# Fungi and Arsenic: Tolerance and Bioaccumulation by Soil Saprotrophic Species
## Materials and Methods
### Elemental Chemical Analysis of the Tested Species' Fungal Biomass  
Concentrations of 18 elements (As, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, P, Pb, S, Sb, Sn, and Sr) in the fungal biomass of _A. spinosa, C. nanum, M. marquandii_, and _P. illacium_ were analyzed to evaluate the different accumulation of the elements in the four tested strains grown on MEA and CDA media (control) and on MEA and CDA media added with 10 mg/L, 20 mg/L, and 50 mg/L of sodium arsenite. All treatments, sample preparation, and chemical analyses were carried out in quadruplicate. Four samples for each of the four fungal species subjected to the eight different treatments (MEA and CDA media added or not with 10 mg/L, 20 mg/L, and 50 mg/L of sodium arsenite), for a total of 128 samples, were prepared and chemically analyzed in accordance with the following procedure. Each sample was transferred to a quartz vessel and subjected to microwave assisted acid digestion (Ethos Touch Control with Q20 rotor, Milestone, Bergamo, Italy) for 30 min, using 2 mL of ultrapure concentrated HNO\({}_{3}\) (assay \(>\) 67%; residue \(<\) 1 mg/L) and 1 mL of H\({}_{2}\)O\({}_{2}\) (assay \(>\) 30%; residue \(<\) 20 mg/L); HNO\({}_{3}\) and H\({}_{2}\)O\({}_{2}\) were high purity solvents for trace analysis and were purchased from Promochem, LGC Standards GmbH (Wesel, Germany). The vessel was irradiated with a maximum power of 1000 W and microwave digestion was carried out at maximum temperature (180 \({}^{\circ}\)C) and pressures \(\leq\) 40 bar. The digested solution was then diluted to 50 mL with deionized water (Arioso UP 900 Integrate Water Purification System) and filtered through syringe filters (25 mm diameter, 0.45 \(\upmu\)m pore size, GVS Filter Technology, Morecambe, UK). Concentrations of Ca, Fe, K, Mg, Na, P, and S were determined by inductively coupled plasma optical emission spectrometry (ICP-OES; Vista MPX CCD Simultaneous; Varian, Victoria, Mulgrave, Australia) in axial view mode and equipped with a cyclonic spray chamber. Determination of Cd, Co, Cr, Cu, Mn, Ni, Pb, Sb, Sn, and Sr concentrations was performed by quadrupole inductively coupled plasma mass spectrometer (ICP-MS; model 820-MS; Bruker, Bremen, Germany) equipped with a glass nebulizer (0.4 mL/min; Analytik Jena AG, Jena, Germany). Overall, ICP-OES was used for the determination of elements present in higher concentrations, while ICP-MS was used for the analysis of trace and ultra-trace elements. For both ICP-OES and ICP-MS determinations, external standard calibration curves were performed by serially diluting multi-element standard stock solutions (1000 \(\pm\) 2 mg/L; Exaxol Italia Chemical Manufacturers Srl, Genoa, Italy). To control the nebulizer efficiency, rhodium was set at 5 \(\upmu\)g/L as the internal standard for all of the measurements and was prepared from standard stock solutions (1000 \(\pm\) 2 mg/L; Panreac Quimica, Barcelona, Spain). For ICP-MS analysis, a standard solution containing 5 \(\upmu\)g/L of Ba, Be, Ce, Co, In, Pb, Mg, Tl, and Th was prepared daily in 1% HNO\({}_{3}\) from a multi-standard stock solution (10.00 \(\pm\) 0.05 mg/L; Spectro Pure, Ricca Chemical Company, Arlington, TX, USA) to select the best ICP-MS performance. Further details about the ICP-OES and ICP-MS conditions are reported in [PERSON] et al. (2014, 2015) and in [PERSON] et al. (2017), respectively. Atomic fluorescence spectrometry with hydride generation (HG-AFS; 8220 Titan HG-AFS, FullTech Instruments, Rome, Italy) was used for the determination of the total As concentration in fungal biomass, since this technique enables avoiding possible interferences in As determination by ICP-MS due to Cl cluster formation and has a higher sensitivity for As with respect to ICP-OES, thus allowing a higher analytical quality of the data to be obtained ([PERSON], 2015; [PERSON], 2015).