## 3 Results  
### Evaluation of the Effect of the Main Operational Labels in the Biocidal Activity  
To evaluate the operational parameters involved in the use of EW, key variables such as FAC, time, pH, salinity, and the C2 strain were selected (Table 2). Specifically, pH values of 5, 6.5, and 7.5 were tested due to their similarity to real conditions in various aquaculture facilities [12]. Moreover, FAC solutions of 5, 25, and 125 ppm were assessed to cover a broad range of potential applications. Salinities of 1.5% and 3% NaCl were chosen to simulate brackish and marine water conditions, where the bacterium thrives. Importantly, time was limited to 15 min to minimize potential animal exposure to EW. Regarding the initial trials, survival percentages exceeding 100% were considered outliers since differences in colony counts were negligible. The outcomes of these trials are summarized in Table 3.  
In terms of FAC concentration, the highest level tested (125 ppm) demonstrated an almost immediate effectiveness in bacterial reduction (less than 1 min), irrespective of pH. While no significant differences were observed between salinities, a slightly higher reduction was noted at 3%. Conversely, at low FAC concentrations (5 ppm), the effect of EW was minimal. At 25 ppm, the bacterial population decreased significantly, with survival percentages varying according to disinfection time (Table 3). Additional trials conducted with bacterial suspensions in PBS instead of LB-1 medium revealed rapid reductions in bacterial populations at 25 ppm of free chlorine (SAEW, NEW, and SBEW). Remarkably, within the first minute, survival rates dropped below 0.01% in all cases. suggesting that organic matter in the medium influences EW's antibacterial activity. Building on these findings, further studies were conducted with FAC concentrations ranging from 15 to 25 ppm to determine the minimum effective disinfection concentration. These solutions were prepared at pH 7.5 and salinities of 1.5% or 3% (SBEW15, SBEW20, and SBEW25), conditions under which _V. harveyi_ thrives. The results at 5, 10, and 15 min are shown in Table 3. While SBEW15 was ineffective (survival rate > 65%), SBEW20 andSBEW25 exhibited strong bactericidal effects. Notably, higher salinity (3%) enhanced disinfection. The optimal conditions identified were SBEW25 for 10 to 15 min, although extended exposure to SBEW20 also achieved satisfactory results. To further understand the impact of the variables, a multivariate general linear model analysis was performed. This analysis included independent variables such as FAC concentration, pH, salinity, time, and their first-order combinations (Table 1). The results revealed significant contributions (\(p<0.05\)) from FAC, time, and pH-salinity interactions to bacterial reduction (Table 4). Moreover, combinations such as FAC-time and pH-FAC were significant, indicating that free chlorine's effectiveness is influenced by environmental pH and salinity. \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Label** & **Value** & **F** &
\begin{tabular}{c} **Degrees of Freedom of** \\ **Hypothesis** \\ \end{tabular} & **Sig.** \\ \hline pH & 0.085 & 8.523 & 4.000 & \textless{}0.001 \\ FAC & 0.710 & 53.113 & 8.000 & \textless{}0.001 \\ Time & 0.552 & 36.801 & 8.000 & \textless{}0.001 \\ Salinity & 0.023 & 4.470 & 2.000 & 0.012 \\ FAC*salinity & 0.130 & 7.628 & 8.000 & \textless{}0.001 \\ pH*FAC & 0.105 & 6.061 & 8.000 & \textless{}0.001 \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Results on statistical analysis applying a multivariate general linear model.