster analysis (HCA) and principal component analysis (PCA). Correlation and variability were made at a 95% significance level (P ≤ 0.05).