Ethos one

Soil samples of about 0.5 g were digested with 2 mL HCl, 6 mL HNO₃, and 2 mL HF in a microwave digestion system (Milestone ETHOS ONE, Milestone, Milan, Italy). Then, inductively coupled plasma–mass spectrometry (ICP–MS, Agilent, Santa Clara, CA, USA) was used to determine the content of heavy metals (Mo, Cd, Sb, Cu, Zn, Hg, and Pb). Quality assurance/control procedures were performed using standardized reference materials (National Institute of Metrology, Beijing, China) for each batch of samples (one blank and one standard).

The one fully matured fruiting body of Neoboletus luridiformis (Rostk.) Gelardi, Simonini & Vizzini 2014 was sampled and prepared as described in detail in our previous paper [17 (link)] that also includes the basic characteristics of Neoboletus luridiformis (Rostk.) and the sampling location. We chose this species due to its belonging to the Boletaceae family, which includes a wide group of commonly collected and consumed species. The representatives of the family are known for their relatively high bioaccumulation properties and tubular hymenophore.
In total, 17 elements divided into 4 separate groups of elements: Major Essential Elements – MEEs (Ca, K, Mg, and Na), Essential Trace Elements – ETEs (Cr, Cu, Fe, Mn, Ni, Se, and Zn), Trace Elements with Detrimental Health Effect – TEWDHE (Ag, Ba, Cd, Pb) and Nutritionally Nonessential Elements – NNEs (Al and Sr) determined by ICP OES. The detailed preparation of the 101 partial samples of the fruiting body is presented in Fig. 1.
Fig. 1

The N. luridiformis fruiting body cut to determine the spatial distribution of elements with the sampling points (raster 1 × 1 cm)

Concentrated (67%) nitric acid (Honeywell, Saint-Germain-en-Laye, France) and 30% hydrogen peroxide (Supelco Inc., Bellefonte, Pennsylvania, USA) of trace purity were used to analyze the content of the studied elements in mushrooms. Approximately 150 mg of a dried and homogenized partial sample was weighed to four decimal places on analytical balances KERN ABT 120-5DM (KERN & Sohn GmbH, Balinger, Germany) into PTFE digestion tubes, where 8 mL of HNO₃ and 2 mL of H₂O₂ were added. The samples were then prepared in a closed microwave digestion system ETHOS-One (Milestone, s.r.l., Sorisole BG, Italy). The process of digestion was carried out according to the following program: (I) 0–15 min.: increase to 200 °C (pressure increase to 40 Bar); (II) 15–30 min.: temperature fixed at 200 °C (40 Bar, 1.8 kW); (III) 30–50 min.: Cooling. After microwave digestion, the samples were filtered through qualitative filter paper, Grade 595 with a porosity of 4–7 μm (Whatman, Maidstone, UK) into volumetric flasks and filled up to the final volume of 50 mL with deionized water using a LabAqua HPLC device (Biosan, Riga, Latvia). The analysis of the elemental content and quality assurance were carried out on the instrument Agilent ICP OES 720 (Agilent Technologies Inc., Santa Clara, CA, USA) coupled with the autosampler SPS 3 (Agilent Technologies Ltd., Malaysia) according to Shah et al. [18 (link)] and shown in the Table 1. All determined element contents in the text are expressed as mg/kg on dry weight basis (DW).
Table 1

ICP OES determination parameters

Elements	Wavelength[nm]	LoD[µg/L]	LoQ[µg/L]	R
Ag	328.068	0.30	0.99	0.999913
Al	167.019	0.20	0.66	0.999749
Ba	455.403	0.03	0.10	0.999713
Ca	315.887	0.01	0.03	0.999810
Cd	226.502	0.05	0.17	0.999938
Cr	267.716	0.15	0.50	0.999908
Cu	324.754	0.30	0.99	0.999941
Fe	234.350	0.09	0.33	0.999702
K	766.491	0.30	0.99	0.999411
Mg	383.829	0.01	0.03	0.999836
Mn	257.610	0.03	0.10	0.999784
Na	589.592	0.15	0.50	0.999343
Ni	231.604	0.30	0.99	0.999816
Pb	220.353	0.80	2.64	0.999850
Se	196.026	2.00	6.61	0.999974
Sr	407.771	0.01	0.03	0.999490
Zn	206.200	0.20	0.67	0.999881

