## 4 Discussion
[40] reported that gradually transferring bacteria to higher TBT concentrations increases their tolerance, suggesting a \"memory response\" mechanism, which could justify why some bacteria grew in such high concentrations since they were cultured and maintained in TBT-contaminated media. Despite the importance of determining the levels of a toxicant and its by-products in the ecosystems, this does not provide information on the contaminant's toxicity and its degradation products. Such data can be obtained through ecotoxicological assays. In the present study, ecotoxicological testing with _G. umbilicalis_ proved to be a solid approach to determine toxic effects of TBT. Still, the use of sub-lethal, and thus more sensitive endpoints [41] should also be considered in future studies to better estimate the harmful impacts of low levels of butylins on gastropods. _Pseudomonas putida_ (S13) showed the highest biodegradation potential of TBT-contaminated waters, as evidenced by the observed decrease in toxicity mediated by this bacterium. _Pseudomonas putida_ was reported as a TBT-degrading bacteria that uses TBT as a carbon source [27]. The cultivation of _P. putida_ in TBT-contaminated media suggests that the breaking down of TBT by this bacterium generates degradation products with a lower toxicity than the parent compound. The ecotoxicological approach used in this study, allows to show the relevant effect on important species. Nonetheless, follow up studies should be complemented with chemical analyses. An increase in LC\({}_{50}\) was also observed for the treatment with _Seratia marcescens_ (F3), suggesting that remediation of the media had occurred, as the medium became less toxic. _Serratia marcescens_ has been regarded as a potential organotin-resistant bacteria [26] and here its tolerance is confirmed. To our knowledge, this is the first study concerning the potential of _S. marcescens_ to bioremediate TBT-contaminated waters. Nonetheless, despite the decrease in toxicity of the treated media with this bacterium after 96 h of exposure, the media was still toxic to _G. umbilicalis_. This hints the possibility that some TBT may be still present in the media and a longer remediation period (>48 h) may be needed for a more effective TBT degradation. Additionally, toxic by-products may be generated, which should be monitored in future studies, considering that toxic degradation products may hamper the in situ application of TBT-degrading bacteria. As demonstrated in this study, naturally present bacteria from Portuguese ports can tolerate TBT at high levels and eventually transform it into less toxic compounds, contributing towards ecosystem restoration. The results derived from the ecotoxicological testing demonstrated that _P. putida_ and _S. marcescens_ have the potential to step up TBT removal from contaminated marine environments and thus representing candidate remediation agents for application in bioremediation approaches. Since heavy metal and antibiotic resistance is frequent in TBT-resistant bacteria, exploring TBT-tolerant bacteria applications might be vital for the restoration of polluted environments in general. Knowledge of their genetics and physiology is key for their future application as natural decontamination facilitators. Overall, the present study contributed to the growing knowledge of TBT-degrading bacteria. The decrease in toxicity observed strongly suggest that _P. putida_ can transform TBT into less toxic products and that ecotoxicological testing can be a valuable tool in bioremediation studies as a complement of chemical analyses. Future research should focus on determining mechanisms underlying TBT resistance and biodegradation. Despite the decreasing trend observed over the past decades, TBT levels in water and biota are still matters of concern [42].