## 4 Discussion  
The formulation of nanosystems for a multifunctional coating applicable as a preventive tool against the colonization of microorganisms on stone monuments resulted in a real improvement of chemical treatments with biocides [7; 49; 50; 51; 52]. As previously stressed in the literature, the multifunctional coating in relation to their formulation and functionality can condition the release of antifouling products loaded in nanosystems [11; 13; 18; 21; 53; 54]. In this way, it is therefore important as a first step to analyze the biocide efficacy in a controlled environment before incorporating the nanosystems into the coatings. Our study showed that both the biocide chemical composition and the differential nanostructures can give rise to a differential efficiency in the antifouling activity against green algae. Indeed, the presence of CTAB (cetyl trimethyl ammonium bromide) as a surfactant gives nanocontainers an unequivocal biocidal activity against algal cells. This result could be expected because this product is commonly used in DNA extraction protocol  
Figure 4: Comparative evaluation between the fluorescence of accessory pigments (green) and the chlorophyll (red) of the nanocontainers not loaded and loaded with biocides. The changing of the cells’ fluorescence from red to green and the successive complete disappearance highlights the differential efficiency of products. for plants [55]; the negative effect against algal cells seems coherent. The presence of CTAB explains the antifouling activity of the nanosystems in the absence of further biocidal compounds. However, CTAB is not used as a free biocide due to its toxicity and the possibility of it being released into the environment when directly applied on stone surfaces [56]. According to the EU Regulation [57], CTAB is a cetrimonium bromide (C\({}_{19}\)H\({}_{42}\)BrN) and it is classified in the safety data sheet as toxic for the environment (very toxic to aquatic life with long-lasting effects, Risk Category 1), and for restorer (if ingested and inhaled, Risk Category 4, or irritating to skin or mucous membranes, Risk Category 2/1), but in the same way, it is completely biodegradable both in water and in soil. Moreover, it does not bioaccumulate in organisms. Furthermore, in this context, CTAB is used in the synthesis procedure of nanocontainers and the amount in excess is removed by centrifugation and the application of multifunctional coatings containing the nanocontainers prevents its release into the environment [19; 56; 58]. Therefore, in view of an in situ application, even if residue should remain after washing, its quantity and its entrapment inside the coating would not make it toxic for the environment [19; 56; 57; 58]. Furthermore, our results highlighted that the biocides used by direct applications (Figure 2), although in much higher quantities than in encapsulated biocide (Figure 3b), showed lower antifouling activity. In particular, ZS as a free biocide did not show any relevant biocidal activity against the algal cells at the experimented doses. Noteworthy, these data, which are the first on biocidal efficiency against algal cells, showed different results concerning previously reported data on biocidal efficiency against bacterial [37; 59; 60; 61] and fungal species [61; 62], such as in the case of encapsulated product [63; 64]. Despite the promising results of previously detected microorganisms, ZS did not show a broad spectrum of biocidal efficiency. Such data can be related to the mechanisms of biocidal activity of this compound. Indeed, the previous studies demonstrate that this compound does not act by killing the microorganisms but by inhibiting their adhesion, blocking the interaction of the microorganisms with the surface [61; 63; 64; 65]. Furthermore, the different efficiency between NC for the MNP systems highlighted that the second one presents higher efficiency (Figure 3b). This aspect seems to be related to the structure of the nanocontainers [21; 22].