Minimal Bactericidal Concentration

MBC was determined after broth microdilution by subculturing the content of each well that did not show any visible signs of growth on the surface of non-selective agar plates (Muller-Hinton agar, MHa, Merck, Darmstadt, Germany). This allowed for the determination of the number of surviving cells (CFU/mL) after 24 h of incubation at 37 ◦C [45].

#### Time-Kill Test

Portions of more concentrated bacterial strain suspensions (107 CFU/mL), prepared as previously described, were treated with the studied substances at 2× MIC concentration for solutions with MIC values established and 50% concentration for the others and kept in direct contact for 2 h and 24 h, respectively, at 37 ◦C. At each established time, portions of bacterial cultures were serial diluted in PBS and then plated on Muller Hinton agar medium (MHa). After incubation at 37 ◦C for 24 h, bacterial survival was evaluated [51].

#### Efficacy of Biological Contaminant Removal from the Targeted Surfaces

The efficacy of micro-organism removal from the surface was determined by calculating the decontamination factor achieved by polymeric nanocomposite films after exfoliation. Sterile surfaces (sterile Petri dishes) were contaminated with known concentrations of micro-organisms. Two micro-organisms were applied separately: *Staphylococcus aureus* (ATCC 6538) and *Escherichia coli* (ATCC 8739). The contaminated plates were dried, and then the polymer solutions were applied. The plates were kept under airflow at room temperature. After 24 h, the formed polymeric films were exfoliated. After this, the culture media was applied by spreading on the decontaminated surfaces of Petri dishes. The plates were incubated at 37 ◦C for 24 h. After the incubation period, the number of CFUs was

counted. DF was calculated by considering the initial contamination and the number of CFUs counted after the incubation period.

#### 2.3.2. Chemical Warfare Agent Decontamination Tests

Chemical decontamination using one real warfare agent, sulfur mustard (Yperite or **HD**), and one simulant for nerve agents, dimethyl methylphosphonate (**DMMP**), was performed as follows: firstly, controlled contamination was performed, followed by the application of the decontamination solution on the contaminated surface (after 5 min from contamination); following the film-curing process (20–24 h), the nanocomposite films containing the degradation products of **HD** and **DMMP** were peeled off and subjected to extraction in DCM, followed by GC-MS analysis.

In the first step (controlled contamination, 10 g(toxic)/m2), some metallic probes, measuring 10 cm2, were contaminated with 7.87 μL (10 mg) of **HD** (ρ = 1.27 g/cm3) and other metallic probes, also measuring 10 cm2, were contaminated with 8.61 μL (10 mg) of **DMMP** (ρ = 1.145 g/cm3). After 5 min of toxic-metallic probe direct contact, approximately 1.5 mL of decontamination solution was poured over the contaminated area, making sure the surface was completely covered by the liquid. Once the decontamination solution was placed on the contaminated surface, the active ingredients set up the degradation of the toxic agent while being adsorbed by bentonite nanoclay and entrapped in the polymeric matrix of the nanocomposite. After approximately 20 h, the polymeric nanocomposite coating containing the entrapped contaminants could be easily peeled off. Both the decontaminated surface and the film obtained were subjected to extraction in 10 mL of dichloromethane (DCM). The organic extracts were dried over sodium sulfate, filtered on 45 μm a Sartorius filter, and analyzed by the GC-MS technique. To evaluate the decontamination efficiency, the decontamination factor (DF) was calculated by considering the initial concentration of contaminant and the residual toxic found on the metallic probe after decontamination.

$$\text{DF} = 100 \times (\text{C}\_0 - \text{C}\_{\text{f}}) / \text{C}\_0 \tag{1}$$

where DF is the decontamination factor, C0 is the initial toxic concentration, and Cf is the final concentration, reflecting the residual contamination. Measurements were repeated in triplicate, and the average values obtained were reported.
