# Molecular Response of Bacteria Exposed to Wastewaters-Borne Nanoparticles
## 4 Conclusions  
In summary, our study demonstrates that pristine Al\({}_{2}\)O\({}_{3}\) nanoparticles exhibit strong toxicity toward _P. putida_, with an EC\({}_{50}\) of 0.5 mg/L, while bulk Al\({}_{2}\)O\({}_{3}\) shows negligible toxicity (EC\({}_{50}\)\(\geq\) 10 mg/L), confirming the markedly higher biological activity of the nanoparticulate form. Exposure to pristine Al\({}_{2}\)O\({}_{3}\)NPs consistently upregulates oxidative stress-related genes (_katE_, _alpC_) and the energy metabolism gene _ctaD_ in _P. putida_, indicating a sustained molecular stress response. Gene expression changes in _P. putida_ vary over time, with acute effects observed within 48 h and prolonged responses suggesting no full transcriptional recovery during chronic exposure. In wastewater matrices, Al\({}_{2}\)O\({}_{3}\)NPs trigger more moderate but persistent gene expression changes, particularly in oxidative stress markers, indicating sustained biological activity even after treatment. Responses to wastewater-associated Al\({}_{2}\)O\({}_{3}\)NPs differ between raw and treated matrices, reflecting variable bioavailability and the transformation of particles during wastewater treatment. In contrast, bulk Al\({}_{2}\)O\({}_{3}\), whether in pristine suspensions or wastewater, induces only moderate or negligible gene expression changes in _P. putida_, underscoring its lower biological activity compared to the nanoform. Comparative analysis with _D. magna_ suggests that genes such as _katE_ and _alpC_ may serve as robust cross-species biomarkers of nanoparticle-induced effects.  
These findings confirm that wastewater-borne Al\({}_{2}\)O\({}_{3}\) in nanoparticulate form elicits specific biological responses in bacteria, unlike its bulk counterpart, and that gene expression analyses can offer early insight into sublethal exposure effects. Further integration of molecular data with classical endpoints will enhance predictive capacity in the environmental risk assessment of engineered nanomaterials.  
**Supplementary Materials:** The following supporting information can be downloaded at [[https://www.mdpi.com/article/10.3390/app15147746/s1](https://www.mdpi.com/article/10.3390/app15147746/s1)]([https://www.mdpi.com/article/10.3390/app15147746/s1](https://www.mdpi.com/article/10.3390/app15147746/s1)). Table S1. Acute toxicity of Al\({}_{2}\)O\({}_{3}\) (nanoparticles and bulk form) towards _Pseudomonas putida_ (16 h exposure). Table S2. Acute toxicity of untreated and treated wastewater samples towards _Pseudomonas putida_ after 16 h exposure. Table S3. Ecotoxicity categories of chemical compounds/ wastewater for aquatic organisms by EPA (U.S. Environmental Protection Agency). Table S4. Ecotoxicity criteria for wastewater samples developed by [PERSON] et al. (2003) [34].  
**Author Contributions:** Conceptualization, N.D.; methodology, N.D.; validation, K.A. and M.Z.-R.; formal analysis, N.D.; investigation, N.D. and K.A.; resources, N.D. and K.A.; data curation, N.D.; writing--original draft preparation, N.D.; writing--review and editing, K.A. and M.Z.-R.; visualization, N.D.; supervision, M.Z.-R.; project administration, N.D.; funding acquisition, N.D. All authors have read and agreed to the published version of the manuscript.  
**Funding:** This research was funded by the National Science Center through the Miniatura 5 Program (021/05/X/ST10/00884) and was co-financed by the Warsaw University of Technology within the Excellence Initiative: Research University (IDUB) program (1820/106/Z01/2023).  
**Institutional Review Board Statement:** Not applicable.  
**Informed Consent Statement:** Not applicable.  
**Data Availability Statement:** All raw data that are fundamental to the results presented in this study can be provided by the corresponding author on request.  
**Conflicts of Interest:** The authors declare no conflicts of interest.