Unravelling the mechanism of toxicity of alkyltributylphosphonium chlorides in Aspergillus nidulans conidia

Abstract

The mechanism of toxicity of alkyltributylphosphonium chlorides [P_{4 4 4 n}]Cl (n = 1, 3–8, 10, 12 or 14) in conidia of the filamentous fungus Aspergillus nidulans is reported. Systematic elongation of one of the alkyl substituents resulted generally in higher toxicity, as defined by their inhibitory and lethal effects. In this study, fluorescence microscopy is proposed as a direct method for assessing the impact of ionic liquids on the plasma membrane integrity. Data were complemented by microscopic evaluation of the conidia cell wall and morphology. The higher toxicity of phosphonium ionic liquids carrying long alkyl substituents is most likely due to their strong interaction with the conidia cellular boundaries.

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Introduction

The potential of ionic liquids has been thoroughly explored over the last two decades. From estimated millions of possible formulations,¹ several hundred ionic liquids are already well known and characterised.² Ionic liquids are generally defined as salts that are liquids below 100 °C,¹ and some are regarded as green solvents due to their excellent solvation capacity, negligible vapour pressure, and bulk non-flammability.³ Their inherent potential comes also from their structural diversity and tunable physical and chemical properties. However, ionic liquids comprise a very heterogeneous group of fluids that are not intrinsically green: some are toxic and non-biodegradable, as seen in recent reviews on their biodegradability and environmental impact.^4,5

A considerable amount of data is now available on the chemistry¹ and physical properties² of ionic liquids, and numerous applications have been already proposed.⁶Imidazolium ionic liquids are still at the heart of most of these studies, but the focus of interest is moving towards other cationic groups. These include quaternary phosphonium ionic liquids, some of which are currently produced in tonne quantities, particularly by Cytec Industries Inc.⁷ 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.^8,9 Their properties, including biological activity, have inspired numerous patents for a broad range of applications, e.g. intermediates in the chemical synthesis of terpenes,¹⁰ antistatic agents,¹¹ biocides, either alone¹² or combined with other compounds,^13,14plant growth regulators¹⁵ and anti-cancer agents.¹⁶

Amongst the quaternary phosphonium ionic liquids, the tetraalkylphosphonium ones are, at present, the most studied. They have been already investigated for diverse applications, e.g.solvents in separation processes^17,18 and phenol bioremediation,¹⁹ media for chemical reactions,^20,21 lubricants,²² and antimicrobial²³ and anti-cancer agents.²⁴ In the search for benign counterions, tetraalkylphosphonium cations were combined with various amino acids^25,26 or acesulfamates.²⁷

Up to now, there are only few studies regarding the toxicity of phosphonium ionic liquids. Their apparent high toxicity was observed in various aquatic organisms, i.e. Vibrio fischeri and Daphnia magna,^28,29 and Pseudokirchneriella subcapitata.^29,30 To the best of our knowledge, the only systematic study on tetraalkylphosphonium ionic liquids was performed with [P_{6 6 6 n}]Cl (n = 6 to 16).³¹ Toxicity against several bacterial and yeast strains was shown to generally increase with the elongation of the alkyl substituent. This toxicity trend has also been observed for different ionic liquids cations,^32,33 and correlates well with the cation lipophilicity.³⁴

In this work, we present a toxicity and biodegradability assessment of [P_{4 4 4 n}]Cl, where n = 1, 3–8, 10, 12 or 14, towards Aspergillus nidulans. Their inhibitory and lethal effects against fungal conidia were, as expected, determined by the length of the alkyl substituent in the cation. To better understand their mechanism of toxicity, we have also observed their effects on the integrity of conidia boundaries and morphology. Fluorescence microscopy has been used previously to illustrate the effect of a single imidazolium-based ionic liquid on eukaryotic cells (HeLa cells).³⁵ However, this is the first systematic study where the interaction of ionic liquids with cellular boundaries is being accounted for with fluorescence microscopy. This technique allowed a direct measure of their effect, strongly suggesting that narcosis (nonspecific disruption of membranes, not to be confused with necrosis)^36–38 is the basis of their toxic action.

