# Molecular Response of Bacteria Exposed to Wastewaters-Borne Nanoparticles
## 2 Materials and Methods
### Growth Inhibition and Toxicity Assessment in Pseudomonas putida  
The bacterial growth inhibition assay was conducted using _P. putida_ KT2440 (DSM 6125), obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ) and Life Technologies GmbH (Karlsruhe, Germany). All necessary components for preparing the bacterial culture and test media were sourced from Sigma-Aldrich (Poznan, Poland).  
\begin{table}
\begin{tabular}{c c c} \hline \hline
**No.** & **Samples Collected for Analysis** & **Sample Name** \\ \hline
1 & Al\({}_{2}\)O\({}_{3}\)NPs (10 mg/L) & NPs \\ \hline
2 & Bulk Al\({}_{2}\)O\({}_{3}\) (10 mg/L) & Bulk \\ \hline
3 & Control (water) & Control \\ \hline
4 & Synthetic domestic wastewater-borne 10 mg/L & \\  & Al\({}_{2}\)O\({}_{3}\)NPs, fed into the SBR reactor (Influent) & WW-NPs-I \\ \hline
5 & Synthetic domestic wastewater-borne bulk Al\({}_{2}\)O\({}_{3}\) & \\  & (10 mg/L), fed into the SBR reactor (Influent) & WW-Bulk-I \\ \hline
6 & Synthetic domestic wastewater (control), fed into the & \\  & SBR reactor (Influent) & WW-Control-I \\ \hline
7 & Synthetic domestic wastewater-borne 10 mg/L & \\  & Al\({}_{2}\)O\({}_{3}\)NPs, treated by activated sludge method in & WW-NPs-E \\  & SBR reactor (Effluent) & \\ \hline
8 & Synthetic domestic wastewater-borne bulk Al\({}_{2}\)O\({}_{3}\) & \\  & (10 mg/L), treated by activated sludge method in & WW-Bulk-E \\  & SBR reactor (Effluent) & \\ \hline
9 & Synthetic domestic wastewater (control), treated by & WW-Control-E \\  & activated sludge method in SBR reactor (Effluent) & \\ \hline \hline \end{tabular}
\end{table}
Table 1: Samples used in the exposure experiments with _P. putida_.  
The assay followed the ISO Guideline 107122 (1994) [32]. Initially, the bacterial culture was transferred into 200 mL of sterilized culture medium. The culture was placed in sterilized Erlenmeyer flasks, sealed with cotton wool, and placed on a magnetic stirrer to ensure proper mixing and aeration throughout the experiment. The bacterial suspension was adjusted to an initial optical density (OD\({}_{610}\)) of 0.2 prior to exposure to the tested solutions. The assessment of growth inhibition was conducted on the basis of measurement of the optical density of samples with \(\lambda\) = 610 nm at the beginning and at the end of the 16-h test. The growth inhibition (\(I\)) was calculated using the following formula:  
\[I=\frac{B_{c}-B_{n}}{B_{c}-B_{0}}\times 100\% \tag{1}\]  
where \(B_{c}\) is the optical density of suspension in the control sample after time t; \(B_{n}\) is the optical density of suspension in the sample examined after time t; and \(B_{0}\) is the optical density of suspension in the control at time 0.  
The effective concentration (EC\({}_{50}\)) values were calculated using probit analysis, a statistical method that estimates concentration-response relationships [33]. The 95% confidence intervals were also calculated to ensure precision in determining the concentration at which 50% of the organisms exhibit a specific effect. To quantify acute toxicity, toxicity units (TU\({}_{a}\)s) were calculated from the EC\({}_{50}\) values according to the following standard formula:  
\[TU_{a}=\frac{1}{EC_{50}}\times 100 \tag{2}\]  
where \(EC_{50}\) is expressed in %. Higher \(TU_{a}\) values indicate greater acute toxicity. This approach enables the direct comparison of toxicity levels across different treatments or exposure conditions [34].