Use of microalgae to report on the environmental impact of metallic nanoparticles

Resumen

Nanotechnology has become one of the most successful technology nowadays. Many nanoparticles (NPs) have been synthesized and used in numerous applications including medicine, industry and remediation processes. Due to this increasing production and widespread use of NPs, huge amounts of them are discharged in the aquatic environment daily. Their release in aquatic environment constitutes a potential harmful effect to aquatic organisms. Among that toxic effect, reactive oxygen species (ROS) production stands out. Within aquatic organisms the primary producers play an important role since any deleterious effect on them will affect the rest of the trophic chain. Therefore, the study of the mechanisms of toxic action of NPs in these aquatic organisms is necessary to evaluate the potential ecotoxicological effects of these NPs not only for these organisms but for the rest of the aquatic life. Several of these ecotoxicological studies are performed by using bioassays with different organisms which give information about the toxicity of a substance (such as NPs). A further evolution of bioassays is the use of bioreporters which allow to know the toxic information with a less laborious, cost-effective and more rapid toxicity bioassays. Furthermore, the biological effect of NPs will change once they are in the aquatic environment considering their possible physical and chemical interactions with co-occurring pollutants and organic matter in the environment; so that, experiments in real matrices are necessary to understand in a real manner NPs toxicity. The overall aim of this Thesis was to address the mechanisms of toxic action of different metallic NPs by focusing on their ROS production and the construction and use of cyanobacterial bioreporters as a useful tool in ecotoxicology. Chapter 1 is a general introduction which includes some basic concepts about emerging pollutants (EPs) and NPs and their role in aquatic environments. The main characteristics of NPs and their potential mechanisms of action to aquatic organisms are described in this section. Furthermore, the use of bioassays and biosensors in ecotoxicity is reviewed, with special focus on ROS-detecting-bioreporters. it also includes a section dedicated to cyanobacteria as environmentally relevant organisms and the cyanobacterial bioreporters constructed to date. Chapter 2 describes the construction of four luminescent cyanobacterial ROS-detecting bioreporters. In Chapter 2.1 the promoter regions of sodA and sodB genes were fused to luxCDABE genes to construct the ROS-detecting bioreporters Nostoc sp. PCC7120 pBG2154 and Nostoc sp. PCC7120 pBG2165 respectively. In Chapter 2.2 the promoter regions of 2-cys-prx and katA genes were fused to luxCDABE genes to construct the ROS-detecting bioreporters Nostoc sp. PCC7120 pBG2172 and Nostoc sp. PCC7120 pBG2173 respectively. All the bioreporters were characterized both in growth medium and in environmental water samples. The results showed a specific character of Nostoc sp. PCC7120 pBG2154, Nostoc sp. PCC7120 pBG2165 and Nostoc sp. PCC7120 pBG2173 detecting only superoxide anion, while Nostoc sp. PCC7120 pBG2172 was induced by both superoxide anion and hydrogen peroxide. The cyanobacterial bioreporters were able to detect these ROS in pollutants-spiked real water samples although they were less sensitive in polluted waters where the pollutants may suffer a complexation process with the organic matter or other present substances in the water. Their low limits of detection make them the most sensitive ROS-detecting bioreporters constructed to date. In the last decade whole-cell bioreporters have been used in ecotoxicity studies, giving information not only about the toxicity of different pollutants but furthermore about their bioavailability. In this Thesis (Chapter 3) a battery of cyanobacterial bioreporters has been used to report the toxicity of AgNPs, TiNPs, ZnNPs and CuNPs in aquatic environment. The toxicity of these NPs was measured by using the global toxicity bioreporter Nostoc sp. CPB4337 and calculating their EC50. As one of the mechanisms of toxic action of metallic NPs is the toxicity associated to the release of free-ions, the strain Synechococcus sp. PCC7942 pBG2120 (capable to detect bioavailable metal ions) was used. The results confirm that the presence of rereleased free-ions was negligible. However, this strain was capable to detect the metallic NPs per se so giving a new application to this cyanobacterial bioreporter. As oxidative stress is a primary toxic mechanism of metallic NPs, the ROS-detecting bioreporters constructed in Chapter 2 were also used in this study in order to evaluate the ROS formation by the metallic NPs. Firstly, the results confirmed that ROS production was due to the NPs per se and not by the released free-ions. The cyanobacterial bioreporters showed that ROS production varied depending on the growth medium or environmental matrices conditions and on the metallic NPs type. This work demonstrated the different levels of ROS production induced by metallic NPs and the importance of nanotoxicology studies in real environmental matrices. Superparamagnetic iron nanoparticles (SPION) have become one of the most used NPs due to their magnetic properties. They are used in biomedical fields and remediation processes and they may become widespread in the environment. Several studies have been performed to elucidate SPION toxicity in animal cells but only a few of them have been done to study their ecotoxicity so more studies in relevant organisms of the aquatic environment are necessary. In Chapter 4 the toxicity and potential mechanisms of toxic action of two SPION to the microalgae Chlamydomonas reinhardtii were evaluated. The results showed a dose-dependent toxicity. The potential mechanisms of action were measured mostly by flow cytometry indicating that both SPION produced ROS generating an oxidative stress which triggered a series of physiological changes as a decrease in metabolic activity and changes in their plasma membrane potential and in their mitochondrial membrane potential. Furthermore, and for the first time in this study a shading effect and a clear internalization via endocytosis were observed. These results confirm a potential risk for the environment as the potential concentrations of SPION in the environments may surpass those that cause toxicity. Finally, Chapter 5 consists of a general discussion highlighting the main findings of the Thesis in the context of a global discussion. The Thesis ends with general conclusions both in English and Spanish. Overall, this Thesis aims to understand the mechanisms of toxic action of metallic NPs focusing on ROS production which may trigger different alterations in the organisms. Furthermore, novel cyanobacterial ROS-detecting bioreporters have been constructed and characterized as a tool in the study of the ecotoxicity of EPs and therefore NPs in aquatic environments. These new tools contribute to increase the knowledge about the toxicity of pollutants in these environments and aim to solve the surface water contamination problems and a responsible use of nanotechnology.