Periodic Reporting for period 1 - NANOREM (A novel isotope tool to assess nanoparticle toxicity in wetland plants) Reporting period: 2016-09-01 to 2018-08-31 Summary of the context and overall objectives of the project NANOREM is a research project aiming to provide the first comprehensive insight into the toxicity of zinc oxide nanoparticles (ZnO-NPs) in wetland plants. This information is essential to evaluate correctly the risk of releasing large quantities of NPs to the environment. In this project we made use of cutting edge isotope techniques to trace the sources of NP pollution, and to understand how NPs might change the current paradigm of metal cycling and assimilation by plants. Furthermore, NPs were synthesized from plants and plant extracts using novel methods and characterized. The four research objectives designed were the following:i) To evaluate the uptake, accumulation and toxicity of ZnO-NPs in Phragmites australis and Iris pseudacorus as compared with bulk ZnO and free Zn2+.ii) To purify ZnO-NPs from Iris pseudacorus extracts and characterize themiii) To determine the effect of urban run-off water on the solubility, stability, aggregation, assimilation and phytotoxicity of ZnO-NPsiv) To measure the Zn isotopic effects associated with the response of macrophytes to ZnO NPs, bulk ZnO and free Zn2+.The conclusions of this action were the following. Zinc oxide is toxic to wetland plants, and the smallest NPs pose a greater environmental risk. NPs smaller than 50 nm released more Zn in solution, and this was taken up by plant roots. Plants treated with NPs<50 nm showed higher Zn concentration in their tissues and more toxic effects than in response to other ZnO materials. Isotope data indicated that a significant portion of NPs < 50 nm reaches the aerial parts of the plant undissolved and could potentially be transmitted through the food chain. Zinc stable isotope analysis was established as a useful technique for the study of NP metabolization by plants. In addition, this action demonstrated that ZnO-NP can be obtained from plant extracts of I. pseudacorus. This development opens the possibility of recovering metals from polluted plant biomass. Finally, a green system for the remediation of run-off water was developed, which effectively removed Zn and Cu. Carex vulpina was the best species in this system, showing the highest biomass production and Zn concentration. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far NANOREM has been a very successful action and all the goals and milestones proposed were completed. The four research objectives designed have been achieved:i) The uptake, accumulation and toxicity of ZnO-NPs in Phragmites australis and Iris pseudacorus as compared with bulk ZnO and free Zn2+ has been evaluated (M2). Over 80 parameters were recorded and analyzed for each species. Key variables for studying NP phytotoxicity have been identified. Our results show the importance of NP size in their toxicity, uptake, and transport. This information will be crucial to evaluate the environmental risks of ZnO NPs and their potential transmission to the food chain accurately.ii) ZnO-NPs have been obtained from Iris pseudacorus leaf extracts (M3). This achievement has the potential to make a major break-through in metal recycling from wastewater. Our current goal after NANOREM is to protect this technology and continue developing it until it is available to European society.iv) The Zn isotopic effects associated with the response of macrophytes to ZnO NPs, bulk ZnO and free Zn2+ were measured (M5). This pioneering development has allowed to determine which is the main route of ZnO NP uptake by the root and transport to the shoot. Besides, a relationship between NP size and root to shoot Zn isotopic fractionation has been noted. Our results will allow to quantify the proportion of NPs that reach the shoots depending on the size of the NPs. This information is key to estimate the rate of transmission to the food chain of each nanomaterial. The interest of Zn isotope techniques to study NP uptake, transport, and accumulation in plants has been fully established, and has implications for understanding NP fate in the environment.Besides the above research milestones (M2-M5), accreditation for professorships in Spain has been obtained (M1) . A total of 17 deliverables were completed, and 4 additional deliverables were included. Only four deliverables will be completed after the end of this action: the writing-up and publishing of four research articles containing the research results. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) NANOREM has met all the goals proposed and generated a diversity of positive impacts which will go beyond the end of the project.i) For the Experienced ResearcherNANOREM allowed the fellow to acquire a set of new major concepts and analytical procedures in nanoparticle research. Dr. Caldelas learnt new protocols for the digestion of plant samples and a new Zn purification protocol, and could use a Neptune MC-IPC-MS. During the collaboration with Naturalea Dr. Caldelas gained skills in the design, setting up, and maintenance of a pilot-scale plant-based water purification system. Data have been collected for four publications and a patentable technology, which will boost her career prospects. A strong collaboration was established with researchers from the GET and Naturalea, that we plan to continue in future projects (i.e. ERC Starting Grant). Work in the Urban River Lab also offered great possibilities for networking local research groups (Sabater in UB, Ribot and Gacia in CREAB-CSIC). During the two conferences Dr. Caldelas established contact with two more groups (Sanità di Toppi in U. di Pisa, Italy, and Küpper in Institute of Plant Molecular Biology ASCR, Czech Republic). Finally, she acquired experience managing her own research project, supervising students, communicating her work to a wider audience, and transferring her research into practical and marketable solutions. In this manner Dr Caldelas has prepared herself for the role of group leader and made the most of this opportunity to advance her career.ii) For the European society – On the first place, NANOREM has had a major impact in the European Research Area by bringing together a multidisciplinary and intersectoral team with the capacity to produce world-class research in NP ecotoxicology. The composition of the team warranted a fluid exchange between disciplines and sectors, maximized the results, and strengthened cross-border links. This positive impact will continue after the end of the project by means of the on-going collaborations that have been established with GET Toulouse and Naturalea. At the same time, this project contributed to retain and train European talent, and to promote gender equality in the ERA. In line with the Innovation Union initiative, the outcomes of this project will enhance the competitiveness of the European companies by means of the new products and practical solutions to the real challenges of the industry that we have obtained. We have developed a compact green filter system for the clean-up of urban run-off water, and a new procedure for NP biosynthesis from Zn-polluted plant extracts. Most importantly, our project has contributed to fill in a gap of knowledge that is hampering the accurate risk assessment of NP as a potential hazard for human and environmental health. In this area, the impact of the action was boosted by increasing the number of species from the original four to six. We hope thus to better contribute to maintain the safety of the European citizens and the quality of our water bodies, in line with the Water Framework Directive. Zinc oxide nanoparticles biosinthesized from I. pseudacorus extracts.
