## 3 Results and Discussion
### Ecotoxicity Studies

Table 1: Kinetic parameters from 4-n-NP biodegradation in solution after inoculation with several bacterial strains. To observe if the increase in toxicity was related to the presence of certain transformation products, those generated after 28 days of 4-n-NP degradation in the solution in the case of the three bacterial strains that reached higher NP degradation (_A. faecalis_ CN8, _S. putrefaciens_ CN17, and _P. putida_ CN14) were analyzed. Three transformation products (TPs) were identified, which are shown in Figure 2, and their chemical structures are included in Figure S2. TP1 (4-(6-oxo-1,1-dimethylheptyl) phenol) and TP2 (4-nonyl-4-hydroxycyclohex-2,5-dienoene) were detected as degradation products using the three strains studied, and TP3 (4-nonyl-4-hydroxycyclohex-2-enone) appeared only in 4-n-NP degradation using _A. faecalis_ CN8. The three TPs found involved the basic structure of 4-n-NP, indicating that none of the three bacterial strains under study could provoke the cleavage of the aromatic ring. As far as we know, TP1 (4-(6-oxo-1,1-dimethylheptyl) phenol) had not been previously detected from NP degradation until the study by [PERSON] et al. [36] by _B. safensis_ CN12, and now it is detected in this study for _A. faecalis_, _S. putrefaciens_, and _P. putida_. \begin{table}
\begin{tabular}{c c c c} \hline
**Bacterial Strain** & **4-n-NP Remaining (mg L\({}^{-1}\))** & **TU** & **Toxicity** \\ \hline Control & 10.0 & 5.5 a & Acute toxicity \\ \hline _B. safensis_ CN12 \({}^{*}\) & 0.00 & 49.4 b & High acute toxicity \\ _A. faecalis_ CN8 & 2.46 & 17.6 ab & High acute toxicity \\ _S. putrefaciens_ CN17 & 2.62 & 19.6 ab & High acute toxicity \\ _P. putida_ CN14 & 3.35 & 28.6 ab & High acute toxicity \\ _E. bugandensis_ CN13 & 3.70 & 31.6 ab & High acute toxicity \\ _O. anthropi_ CN16 & 4.92 & 10.9 ab & High acute toxicity \\ _A. salmonicida_ CN1 & 5.89 & 32.3 ab & High acute toxicity \\ _P. saccharolyticum_ CN15 & 5.92 & 9.20 a & Acute toxicity \\ \hline \end{tabular}  
* From [PERSON] et al. [36].  
\end{table} 
Table 2: The acute toxicity test towards _A. fischeri_ (Microtok assay) in the 4-n-NP solution (10 mg L\({}^{-1}\)) before inoculation and 28 days after inoculation with different isolated bacterial strains. TU values followed by the same letter are not significantly different (_p_ < 0.05). Figure 2: Transformation products (TPs) detected in solution after 28 days of degradation of 4-n-NP by various bacterial strains. On the contrary, TPs 2 and 3 were detected previously by [PERSON] et al. [22; 23] and more recently by [PERSON] et al. [39], who also commented that their toxicity had not been discussed in previous studies. According to [PERSON] et al. [22; 23], TP3 was generated from TP2 and served as the substrate, but only in the case of _A. faecalis_ such a reaction takes place, since in the case of _S. putrefaciens_ CN17 and _P. putida_ CN14, TP2, as well as TP1, is accumulated in the system. Regarding the increased toxicity observed, these results seem to indicate that the TPs generated and/or other metabolites that we have not been able to detect are more toxic than the parent compound. The observed increase in toxicity highlights the importance of considering TPs in ecological risk assessments, as focusing solely on the parent compound may lead to an underestimation of the actual environmental risk. Therefore, the bacterial strains tested in the present study can be used to degrade 4-n-NP, but degradation should be improved using consortia with other bacterial strains capable of degrading the toxic TPs produced, since the limited metabolic functions of these pure strains resulted in the incomplete degradation of 4-n-NP or its degradation products.