This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013 New J. Chem., 2013, 37, 1569--1577 1569 Cite this: New J. Chem., 2013, 37, 1569 A molecular analysis of the toxicity of alkyltributylphosphonium chlorides in Aspergillus nidulans† Diego O. Hartmann and Cristina Silva Pereira* Investigating ionic liquids in vivo effects at a molecular level is crucial for the deeper understanding of their toxicity and the development of new biological applications. In the present study, we propose the use of qRT-PCR to analyse the expression of Aspergillus nidulans genes after exposure to alkyltributylphosphonium chlorides ([P 4 4 4 n ]Cl, where n = 1, 4, 8 or 12). The selected genes are involved in plasma membrane and cell wall biosynthesis and repair mechanisms. The data strongly indicate cell wall damage as the common mechanism of toxicity amongst these ionic liquids, while plasma membrane permeabilisation is dependent on the alkyl substituent length. Considering their effects on the fungal cell walls, the knowledge herein produced opens doors for several possible applications of quaternary phosphonium ionic liquids, in particular their potential use in antifungal formulations. Introduction Over the last two decades, the potential of ionic liquids has been extensively explored. Their inherent potential comes from their structural diversity and tunable physical and chemical prop- erties. From the estimated millions of possible formulations, 1 several hundred ionic liquids are already well known and characterised. 2 A great amount of data is available on their chemical 1 and physical properties, 2 and numerous applications have been proposed. 3 Ionic liquids are generally defined as salts that are liquid below 100 1C, 1 and some are regarded as green solvents due to their excellent solvation capacity, negli- gible vapour pressure, and bulk non-flammability. 4 However, ionic liquids comprise a very heterogeneous group of fluids that are not intrinsically green. Recent reviews on their environ- mental impact and biodegradability highlight the toxic nature and recalcitrance of some of these compounds. 5,6 Although great amount of data have been produced focusing on imidazolium-based ionic liquids, in recent years, other cationic groups have been receiving more attention. Amongst them are the quaternary phosphonium ionic liquids. They are generally considered thermally and chemically more stable (the latter due to the absence of an acidic proton) than the quaternary ammonium or imidazolium salts. 7 Within the quaternary phosphonium ionic liquids, the tetraalkylphos- phonium ones are, to present, the most studied. They have already been investigated for diverse applications, 8–15 but there are only few studies regarding their environmental impact. Their apparent high toxicity was observed in various aquatic organisms. 16–18 Recently, our group performed a systematic study on the toxicity and biodegradability of alkyltributyl- phosphonium chlorides, namely [P 444 n ]Cl, where n = 1, 3–8, 10, 12 or 14 (Fig. 1). 19 Their inhibitory and lethal effects on Aspergillus nidulans were, as expected, determined by the length of the alkyl substituent in the cation, and correlated well with the cation lipophilicity. 20 Fluorescence microscopy suggested that membrane permeabilisation and cell wall damage are the basis of the toxic mechanism of action of [P 4 4 4 n ]Cl, when n Z 4, while for n o 4 it remained to be solved. 19 Fig. 1 The structure of the alkyltributylphosphonium cation. Instituto de Tecnologia Quı ´mica e Biolo´gica, Universidade Nova de Lisboa, Av. da Repu ´blica, Oeiras, 2780-157, Portugal. E-mail: spereira@itqb.unl.pt † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c3nj00167a Received (in Porto Alegre, Brazil) 11th February 2013, Accepted 6th March 2013 DOI: 10.1039/c3nj00167a www.rsc.org/njc NJC PAPER Published on 07 March 2013. Downloaded by INSTITUTO DE TECNOLOGIA QUIMICA E BIOLOGICA on 28/10/2014 20:31:07. View Article Online View Journal | View Issue