Spectro genesis

Each soil sample replicate was separated into two subsamples. One subsample was dried at 105 °C for standard chemical assays. The soil pH and electrical conductivity (EC) were measured electrometrically in two different soil–H₂O solutions (1:1, vol/vol and 1:2, vol/vol, respectively) [70 ]. The total carbon and nitrogen content were determined using dry combustion (FLASH EA1112 CHN analyzer; Thermo Fisher, Waltham, MA, USA). The available P was determined with the Olsen method [71 ]. Extractable microelements were quantified following digestion with diethylene-triamine-penta-acetic acid [72 (link)]. We determined the concentration of seven microelements: manganese (Mn), zinc (Zn), iron (Fe), chromium (Cr), cobalt (Co), copper (Cu), and nickel (Ni). The total microelement content was quantified following digestion with nitric acid [73 (link)]. The analysis was conducted using inductively coupled plasma optical emission spectrometry (Spectro Genesis; SPECTRO Analytical Instruments, Kleve, Germany). The second soil subsample was used to determine nitrate and ammonium colorimetrically, following 2 M potassium chloride extraction [74 (link)]. Nitrate and ammonium were quantified at the end of the incubation period.

Pseudo total heavy metal(loid) (As, B, Cr, Cu, Mn, Ni, Se and Zn) concentrations in FA (CFA) and soil (CSoil), and their total concentrations in root (CRoot) and leaf (CLeaf) samples were determined after wet digestion in a microwave oven (CEM, Mars 6 Microwave Acceleration Reaction System, Matthews, NC, USA) [97 ,98 ]. Their bioavailable concentrations (CDTPA) in FA (soil) were determined using Lindsay and Norvell’s method [99 (link)], while the bioavailable content of B was determined by extraction in warm water. Certified reference materials were analysed to test the accuracy of the analytical procedures: FA (ash from coal BCR—038), soil (clay ERM—CC141) and plant material (Beech leaves BCR—100), provided by the IRMM (Institute for Reference Materials and Measurements, Geel, Belgium), certified by EC-JRC (European Commission—Joint Research Centre). Chemical element concentrations (mg kg⁻¹) in the examined samples, obtained after these extractions, were determined using inductively coupled plasma optical emission spectrometry (ICP-OES, Spectro Genesis, Spectro-Analytical Instruments GmbH, Kleve, Germany). The detection limits for the analysed elements were as follows (mg kg⁻¹): As—0.005, B—0.005, Cr—0.001, Cu—0.001, Mn—0.001, Ni—0.009, Se—0.007 and Zn—0.005. The average recovery values for elements in the standard reference materials were in the range of 100 ± 20%.

Soil physicochemical characteristics (Supplementary Table 1) were determined using protocols outlined by AgriLASA (2004). Soil pH was measured using the slurry method at a 1:2.5 soil/water ratio, and the pH of the supernatant was recorded with a calibrated bench top pH meter (Crison Basic, + 20, Crison, Barcelona, Spain). The concentrations of soluble and exchangeable of sodium (Na), potassium (K), carbon (C), magnesium (Mg), and phosphorus (P) were determined using Mehlich 3 test^{32 (link)}. The extractable ion concentration was quantified using ICP-OES (Inductively Coupled Plasma Optical Emission Spectrometry, Spectro Genesis, SPECTRO Analytical Instruments GmbH & Co. KG, Germany). Soil particle size distribution (sand/silt/clay percent) was measured using the Bouyoucos method³³ . Total nitrogen (TN) and soil organic carbon (TOC) were measured using the catalyzed high temperature combustion method (Dumas method)³⁴ . The Enhanced Vegetation Index-2 (EVI2) was obtained from the NASA Land Processes Distributed Active Archive Center’s (LP DAAC) VIIRS Vegetation Indices dataset^{35 (link)} at a 500-m resolution.

