## 4 Discussion  
Naturally occurring marine bacteria from Portuguese ports were grown and isolated in high concentrations of TBT and their decontamination potential was evaluated. The results suggest that TBT is highly selective and can negatively affect bacterial growth. A decrease in the CFUs abundance with increasing TBT concentrations was observed for all sampled sites. At 0.1 mM, the bacterial growth was affected, with a decrease of CFUs over 50% compared to the control, suggesting this low concentration is highly toxic. In Peniche, the percentage of tolerant bacteria was constant with increasing TBT concentrations. Peniche was also the location with the highest percentage of tolerant bacteria at 3 mM (7.7% compared to control). These results may be explained by the initial pool of bacteria, which was the highest from all sampling sites. Butyltin's levels in collected waters were below the limit of quantification (<50 ng L\({}^{-1}\)) in all sampling sites. Marinas and ports are hotspots of TBT contamination due to boat traffic and shipyard activities [35; 36]. Thus, it is expected that these sampling sites would be contaminated with TBT and that smaller concentrations of this highly toxic chemical might be present, particularly in sediments, where it persists [8; 37]. Previous studies reported levels of butyltin in Portuguese waters. In 1999/2000, samples collected along Portuguese rivers and coastal waters revealed butyltin levels ranging from 23 to 79 ng L\({}^{-1}\)[35]. In 1999, on Tagus estuary, TBT levels ranged from 1.1 to 21.1 ng L\({}^{-1}\)[38]. Although those studies date back to the pre-ban period, a more recent study revealed that in 2014 TBT was still present in Portuguese coastal waters, since quantifiable amounts were detected in dog-whelks (_Gastropoda_) tissue samples [16]. Additionally, some studies refer the occurrence of TBT-tolerant bacteria to be related to the occurrence of other contaminants such as metals, making them cross-resistant to TBT [39]. Seaports are traditionally contaminated sites, making these heavily impacted sites more selective for bacteria that develop mechanisms to adapt to such conditions. The REP-PCR method was used to characterize the 157 isolates tolerant to 3 mM of TBT. One hundred and eleven different patterns were identified. Peniche port samples had the lowest level of diversity, with just 5 distinctive patterns identified using REP-PCR, sharing a similarity of more than 79%, suggesting that the isolates from this location were genetically close to each other. This low diversity and higher initial pool of microorganisms (5000 \(\pm\) 513 CFU mL\({}^{-1}\)), may explain the high percentage of tolerant CFUs observed for this location. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Exposure Period** & **Treatment** & **LC\({}_{50}\) (\%)** & **95\% Confidence Interval** \\ \hline \multirow{3}{*}{48 h} & TBT & 17.02 & 16.20–17.94 \\  & S13 + TBT & \textgreater{}20 & n.d. \\  & F3 + TBT & \textgreater{}20 & n.d. \\ \hline \multirow{3}{*}{72 h} & TBT & 8.40 & 8.19–8.62 \\  & S13 + TBT & \textgreater{}20 & n.d. \\  & F3 + TBT & 19.90 * & 13.89–45.32 \\ \hline \multirow{3}{*}{96 h} & TBT & 5.91 & 5.89–5.93 \\  & S13 + TBT & 19.97 * & 19.63–20.34 \\ \cline{1-1}  & F3 + TBT & 10.21 * & 8.04–13.45 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Lethal concentration values estimated for _Gibbula umbilicalis_ exposed to untreated and bioremediated media. Strain S13: _Pseudomonas putida_; Strain F3: _Serratia marcescens_. Asterisk (*) indicates significant differences in LC\({}_{50}\) between bioremediated treatments and untreated media. n.d.-not determined. To select the most suitable strains for bioremediation assays, bacteria were exposed to 6 mM TBT and two different strains were selected based on their growth in this condition. Although TBT inhibits the growth of these isolates, there were no apparent differences in growth between 1 mM, 3 mM, and 6 mM. [PERSON] et al.