## 3 Results and Discussion
### Ecotoxicity Studies  
Toxicity measurements using Microtax(r) bioassays were carried out in the solution of 4-n-NP (10 mg L\({}^{-1}\)) before inoculation with the selected bacterial strains under study and after finishing the bioremediation process (28 days). The results obtained are shown in Table 2. It can be observed that the non-inoculated 4-n-NP solution presented a TU (toxic unit) value of 5.5, corresponding to a level of _Acute Toxicity_ according to [PERSON] et al. [48]. However, when the 4-n-NP concentration in the solution decreased due to biodegradation (Figure 1), the toxicity increased, reaching levels of High Acute Toxicity (10 \(<\) TU \(<\) 100) for all the isolates except for _P. saccharolyticum_ CN15, which remained as Acute Toxicity (1 \(<\) TU \(<\) 10). It seems to indicate that the transformation products (TPs) generated by the biodegradation of 4-n-NP must be more toxic than the parent compound, increasing the toxicity of the solution [49]. In Table 2, the isolated bacterial strains are shown in increasing order of 4-n-NP concentration remaining in the solution, but no direct correlation was observed between toxicity increase and the reduction of 4-n-NP in the solution. This suggest that toxicity is more closely related to the presence and concentration of the different TPs generated. It should also be considered that the different bacterial strains could show different degrees of biodegradation towards each specific metabolite produced. In addition, those strains with a lower extent of degradation, as previously mentioned for _A. salmonicida_, which showed the highest TU (Table 2), are those that showed kinetics of the HS type (Table 1). This kinetic model consists of two sequential first-order curves and a faster stage, followed by another with a slower degradation rate. This second slower stage could be provoked by the generation and accumulation of TPs whose toxicity inhibits, to different degrees for each strain, their degradation activity, making it difficult to continue with 4-n-NP degradation. The toxicity of NP is discussed in various studies. It is widely known that NP (the last end product from NPEO biodegradation) is highly toxic in its recalcitrant form and can have a long half-life in the environment [50], including harmful effects on the nervous, reproductive, immune, endocrine, and metabolic systems [51]. However, as far as we know, the toxicity of the different TPs detected after NP biodegradation has not been discussed in previous studies [39], except in the study of [PERSON] et al. [36] where four metabolites were detected throughout the biodegradation process by _B. safensis_ CN12 and were shown to have higher toxicity than 4-n-NP. \begin{table}
\begin{tabular}{c c c c c c c c c c c} \hline \hline  & **Kinetic Model** & **K\({}_{\textbf{1}}\)** & **K\({}_{\textbf{2}}\)** & **tb** & \(\alpha\) & \(\beta\) & **DT\({}_{\textbf{50}}\)** & **Degradation** & **R\({}^{2}\)** & \(\chi^{2}\)** \\  & **Model** & **(d\({}^{-1}\))** & **(d\({}^{-1}\))** & **(d)** & **(d\({}^{-1}\))** & **(d\({}^{-1}\))** & **(days)** & **(\%)** & **R\({}^{2}\)** & \(\textbf{Err}_{scaled}^{2}\)** \\ \hline Control & SFO & 0.005 & - & - & - & - & 129 & 14.7 & 0.767 & 2.27 \\ \hline _B. safensis_ CN12 * & SFO & 0.790 & - & - & - & - & 0.90 & 100 & 0.986 & 5.39 \\ _A. faecalis_ CN8 & FOMC & - & - & - & 0.785 & 6.265 & 8.9 & 75.4 & 0.969 & 3.91 \\ \hline _S. putrefacients_ CN17 & FOMC & - & & - & 1.182 & 13.02 & 10.4 & 73.8 & 0.997 & 1.36 \\ _P. putida_ CN14 & FOMC & - & - & - & 0.643 & 5.404 & 10.5 & 66.5 & 0.980 & 2.86 \\ _E. bugandensis_ CN13 & HS & 0.059 & 0.003 & 14.0 & - & - & 11.7 & 63.0 & 0.952 & 0.17 \\ _O. anthropi_ CN16 & HS & 0.093 & 0.000 & 7.7 & - & - & 7.4 & 50.8 & 0.973 & 0.02 \\ _A. salmonicida_ CN1 & HS & 0.202 & 0.001 & 2.4 & - & - & 203 & 41.1 & 0.936 & 0.78 \\ _P. saccharolyticum_ CN15 & FOMC & - & - & - & 0.142 & 1.031 & 136 & 40.8 & 0.879 & 3.89 \\ \hline \hline \end{tabular}  
* From [PERSON] et al. [36].  
\end{table}