# Fungi and Arsenic: Tolerance and Bioaccumulation by Soil Saprotrophic Species
## Materials and Methods  
### Screening of Fungal Strains for Arsenic Tolerance and Accumulation  
Four strains of soil saprotrophic fungi, previously isolated from environments with high natural concentrations of arsenic and currently preserved at the culture collection of the Fungal Biodiversity Laboratory (FBL) (Sapienza, University of Rome), were screened to evaluate growth responses and assess As tolerance and As bioaccumulation. The tested strains, belonging to the Mucoromycota and Ascomycota phyla, were _Absidia spinosa_ (FBL 69), _Cephalotrichum nanum_ (FBL 73), _Metarhizium marquandii_ (FBL 484), and _Purpureocillium lilacinum_ (FBL 478). _A. spinosa_ and _C. nanum_ were isolated from different soils of the geothermal area of Travale-Radicondoli, characterized by high concentrations of potentially toxic elements including As [42], while _P. illacinum_ and _M. marquandii_ were isolated from the soils of Latin Valley, which are characterized by high background concentrations of As [43]. The strains were reactivated and maintained at 25 \({}^{\circ}\)C in the dark on Malt Extract Agar (MEA), prepared according to the following composition (g/L in distilled water): malt extract, 20; peptone, 1; glucose, 20; agar, 20. All components were purchased from Becton Dickinson (Sparks, MD, USA). Prior to the experiments 7-day old stock cultures of the fungal strains were established. Tolerance screenings to sodium arsenite (NaAsO\({}_{2}\)) (assay \(\geq\) 90%; antimony (Sb) \(\leq\) 0.5%; Sigma-Aldrich) were carried out in Petri plates containing solid culture medium supplemented with a sodium arsenite solution and mixed to homogenize before solidification. In order to evaluate the influence of nutritional conditions on the strains' ability to tolerate and accumulate arsenic, the plate test was performed on two cultural media, Malt Extract Agar (MEA) and Czapek-Dox Agar (CDA). The composition of CDA was as follows (g/L distilled water): sucrose, 30; NaNO\({}_{3}\), 3; K\({}_{2}\)HPO\({}_{4}\), 1; MgSO\({}_{4}\)-7H\({}_{2}\)O, 0.5; KCl, 0.5; and FeSO\({}_{4}\)-7H\({}_{2}\)O, 0.01. All chemicals were purchased from Merck (Darmstadt, Germany). The pH of CDA was adjusted to 5.8 by introducing HCl, while MEA pH was left unmodified (pH 5.4). To assess a possible dose-dependent response, three different concentrations 10 mg/L, 20 mg/L, and 50 mg/L of NaAsO\({}_{2}\) were tested, which were equivalent to 5.8 mg/L, 11.5 mg/L, and 28.8 mg/L of As(III), respectively. The concentrations were selected considering the As concentrations tested in previous papers such as [PERSON] et al. [44] and the environmental As concentrations of the soils from which the fungi were isolated. Controls and chemical blanks were also set up. All assays were carried out in quadruplicate. Growth responses and fungal tolerance to As were investigated by tolerance indexes based on the growth data of diametric extension and dry weights. Two indexes were calculated: Rt:Rc (%) defined as the ratio of the colony extension rates in the presence (Rt) or absence (Rc) of NaAsO\({}_{2}\) and the Tolerance Index (T.I.) based on the dry weights (DW) of fungal biomass (T.I. (%) = (DW of treated mycelium/DW of control mycelium) \(\times\) 100) [23]. To facilitate the recovery of the mycelium, sterile cellophane membranes, allowing the passage of nutrients and metabolites between the medium and the colony, were placed on the surface of the culture medium [22]. Inoculation was carried out placing at the center of the plate a 6-mm-diameter plug taken from the actively growing margin of the stock cultures using a sterile cork borer. During the seven days of incubation, measures of diametric extension were carried out daily. After incubation for seven days at 25 \({}^{\circ}\)C in the dark, using a sterile razor blade,the mycelia were recovered from the membrane and oven dried at 100 \({}^{\circ}\)C until reaching a constant weight for at least two days.