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

Article abstract of DOI:10.1039/c1nj20470j

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. The Royal Society of Chemistry and the Centre National de la Recherche Scientifique.

Full text of DOI:10.1039/c1nj20470j

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New Journal of Chemistry
A journal for new directions in chemistry
www.rsc.org/njc
Volume 36 | Number 1 | January 2012 | Pages 1–180
ISSN 1144-0546
COVER ARTICLE
Pereira et al.
Unravelling the mechanism of toxicity
of alkyltributylphosphonium chlorides
in Aspergillus nidulans conidia
1144-0546(2012)36:1;1-J

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PAPER
Unravelling the mechanism of toxicity of alkyltributylphosphonium
chlorides in Aspergillus nidulans conidia
a
a
b
´
Marija Petkovic,w Diego O. Hartmann,w Gabriela Adamova,
Kenneth R. Seddon,ab Luıs Paulo N. Rebeloa and Cristina Silva Pereira*abc
´
Received (in Montpellier, France) 1st June 2011, Accepted 13th September 2011
DOI: 10.1039/c1nj20470j
The mechanism of toxicity of alkyltributylphosphonium chlorides [P4 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.
inspired numerous patents for a broad range of applications,
Introduction
e.g. intermediates in the chemical synthesis of terpenes,10 anti-
static agents,11 biocides, either alone12 or combined with other
compounds,13,14 plant growth regulators15 and anti-cancer
agents.16
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 1C,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 environ-
mental impact.4,5
Amongst the quaternary phosphonium ionic liquids, the
tetraalkylphosphonium ones are, at present, the most studied.
They have been already investigated for diverse appli-
cations, e.g. solvents in separation processes17,18 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 acids25,26 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
[P6 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
A considerable amount of data is now available on the
chemistry1 and physical properties2 of ionic liquids, and numer-
ous applications have been already proposed.6 Imidazolium
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
In this work, we present a toxicity and biodegradability
assessment of [P4 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 under-
stand 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 illu-
strate the effect of a single imidazolium-based ionic liquid on
a
´
Instituto de Tecnologia Quımica e Biologica, Universidade Nova de
Lisboa, Av. da Repu´blica, 2780-157, Oeiras, Portugal
´
b The Queen’s University Ionic Liquid Laboratories, QUILL,
The Queen’s University of Belfast, Belfast BT9 5AG, UK
c
´
Instituto de Biologia Experimental e Tecnologica (IBET),
Apartado 12, 2781-901, Oeiras, Portugal.
E-mail: spereira@itqb.unl.pt
w Equally contributing authors.
c
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2012
56 New J. Chem., 2012, 36, 56–63

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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.
from 10 mM to 95 mM (distributed stepwise from 1.25 mM 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 1C, for seven days. Fungal
growth (or lack thereof) was evaluated at the end of incuba-
tion, 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 ml loop, onto a malt extract agar medium
(Oxoid). The plates were incubated in the dark, at 27 1C, 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.
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, ZnSO4ꢀ7H2O,
CuSO4ꢀ5H2O, FeSO4ꢀ7H2O, MgSO4ꢀ7H2O, NaNO3 and KCl;
and dimethylsulfoxide (dmso; 99.5%), propidium iodide
(Z 94%, stock solution 20 mM in dmso), Calcofluor White
M2R (Fluorescent Brightener 28, stock solution 5 mM in
water) and glycerol ( Z 99.5%), respectively. Deuteriated
propanone used in the biodegradability assessment was pur-
chased from EURISO-TOP (France).
Membrane and cell wall integrity assays
Four ionic liquids were chosen for the plasma membrane and
cell wall integrity assays, namely [P4 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).
Ionic liquids
All ionic liquids used in this study were prepared by QUILL
(Queen’s University Ionic Liquids Laboratory, Belfast, UK),
except for [P4 4 4 4]Cl and [P4 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 [P4 4 4 n]Cl, where n = 3, 5–8,
10 or 12, were prepared by nucleophilic (SN2) addition of
tributylphosphine to the respective 1-chloroalkane. [P4 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.
Aspergillus nidulans was cultivated on DG18 agar, at 27 1C,
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 1C 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 [P4 4 4 1]Cl and
[P4 4 4 4]Cl. In both assays, conidia suspension was centrifuged
(15 000 rpm, 4 1C) and washed three times with a saline solution
in order to remove the ionic liquid.
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 1C in
cryoprotective solution containing 0.85% w/v NaCl and 10%
v/v glycerol.
For the membrane integrity assay, conidia were incubated
afterwards with propidium iodide (PI) (lex = 538 nm, lem
=
617 nm, red), at the final concentration of 20 mM, for 15 min,
at 27 1C in the dark and under agitation. Residual dye was
removed by centrifugation (15 000 rpm, 4 1C) and washing
(3ꢂ) and conidia were resuspended in 100 ml of saline solution
with 10% v/v glycerol. Slides were mounted with 10 ml 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 40ꢂ
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) ꢂ 100. The experiment was repeated
three times. When membrane damage was higher than 50%, the
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 inhibi-
tion and death, respectively.
The minimal culture medium containing glucose (1.0 g lꢁ1
)
and K2HPO4 (1.0 g lꢁ1) was dissolved in distilled water,
sterilised in an autoclave (20 min; 121 1C), and supplemen-
ted with the mixture of essential salts, previously sterilised by
filtration: NaNO3 (3.0 g lꢁ1), ZnSO4ꢀ7H2O, (0.01 g lꢁ1),
CuSO4ꢀ5H2O (0.005
g
l
ꢁ1), MgSO4ꢀ7H2O (0.5
g l
ꢁ1),
FeSO4ꢀ7H2O (0.01 g lꢁ1) and KCl (0.5 g lꢁ1). The ionic liquids
were added to the minimal culture media with final concentrations
c
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New J. Chem., 2012, 36, 56–63 57