Experimental

Chemicals

All compounds used in the preparation of minimal media and in the plasma membrane and cell wall integrity assays, with the exception of NaCl (Panreac, 99.5%), were purchased from Sigma Aldrich: D(+)-glucose, K₂HPO₄, ZnSO₄·7H₂O, CuSO₄·5H₂O, FeSO₄·7H₂O, MgSO₄·7H₂O, NaNO₃ and KCl; and dimethylsulfoxide (dmso; 99.5%), propidium iodide (≥94%, stock solution 20 mM in dmso), Calcofluor White M2R (Fluorescent Brightener 28, stock solution 5 mM in water) and glycerol (≥99.5%), respectively. Deuteriated propanone used in the biodegradability assessment was purchased from EURISO-TOP (France).

Ionic liquids

All ionic liquids used in this study were prepared by QUILL (Queen's University Ionic Liquids Laboratory, Belfast, UK), except for [P_{4 4 4 4}]Cl and [P_{4 4 4 14}]Cl which were supplied by Cytec Industries, Canada. The comprehensive study on the synthesis and physicochemical properties of these ionic liquids will be published elsewhere.³⁹ [P_{4 4 4 n}]Cl, where n = 3, 5–8, 10 or 12, were prepared by nucleophilic (S_N2) addition of tributylphosphine to the respective 1-chloroalkane. [P_{4 4 4 1}]Cl was synthesised by a neutralisation reaction of methanolic tributylmethylphosphonium methylcarbonate with concentrated aqueous hydrochloric acid. Ionic liquids were characterised by ¹H, ¹³C and ³¹P NMR spectroscopy, mass spectrometry, CHN elemental analysis, and halide and water content analyses.

Fungal strain

Aspergillus nidulans strain FGSC A4 was cultivated on dichloran-glycerol (DG18) agar (Oxoid), and a suspension of fungal conidia, prepared as previously described,⁴⁰ was stored at −80 °C in cryoprotective solution containing 0.85% w/v NaCl and 10% v/v glycerol.

Toxicity tests

The toxicity of ionic liquids to A. nidulans was evaluated by determining their minimal inhibitory (MIC) and fungicidal concentrations (MFC), distinguishing between growth inhibition and death, respectively.

The minimal culture medium containing glucose (1.0 g l⁻¹) and K₂HPO₄ (1.0 g l⁻¹) was dissolved in distilled water, sterilised in an autoclave (20 min; 121 °C), and supplemented with the mixture of essential salts, previously sterilised by filtration: NaNO₃ (3.0 g l⁻¹), ZnSO₄·7H₂O, (0.01 g l⁻¹), CuSO₄·5H₂O (0.005 g l⁻¹), MgSO₄·7H₂O (0.5 g l⁻¹), FeSO₄·7H₂O (0.01 g l⁻¹) and KCl (0.5 g l⁻¹). The ionic liquids were added to the minimal culture media with final concentrations from 10 μM to 95 mM (distributed stepwise from 1.25 μM to 2.5 mM). Each liquid medium (1 cm³) was inoculated with a suspension of fungal conidia, in order to obtain the final concentration of 10⁵ conidia per cm³, and divided into four wells (0.25 cm³ each) of a 96-well microtitre plate. Cultures were incubated in the dark, at 27 °C, for seven days. Fungal growth (or lack thereof) was evaluated at the end of incubation, gauging by eye the formation of mycelium (turbidity) and/or conidia. The lowest concentration that inhibited growth was taken as the MIC. Additionally, all the samples where no active growth was detected were used as inocula and spread, with a 1 μl loop, onto a malt extract agar medium (Oxoid). The plates were incubated in the dark, at 27 °C, for seven days. The lowest concentration of the testing compound which resulted in unviable conidia was taken as the MFC. MIC and MFC values should not be interpreted as absolute ones, but rather as an indication of the inhibitory and the fungicidal upper concentration limits.

Membrane and cell wall integrity assays

Four ionic liquids were chosen for the plasma membrane and cell wall integrity assays, namely [P_{4 4 4 n}]Cl, where n = 1, 4, 8 or 12. The selected testing concentrations (0.01, 0.1, 1, 10 and 100 mM) range below and above the previously obtained MIC and MFC values in order to facilitate the comparison of ionic liquids effects. Sodium chloride solutions (0.05, 0.5, 1 and 2 M) were included as osmotic stress controls (membrane integrity assays).