LoD: limits of detection; LoQ: limit of quantification; R: correlation coefficient of calibration curves

The oven-dried OP biomass and hydrochar were digested using a mixture of HNO₃ and H₂O₂ at 140 °C, 15 min holding time, and 1000 W energy in a microwave digestion system (ETHOS One, Milestone, Sorisole, Italy) to extract metals and their quantification by ICP-OES analysis. The metal oxides were measured by multiplying the total metal contents with conversion factors [42 ]. A variety of empirical indicators, including the alkali index, base-to-acid ratio, slagging index, fouling index, slag viscosity index, and bed agglomeration index, were used to assess the slagging and fouling risks of raw OP biomass and hydrochars [43 (link),44 (link)]. Table 1 shows the procedures used to compute the slagging and fouling indices. The alkali index (AI) of a fuel is an indicator of slagging–fouling propensities, which was estimated using alkali oxides per heat unit (Kg·GJ⁻¹). The base-to-acid ratio (B/A) reflects the ash fusion temperature and slag viscosity. It was calculated based on the concentrations of the basic (Fe₂O₃, CaO, MgO, Na₂O, K₂O, and P₂O₅) and acidic (SiO₂, Al₂O₃, and TiO₂) minerals. The slagging index (SI) forecasts the propensity for fused slag deposition on furnace walls by considering the sulfur concentration and base-to-acid ratio. The tendency for corrosive alkali minerals to build on the furnace walls is predicted by the fouling index (FI). The slag viscosity index (SVI) predicts the slagging tendency of the metal oxides by considering the silica concentration and the probable creation of metal silicates with low melting temperatures. Metal oxide agglomeration in combustion furnaces is forecasted by the bed agglomeration index (BAI). A higher risk of bed agglomeration is indicated by a fuel BAI value of less than 0.15 [43 (link)].

After 5 days treatment with 0 (control) or 100 μM Cd, Salvia plants were harvested, separated in roots and aboveground (shoots-leaves) tissues, washed three times in deionized water, and then dried at 65 °C to constant biomass, milled and finally sieved. Dried sieved samples of 0.3 g were transferred in 10 mL quartz vessels with 65% (v/v) nitric acid (Suprapur, Merck, Darmstadt, Germany) and 30% (v/v) hydrogen peroxide (Suprapur, Merck, Darmstadt, Germany) in 3:1 ratio. Digestion was carried out in the microwave assisted digestion system Ethos One (Milestone Srl, Sorisole, BG, Italy). The process run out in 3 stages: ramp time—20 min to reach 200 °C and 1500 W; hold time—30 min at 200 °C and 1500 W; cooling—30 min. The next step was the quantitative transfer of digested samples into polypropylene tubes and dilution with demineralized water (Direct-Q 3 UV, Merck, Darmstadt, Germany). All prepared samples were diluted immediately prior to inductively coupled plasma mass spectrometer (ICP-MS) analysis. Samples were analyzed in an ICP-MS model ELAN DRC II (PerkinElmer Sciex, Toronto, Canada) [127 (link)]. ICP-MS operational conditions, instrumental settings calibration solutions, data validation, and validation parameters are given in Appendix A. Elemental analysis was performed for Cd, Cu, Ca, Mg, Mn, Fe, and Zn.

Before digestion, the samples were dried in 65 °C for 12 h using an electrically heated laboratory oven. Then, subsamples of dried and powdered mushrooms (∼0.5000 g) were digested with 5 mL of 65% HNO₃ (Suprapure, Merck, Germany) under pressure in a model Ethos One (Milestone Srl, Italy) microwave oven. The rotor body holds up to ten digestion vessels made of high-purity TFM™ polytetrafluoroethylene (PTFE) with capacity of 50-mL per vessel. The heating program was performed in one step: the power of the process was 1500 W, ramp time 20 min, temperature 200 °C, and hold time 30 min. Blank reagent solutions were prepared in the same way. For every set of ten digested mushroom samples, two blank samples were run. The digests were diluted to 10 mL using deionized water (TKA Smart2Pure, Niederelbert, Germany) in volumetric flasks. If this was necessary, a specific digest was further diluted (about ten times) to keep a linearity of measurements or acid concentration below 3%.