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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) (lex = 346 nm, lem = 460 nm, blue),
at a final concentration of 25 mM, incubated for 30 min at
27 1C, 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 63ꢂ 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.
+
] ,
Fig. 1 The structure of the alkyltributylphosphonium cation, [P4 4 4 n
where n = 1, 3–8, 10, 12 or 14.
alkyl chain substituent (Table 1 and Fig. 2A). MIC and MFC
values were distributed over a broad range (Table 1),
The water activity (aw) of some testing solutions was deter-
mined with a portable water activity indicator (HydroPalm
AW1) following the manufacturer’s instructions at 27 1C.
e.g. MICs of [P4
4]Cl and [P4
14]Cl were 37.6 and
4
4
4
4
0.011 mM, respectively. The toxicity of [P4 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 coeffi-
Scanning electron microscopy
cient, log10(k0). For [P4 4 4 4
were 2.5 and 6.9, respectively.34 They suggest a relative scale of
hydrophobicity for [P4 4 4 n
+ when n Z 4, which is expected
]
+ and [P6 6 6 14]+, log10(ko) values
Freshly harvested conidia of A. nidulans (as described in the
previous subsection) were incubated with a 100 mM solution
of [P4 4 4 n]Cl, where n = 1, 8 or 12, at a final concentration of
106 conidia per cm3. After two hours at 27 1C under agitation,
conidia were centrifuged and washed (4ꢂ) 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 (5000ꢂ)
and individual conidia (15 000ꢂ).
]
to increase linearly with the increase of the number of carbon
atoms in the alkyl substituent.
While considering the MFC values, [P4 4 4 n]Cl, where n = 1,
3 or 4, reported similar lethal effects. Their inhibitory effects
showed that [P4 4 4 3]Cl was an exception, since the MIC value
was higher than expected, when compared with [P4 4 4 1]Cl and
[P4 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 [P4 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 intra-
cellular 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
Biodegradability assessment
Concentrations of ionic liquids used in the biodegradability
assay were approximately one half of the previously deter-
mined MIC values. Fungal cultures of 20 cm3 were inoculated
as described in the subsection Toxicity tests, and incubated in
the dark at 27 1C under agitation (90 rpm) for 21 days. The
culture extracts were filtered (glass fibre prefilters) and lyophi-
lised 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 [P4
n]Cl,
4
4
Table 1 Numerical values of minimal inhibitory and fungicidal
concentrations (MIC and MFC, respectively) of the alkyltributyl-
phosphonium chlorides, [P4 4 4 n]Cl, defined for Aspergillus nidulans.
where n = 1, 3–8, 10, 12, or 14 (Fig. 1), was performed. While
+
the [P6 6 6 n
+
]
series of ionic liquids,31 and some based on the
[P4 4 4 n
]
cation,41 have already been investigated for their
n
MIC / mM
MFC / mM
ecotoxicity, this is the first systematic study on [P4 4 4 n]Cl.
Filamentous fungi of the Penicillium genus show high
tolerance and biodegradation ability towards different groups
of ionic liquids.42,43 Aspergillus 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 [P4 4 4 n]Cl (n = 1, 3–8, 10, 12 or 14)
was defined solely by the cation structure, and for n Z 4
increased, almost exponentially, with the elongation of the
1
3
4
5
6
7
8
10
12
14
32.5
77.6
37.6
7.53
4.52
1.91
0.8
0.24
0.021
0.011
92.5
87.6
85.23
17.56
7.03
2.41
0.9
0.24
0.021
0.011
c
58 New J. Chem., 2012, 36, 56–63
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Fig. 2 (A) Minimal inhibitory and fungicidal concentrations (MIC and MFC, respectively) of the alkyltributylphosphonium chlorides,
[P4
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.
4
4
(B) Percentage of membrane-damaged conidia after one hour of incubation with alkyltributylphosphonium chlorides, [P4 4 4 n]Cl (n = 1, 4, 8 or 12),
obtained as (number of propidium iodide-stained conidia/total number of conidia) ꢂ 100.
liquid bound to the bacterial cells.47 When applied to whole
cells, however, the bioaccumulated and the bioadsorbed frac-
tions 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
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 con-
centrations of [P4 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 approxi-
mately 5% of conidia with membrane damage, considered as a
basal level of injured cells or a consequence of the harvesting
procedure. The water activity (aw) of the ionic liquid solutions
was equal to that of distilled water (aw = 1). In addition, after
exposing conidia to sodium chloride solutions of up to 2 M
(aw Z 0.937), the level of membrane damage was similar to
In this study, a rapid method to detect the effects of ionic
liquids on cellular boundaries is proposed, instead of deter-
mining their fate in loco. Alterations of the plasma membrane
integrity are commonly identified by microscopy or detec-
tion of intracellular components leakage.49 While considering
the former, fluorescence microscopy is a reliable technique
for observation of cellular structures of filamentous fungi.50
Propidium 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
c
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New J. Chem., 2012, 36, 56–63 59