Aspergillus nidulans was cultivated on DG18 agar, at 27 °C, in the dark, for 5–6 days prior to harvest. Conidia were harvested with a saline solution (0.85% w/v NaCl) and used immediately. A suspension of 10⁶ conidia per cm³ of the testing ionic liquid solution was incubated for one hour at 27 °C under agitation (90 rpm). The incubation time for the cell wall integrity assay was prolonged to two and four hours, since within the first hour no significant alterations were detected for [P_{4 4 4 1}]Cl and [P_{4 4 4 4}]Cl. In both assays, conidia suspension was centrifuged (15 000 rpm, 4 °C) and washed three times with a saline solution in order to remove the ionic liquid.

For the membrane integrity assay, conidia were incubated afterwards with propidium iodide (PI) (λ_ex = 538 nm, λ_em = 617 nm, red), at the final concentration of 20 μM, for 15 min, at 27 °C in the dark and under agitation. Residual dye was removed by centrifugation (15 000 rpm, 4 °C) and washing (3×) and conidia were resuspended in 100 μl of saline solution with 10% v/v glycerol. Slides were mounted with 10 μl of the obtained suspension, in triplicates (technical replicates). Conidia were observed with an Axio Imager.M1 fluorescence microscope (Zeiss) using a 15 AlexaFluor 546 filter set (PI-stained conidia) and differential interference contrast (DIC, total number of conidia). The objective was an EC Plan-Neofluar with 40× magnification and images were captured with an ORCA-ER digital camera (Hamamatsu). Three fields of view from each slide were chosen randomly. Counting of conidia was carried out manually in JMicroVision v1.27. The percentage of membrane-damaged cells was obtained as (number of PI-stained conidia/total number of conidia) × 100. The experiment was repeated three times. When membrane damage was higher than 50%, the assay was repeated with shorter exposure periods (15, 30 and 45 minutes).

In the cell wall integrity assay, conidia were stained with Calcofluor White (CFW) (λ_ex = 346 nm, λ_em = 460 nm, blue), at a final concentration of 25 μM, incubated for 30 min at 27 °C, in the dark, under agitation and further processed as described above. In this assay, a 49 DAPI filter set (CFW-staining) and DIC were used with a Plan-Apochromat 63× oil immersion objective. Three fields of view, chosen randomly from each slide, were imaged. The experiment was repeated twice. This assay provided only qualitative analysis of the alterations of the cell wall.

The water activity (a_w) of some testing solutions was determined with a portable water activity indicator (HydroPalm AW1) following the manufacturer's instructions at 27 °C.

Scanning electron microscopy

Freshly harvested conidia of A. nidulans (as described in the previous subsection) were incubated with a 100 mM solution of [P_{4 4 4 n}]Cl, where n = 1, 8 or 12, at a final concentration of 10⁶ conidia per cm³. After two hours at 27 °C under agitation, conidia were centrifuged and washed (4×) with saline solution in order to remove the ionic liquid. Water was removed from the samples by lyophilisation and coated with a 15 nm layer of Au/Pd using a sputter coater (ex-Polaron E5100). Electron micrographs were recorded using an analytical field emission gun-scanning electron microscope (FEG-SEM: JEOL JSM7001F with Oxford light elements EDS detector) operated at 2 kV. The selected micrographs, from the biological triplicates, are regarded as representative ones of the overall sample (5000×) and individual conidia (15 000×).

Biodegradability assessment

Concentrations of ionic liquids used in the biodegradability assay were approximately one half of the previously determined MIC values. Fungal cultures of 20 cm³ were inoculated as described in the subsection Toxicity tests, and incubated in the dark at 27 °C under agitation (90 rpm) for 21 days. The culture extracts were filtered (glass fibre prefilters) and lyophilised to remove water. Solid remains were dissolved in 1 cm³ of deuteriated propanone, filtered and analysed by ¹H and ³¹P NMR spectroscopy.

Results and discussion

In the present work, the toxicological assessment of a series of quaternary phosphonium ionic liquids, namely [P_{4 4 4 n}]Cl, where n = 1, 3–8, 10, 12, or 14 (Fig. 1), was performed. While the [P_{6 6 6 n}]⁺ series of ionic liquids,³¹ and some based on the [P_{4 4 4 n}]⁺ cation,⁴¹ have already been investigated for their ecotoxicity, this is the first systematic study on [P_{4 4 4 n}]Cl.

