## 4 Discussion
### Tolerance to Biocides  
Despite the significant impact of the green revolution in the biocides market [92], traditional biocidal products are still widely used [93]. This is due to the consolidated protocols used on different materials, the wide knowledge on the pros and cons of their use, and the non-negligible economic aspects. In situ trials are often used in restoration practice to customize treatments as they confidently reproduce, in a short time, the interaction of biocides with biota and materials, and provide a more reliable indication of the in situ persistence of the biocide [94; 95; 96; 97]. However, several factors can negatively affect the efficiency of any restoration protocol. The metabolic state of the organism is important for their responsiveness to biocides treatments. Metabolically active colonies are, indeed, more sensitive to chemical and physical stresses [98]. BF, being pokilohydric organisms, withstand the unfavorable conditions in a dormant state mainly induced by desiccation (anhydrobiosis) [12; 99]. In addition, fungi growing in stone cracks and fissures may be not easily reached by biocides applied on a monument surface during restoration treatments. Even the typical clump-like BF colonies, ensuring the optimal surface-volume ratio, minimizes the direct exposure to external stressors, including biocides; moreover, external cells in the colony shield the inner layers from external injuries (shadow effect) [100]. Conversely ADT plate assays have an undeniable advantage as they reproduce the best conditions to record the sensitivity to biocides of single metabolically active cells in a medium that cannot seize the biocide as stones frequently do. For these reasons, the tolerance/resistance recorded in the most responsive condition should warn about their possible survival after treatments, as well as their spread. The used biocides have different formulations, mechanisms of action, and thus different outcomes. BZC (CAS No 8001-54-5) is a quaternary ammonium compound (QAC) routinely used for chemical antisepsis and disinfection and widely applied in cultural heritage preservation treatments. According to the concentration used, BZC acts as a highly active detergent causing cell membrane permeability alterations, irreversible damage to the barrier function and proteins denaturation [101]. Benzalkonium chloride is also the only active compound in the PREV patent, albeit in a lower concentration (50%) than in BZC (90%). Despite the lower concentration of QAC salt in PREV, the inhibition halos produced were frequently larger than BZC, such as for _Sal. thailandicus_ CCFEE 5723, _K. petricola_ CCFEE 5776, and _S. tyrhenica_ CCFEE 5935. This apparent incongruence, already evidenced in previous studies [36], has been explained with the possible different composition in terms of n-alkyl groups length and/or degree of C-C saturation in the two chemicals [102], and by additives, not specified in the composition of biocides but included in the patent of the commercial formulas [21; 103]. The other two biocides tested have instead intracellular targets. As a matter of fact, \(N\),\(N\)-dimethyl-\(N^{\prime}\)-phenyl-\(N^{\prime}\)-(fluoro-dichloromethylthio)-sulphamide (CAS No 1085-98-9), the active principle of LICH, is supposed to affect a number of enzymes by reacting with -SH bonds [104]. While the fungicidal and antimicrobial action played by IPBC (3-iodo-2-propynyl butyl carbamate, CAS No 55406-53-6) and OIT (2-\(n\)-octyl-4-isothiazolin-3-one; CAS No 26530-20-1), the active principles of BioR, are probably due to iodine toxicity, and inhibition of cell respiration/ATP production, respectively [105; 106]. The achieved results evidenced an increasing biocidal power, in terms of inhibition halos and number of strains completely inhibited, as follows: BZC-PREV, LICH, and BioR. The thick multilayered and strongly melanized cell wall typical of these fungi serve as an efficient barrier for toxic substances with positive influence on resistance.