The macro and microelements contents in microgreens were determined by microwave digestion technique combined with inductively coupled plasma optical emission spectrometry [48 (link),49 (link)]. Complete digestion of dry microgreen material (0.5 g) was achieved with 100% HNO₃ using microwave digestion system Multiwave GO (Anton Paar GmbH, Graz, Austria). The digestion program was as follows: 170 °C was reached within 5 min, digested for 20 min; the mineralized samples were diluted to 50 mL with deionized water; and the elemental profile was analyzed using an ICP-OES spectrometer (Spectro Genesis, SPECTRO Analytical Instruments, Kleve, Germany). The operating conditions employed for ICP-OES determination were 1300W RF power, 12 L min⁻¹ plasma flow, 1.0 L min⁻¹ auxiliary flow, 0.8 L min⁻¹ nebulizer flow, 1.0 mL min⁻¹ sample uptake rate. The analytical wavelengths (nm) chosen were: B ⁺ 249.773 nm, Ca²⁺ 445.478 nm, Cu⁺ 324.754 nm, Fe²⁺ 259.941 nm, K⁺ 766.491 nm, Mg²⁺ 279.079 nm, Mn²⁺ 259.373 nm, Na⁺ 589.592 nm, P⁺ 213.618 nm, S⁺ 182.034 nm, and Zn⁺ 213.856 nm. The calibration standards were prepared by diluting a stock multi-elemental standard solution (1000 mg L⁻¹) in 6.5% (v/v) nitric acid, and by diluting stock phosphorus and sulfur standard solutions (1000 mg L⁻¹) in deionized water. The calibration curves for all the studied elements were in the range of 0.01–400 mg L⁻¹.

Inductively coupled plasma optical emission spectrometry (ICP-OES, Spectro Genesis, Spectro-Analytical Instruments GmbH, Kleve, Germany) was used to determine PTE concentrations (mg kg^-1) in FA, soil, and plant (root and leaf) samples. Prior to analysis, samples were subjected to wet digestion in a microwave oven (CEM, Mars 6 Microwave Acceleration Reaction System, Matthews, NC, USA). The US EPA acid digestion method 3051 [U.S. Environmental Protection Agency (USEPA), 1998 ] was used to determine the pseudo total concentrations of PTEs in FA and soil samples, i.e. their maximum content that can be available to plants, as this method does not ensure complete digestion of the elements bound to silicate mineral fraction. The total content of PTEs in root and leaf samples was obtained by means of US EPA acid digestion method 3052 [U.S. Environmental Protection Agency (USEPA), 1996 ]. The detection limits for the analysed PTEs were as follows: As - 0.005, B - 0.005, Cr - 0.001, Cu - 0.001, Mn - 0.001, Ni - 0.009, Se - 0.007, and Zn - 0.005. To verify the accuracy of the analytical procedures, certified reference materials were analysed: FA (coal ash BCR - 038), soil (clay ERM - CC141) and plant material (beech leaves BCR - 100) provided by IRMM (Institute for Reference Materials and Measurements, Geel, Belgium) and certified by EC-JRC (European Commission - Joint Research Centre).

Analysis of soil samples was conducted at the Soil Science Laboratory of the University of Pretoria, South Africa in accordance with the standard procedures (SSSA, 1996). Prior to analysis, soil samples were sieved (2 mm) and dried overnight at 50 °C.
The slurry technique was used to measure pH (1:3 soil/deionised water) with a Crison Bench pH meter (Crison Instruments, Barcelona, Spain) after allowing soil to settle for 30 min. Soil N-NO₃^-, N-NH₄⁺ and cation exchange capacity (CEC) were determined by extraction (2M KCl) with subsequent titration. Total P was measured using the P Bray method and total C was measured using the Walkley-Black method. Ammonium acetate extraction was used to measure salt concentrations (K⁺, Ca²⁺, Mg²⁺, Na⁺), analysed using inductively coupled plasma atomic emission spectroscopy (ICP-OES; Spectro Genesis, Spectro Analytical Instruments GmbH, Germany). Moisture content was determined by oven drying 10 g of soil at 60 °C for 48 h and comparing the weight of soil pre- and post-drying.