Fig. 1 The structure of the alkyltributylphosphonium cation, [P_{4 4 4 n}]⁺, where n = 1, 3–8, 10, 12 or 14.

Filamentous fungi of the Penicillium genus show high tolerance and biodegradation ability towards different groups of ionic liquids.^42,43Aspergillus nidulans (whose genome is fully sequenced), due to its high phylogenetic correlation with Penicillium spp.,⁴⁴ was therefore selected for this study.

The toxic effect of the [P_{4 4 4 n}]Cl (n = 1, 3–8, 10, 12 or 14) was defined solely by the cation structure, and for n ≥ 4 increased, almost exponentially, with the elongation of the alkyl chain substituent (Table 1 and Fig. 2A). MIC and MFC values were distributed over a broad range (Table 1), e.g. MICs of [P_{4 4 4 4}]Cl and [P_{4 4 4 14}]Cl were 37.6 and 0.011 mM, respectively. The toxicity of [P_{4 4 4 n}]Cl (n = 4–8, 10, 12 or 14) as a function of the cation might be explainable, despite its limitations, by the 1-octanol/water partition coefficient, log₁₀(k₀). For [P_{4 4 4 4}]⁺ and [P_{6 6 6 14}]⁺, log₁₀(k_o) values were 2.5 and 6.9, respectively.³⁴ They suggest a relative scale of hydrophobicity for [P_{4 4 4 n}]⁺ when n ≥ 4, which is expected to increase linearly with the increase of the number of carbon atoms in the alkyl substituent.

Table 1 Numerical values of minimal inhibitory and fungicidal concentrations (MIC and MFC, respectively) of the alkyltributylphosphonium chlorides, [P_{4 4 4 n}]Cl, defined for Aspergillus nidulans.

n	MIC / mM	MFC / mM
1	32.5	92.5
3	77.6	87.6
4	37.6	85.23
5	7.53	17.56
6	4.52	7.03
7	1.91	2.41
8	0.8	0.9
10	0.24	0.24
12	0.021	0.021
14	0.011	0.011

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Fig. 2 (A) Minimal inhibitory and fungicidal concentrations (MIC and MFC, respectively) of the alkyltributylphosphonium chlorides, [P_{4 4 4 n}]Cl, where n = 1, 3–8, 10, 12 or 14, defined for Aspergillus nidulans. MIC and MFC values are plotted on a logarithmic scale. (B) Percentage of membrane-damaged conidia after one hour of incubation with alkyltributylphosphonium chlorides, [P_{4 4 4 n}]Cl (n = 1, 4, 8 or 12), obtained as (number of propidium iodide-stained conidia/total number of conidia) × 100.

While considering the MFC values, [P_{4 4 4 n}]Cl, where n = 1, 3 or 4, reported similar lethal effects. Their inhibitory effects showed that [P_{4 4 4 3}]Cl was an exception, since the MIC value was higher than expected, when compared with [P_{4 4 4 1}]Cl and [P_{4 4 4 4}]Cl. No clear explanation exists for this observation. It might be hypothesised that the longer alkyl substituents will lead to toxicity, probably by interaction with the cellular boundaries. Therefore, for [P_{4 4 4 n}]Cl, where n = 1, 3 or 4, the effect will be reasonably similar, i.e. controlled by the three butyl substituents.

The toxicity trend observed herein has been reported before in numerous studies focussing on other common groups of ionic liquids, such as the imidazolium and ammonium derivatives.^32,33 This trend, as aforementioned, is usually correlated with increased lipophilicity of the ionic liquids.³⁴ Through interaction with biological membranes, chemicals may cause a loss of membrane integrity, leakage of intracellular materials and, ultimately, cell death. Apart from studies on artificial models of the cell membrane (i.e. supported phospholipid bilayers⁴⁵ or liposomes),⁴⁶ to the best of our knowledge, hitherto no direct evidence of this effect has been reported in the ionic liquid literature. Infrared spectroscopy has been used as an attempt to directly detect the fraction of ionic liquid bound to the bacterial cells.⁴⁷ When applied to whole cells, however, the bioaccumulated and the bioadsorbed fractions are equally accounted for. Interestingly, using a similar method, it was possible to detect compositional alterations in the cell wall of diatoms after exposure to [C₄mim]Cl.⁴⁸

In this study, a rapid method to detect the effects of ionic liquids on cellular boundaries is proposed, instead of determining their fate in loco. Alterations of the plasma membrane integrity are commonly identified by microscopy or detection of intracellular components leakage.⁴⁹ While considering the former, fluorescence microscopy is a reliable technique for observation of cellular structures of filamentous fungi.⁵⁰Propidium iodide (PI) and Calcofluor White (CFW) fluorescent probes were successfully used in fungi for a rapid assessment of viability and membrane integrity⁵¹ and cell wall detection,^52,53 respectively. PI can only enter membrane-damaged cells where it binds to nucleic acids (detected as red fluorescence).

Aspergillus nidulans conidia were exposed to distinct concentrations of [P_{4 4 4 n}]Cl (n = 1, 4, 8 or 12) for one hour and stained with PI. The concentrations selected were 0.01, 0.1, 1, 10 and 100 mM, covering values below and above the MFC for all tested ionic liquids. For each testing condition a percentage of cells with membrane damage was determined. The saline solution control (0.85% w/v NaCl) showed approximately 5% of conidia with membrane damage, considered as a basal level of injured cells or a consequence of the harvesting procedure. The water activity (a_w) of the ionic liquid solutions was equal to that of distilled water (a_w = 1). In addition, after exposing conidia to sodium chloride solutions of up to 2 M (a_w ≥ 0.937), the level of membrane damage was similar to that observed in the saline solution control. Both observations suggest that membrane damage observed after exposure to ionic liquids was not due to osmotic stress.

Percentages of membrane-damaged conidia after exposure to concentrations below MFC values of [P_{4 4 4 n}]Cl, where n = 1, 4, 8 or 12, were reasonably similar to the control (Table 2, Fig. 2B). Obviously, the increase in concentration, still below the MFC, was followed by a slight increase in the number of membrane-damaged cells (∼4 to 14%).

Table 2 Percentage of membrane-damaged conidia after one hour of incubation with alkyltributylphosphonium chlorides, [P_{4 4 4 n}]Cl (n = 1, 4, 8 or 12), obtained as (number of propidium iodide-stained conidia/total number of conidia) × 100. Percentage of membrane-damaged conidia in the saline solution control was 5.11%. Emboldened values were obtained with concentrations above the MFC. The standard deviation of biological triplicates is shown in parenthesis.

n	Ionic liquid concentration / mM
	0.01	0.1	1	10	100
1	4.9 (0.7)	5.7 (1.0)	8.7 (2.9)	14.0 (4.5)	19.7 (3.4)
4	4.3 (0.4)	5.0 (0.4)	8.2 (1.5)	9.8 (4.2)	32.7 (4.3)
8	7.3 (3.2)	11.1 (1.0)	54.8 (0.9)	81.6 (3.0)	93.9 (1.5)
12	11.6 (2.4)	86.2 (4.2)	90.8 (4.6)	93.6 (2.3)	95.3 (0.2)

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While testing ionic liquids concentrations above the MFC value (i.e. leading to cell death), the [P_{4 4 4 n}]Cl with shorter (n = 1 or 4) and longer (n = 8 or 12) alkyl substituents induced moderate and severe membrane damage, respectively. [P_{4 4 4 8}]Cl and [P_{4 4 4 12}]Cl at the lowest ionic liquid concentrations causing death show membrane damage in 55% and 86% of the conidia, respectively. The values for [P_{4 4 4 1}]Cl and [P_{4 4 4 4}]Cl, however, did not exceed 20% and 33%, respectively. This is illustrated in Fig. 3, where the total number of conidia in each field of view obtained at the DIC (A–E) can be compared with the number of conidia stained by PI (A′–E′) in the control or in 100 mM solution of the ionic liquid. An extremely high degree of plasma membrane damage could be observed for [P_{4 4 4 n}]Cl, where n = 8 or 12.

Fig. 3 Membrane integrity assay. Conidia treated with 100 mM of alkyltributylphosphonium chlorides, [P_{4 4 4 n}]Cl (n = 1, 4, 8 or 12), during one hour of incubation, and stained with propidium iodide, PI. Left column (A–E) shows totality of conidia in the differential interference contrast (DIC); right column shows PI-stained conidia (A′–E′). Observed fluorescence indicates membrane-damaged conidia in: (A, A′) saline solution control; (B, B′) n = 1; (C, C′) n = 4; (D, D′) n = 8 and (E, E′) n = 12. Scale bar (E′): 20 μm.

Disruption of membrane permeability, apparently ruling the toxicity of the alkyltributylphosphonium chlorides carrying the longer alkyl substituents (n > 4), is not the main mode of toxicity for the shorter ones. High percentages of membrane damage within a short exposure time suggest that toxicity is probably due to loss of plasma membrane integrity, as reported before for other compounds.⁵¹ Permeabilisation of the plasma membrane might also be associated with the late stage of apoptosis (programmed cell death); however, even after two hours, apoptotic cells of A. nidulans show intact plasma membranes.⁵⁴ In our study, when the percentage of membrane-damaged conidia was higher than 50% ([P_{4 4 4 n}]Cl, where n = 8 or 12), the assay was repeated with shorter exposure periods (15, 30 and 45 minutes). The percentages of membrane-damaged conidia were similar, regardless of the exposure time (data not shown). These data make apparent that the observed membrane damage is a direct effect of the ionic liquids. Severe membrane disruption also explains the similarity between the observed MIC and MFC values, indicating that inhibition of fungal growth is accomplished by cell death.

In order to better understand the interactions of these ionic liquids with the conidia boundaries, the cell wall was also investigated. It is a well-organised complex of glucans, mannoproteins and chitin (constituting ∼20% of cellular biomass),⁵⁵ crucial for maintaining cell morphology and acting as a barrier against mechanical and environmental stress. CFW is a fluorescent probe that binds to chitin and glucans of the cell wall. When stained with CFW, conidia with intact cell walls show a homogenous distribution of fluorescence, when observed under the microscope (see Fig. 4A). If the continuity of the conidia cell wall is disrupted, heterogeneous binding of CFW and irregular distribution of the fluorescence are expected. This fluorescent dye was successfully used to detect cell wall alterations provoked by chemical⁵³ or environmental stress.⁵⁶Aspergillus nidulans conidia were treated with [P_{4 4 4 n}]Cl (n = 1, 4, 8 or 12) for one hour, at the concentrations used in the previously described membrane damage assessment. [P_{4 4 4 n}]Cl with concentrations above the MFC, where n = 8 or 12, led to alterations in the fluorescence distribution on the conidia cell wall classified as moderate and strong, respectively. However, in the case of n = 1 or 4, no alterations were observed. The influence of exposure time, namely one, two or four hours, is exemplified for [P_{4 4 4 n}]Cl (n = 1 or 12) in Fig. 4B and C. After two and four hours, concentrations above the MFC of all tested ionic liquids caused a greater effect in the fluorescence distribution compared with one hour exposure, with the exception of [P_{4 4 4 1}]Cl. Excluding the latter, concentrations above the MFC values appeared to cause cell wall damage. This suggests that, along with membrane damage, cell wall damage plays an important role in the toxicity of [P_{4 4 4 n}]Cl. Sena et al. also suggested this, based on the observation that algae mutants lacking cell walls showed, relatively to the wild type, higher and similar susceptibility to tetrabutylammonium bromide and 1-butyl-3-methylimidazolium bromide, respectively.⁵⁷

Fig. 4 Cell wall damage assay. Conidia treated with 100 mM of alkyltributylphosphonium chlorides, [P_{4 4 4 n}]Cl (n = 1 or 12), during one, two or four hours of incubation and stained with Calcofluor White M2R (CFW). (A) Saline solution control; (B) no obvious alteration of the cell wall in the presence of [P_{4 4 4 1}]Cl (even distribution of fluorescence); (C) significant cell wall damage in the presence of [P_{4 4 4 12}]Cl. Scale bar (C, 4 h): 10 μm.

Fungal cell wall damage is also expected to cause alterations in the overall morphology of conidia. Some of these changes were noted in the DIC of cells treated with ionic liquids for two hours or more (data not shown) and confirmed by FEG-SEM. The A. nidulans conidia from control samples showed a typical spherical morphology, with a convoluted surface.⁵⁸ Amongst the selected ionic liquids for this analysis, only [P_{4 4 4 1}]Cl treatment presented conidia with morphology similar to the control. [P_{4 4 4 8}]Cl and [P_{4 4 4 12}]Cl caused major alterations resulting in an irregular shape (Fig. 5). The conidia of the control sample and treated with [P_{4 4 4 1}]Cl had a characteristic flat-ball shape (Fig. 5A and B) which was not observed in the case of [P_{4 4 4 8}]Cl and [P_{4 4 4 12}]Cl (Fig. 5C and D). Conidia with intact membranes collapse during dehydration under high-vacuum conditions (sample preparation), due to a loss of turgor pressure.⁵⁹Membrane damage, on the contrary, circumvents this effect.

Fig. 5 Scanning Electron Microscopy (SEM) analysis of conidia treated with 100 mM of alkyltributylphosphonium chlorides, [P_{4 4 4 n}]Cl (n = 1, 8 or 12), during two hours of incubation. Micrographs (A–D) present conidia at 5000× magnification, and (A′–D′) show individualised conidium at 15 000×. (A, A′) Saline solution control; (B, B′) n = 1; (C, C′) n = 8; D, D′: n = 12. Scale bars (D, D′): 1 μm.

Quaternary phosphonium ionic liquids were observed to be very resistant to microbial attack, e.g.tricyclohexylphosphine- and trihexylphosphine-derived cations.⁶⁰ The capacity of A. nidulans to biodegrade aerobically [P_{4 4 4 n}]Cl (n = 1, 3–8, 10, 12 or 14) was also assessed. After 21 days, the extracellular extract was collected and analysed by ³¹P and ¹H NMR spectroscopy. Under the testing conditions, no alterations of the spectral peaks of the cations could be observed (data not shown), strongly suggesting null or very weak degradation. Recently, rapid mineralisation of [P_{4 4 4 4}]⁺ and [P_{4 4 4 6}]⁺ by Sphingomonas paucimobilis bacterium was inferred by an indirect impedance test.⁶¹ These data, despite the lack of direct proof of cation depletion from the cultivation media, should stimulate further exploration of the potential of phosphonium ionic liquids.

Conclusions

Toxicity of alkyltributylphosphonium chlorides [P_{4 4 4 n}]Cl (n = 4–8, 10, 12 or 14) towards A. nidulans conidia was observed to increase with the systematic elongation of one alkyl substituent. Analyses of conidia plasma membrane damage and cell wall integrity clearly suggest that the toxicity of [P_{4 4 4 n}]Cl, where n ≥ 4, is being ruled by direct interaction with both cellular structures. This behaviour may also explain the high similarity of their MIC and MFC values. For the shorter alkyl substituents (n = 1 or 3), despite provoking membrane damage in ∼20% of the conidia, inhibition of growth and death are more separated events. [P_{4 4 4 1}]Cl, even after four hours of exposure, had no perceived effects on the cell wall, suggesting that its ability to protect the membrane from damage depends on the ionic liquid structure.

The high potential of fluorescence microscopy to screen membrane damage provoked by other groups of ionic liquids has been demonstrated here. The selected probe is adequate for a broad range of organisms, such as bacteria⁶² and mammalian cells.⁶³ Screening membrane damage at lethal concentrations of ionic liquids will rapidly reveal if narcosis is ruling their toxicity. This robust toxicity screening method should stimulate ionic liquids research towards their conscious design.

Acknowledgements

M. P. and D. O. H. are grateful to Fundação para a Ciência e a Tecnologia (FCT) for the fellowships SFRH/BD/31451/2006 and SFRH/BD/66396/2009, respectively. The work was partially supported by a grant from Iceland, Liechtenstein and Norway through the EEA financial mechanism (Project PT015). G. A. and K. R. S. wish to thank the QUILL Industrial Advisory Board and the EPSRC (Portfolio Partnership Scheme, grant number EP/D029538/1) for their continuing support, and Cytec Industries, Canada, for support of G. A., and the supply of some ionic liquids. The fluorescence microscope used in this study belongs to the Cell Imaging Unit (UIC) at the Gulbenkian Science Institute, Oeiras, Portugal. The authors are thankful to Eng. Isabel Nogueira from Instituto Superior Técnico, Lisbon, Portugal, for the acquisition of SEM micrographs and Eng. Maria Cristina Leitão from ITQB for technical support. The NMR experiments were performed at CERMAX, the ITQB magnetic resonance centre. The NMR spectrometers are part of The National NMR Network (REDE/1517/RMN/2005), supported by “Programa Operacional Ciência e Inovação (POCI) 2010” and FCT.

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Footnote

† Equally contributing authors.

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