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Table 2 Percentage of membrane-damaged conidia after one hour of
incubation with alkyltributylphosphonium chlorides, [P4 n]Cl
4
4
(n = 1, 4, 8 or 12), obtained as (number of propidium iodide-stained
conidia/total number of conidia) ꢂ 100. Percentage of membrane-
damaged conidia in the saline solution control was 5.11%. Embol-
dened values were obtained with concentrations above the MFC. The
standard deviation of biological triplicates is shown in parenthesis.
Ionic liquid concentration / mM
0.01
0.1
1
10
100
n
1
4
8
12
4.9 (0.7)
4.3 (0.4)
7.3 (3.2)
11.6 (2.4)
5.7 (1.0)
5.0 (0.4)
11.1 (1.0)
86.2 (4.2)
8.7 (2.9)
8.2 (1.5)
54.8 (0.9)
90.8 (4.6)
14.0 (4.5)
9.8 (4.2)
81.6 (3.0)
93.6 (2.3)
19.7 (3.4)
32.7 (4.3)
93.9 (1.5)
95.3 (0.2)
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 [P4 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 (B4 to 14%).
While testing ionic liquids concentrations above the MFC
value (i.e. leading to cell death), the [P4 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. [P4 4 4 8]Cl
and [P4 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 [P4 4 4 1]Cl and [P4 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 (A0–E0) in the control or in 100 mM
solution of the ionic liquid. An extremely high degree of plasma
Fig. 3 Membrane integrity assay. Conidia treated with 100 mM of
alkyltributylphosphonium chlorides, [P4 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 inter-
ference contrast (DIC); right column shows PI-stained conidia
(A0–E0). Observed fluorescence indicates membrane-damaged conidia
in: (A, A0) saline solution control; (B, B0) n = 1; (C, C0) n = 4; (D, D0)
n = 8 and (E, E0) n = 12. Scale bar (E0): 20 mm.
membrane damage could be observed for [P4
n]Cl, where
4
4
n = 8 or 12.
Disruption of membrane permeability, apparently ruling the
toxicity of the alkyltributylphosphonium chlorides carrying
the longer alkyl substituents (n 4 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% ([P4 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 accom-
plished by cell death.
also investigated. It is a well-organised complex of glucans,
mannoproteins and chitin (constituting B20% 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.56
Aspergillus nidulans conidia were treated with [P4
n]Cl
4
4
(n = 1, 4, 8 or 12) for one hour, at the concentrations used
in the previously described membrane damage assessment.
[P4 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 [P4 4 4 n]Cl (n = 1 or 12) in Fig. 4B and C.
After two and four hours, concentrations above the MFC of all
In order to better understand the interactions of these
ionic liquids with the conidia boundaries, the cell wall was
c
60 New J. Chem., 2012, 36, 56–63
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Fig. 4 Cell wall damage assay. Conidia treated with 100 mM of
alkyltributylphosphonium chlorides, [P4 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 [P4
1]Cl (even
4
4
distribution of fluorescence); (C) significant cell wall damage in the
presence of [P4 4 4 12]Cl. Scale bar (C, 4 h): 10 mm.
tested ionic liquids caused a greater effect in the fluorescence
distribution compared with one hour exposure, with the excep-
tion of [P4 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 [P4 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. 5 Scanning Electron Microscopy (SEM) analysis of conidia treated
with 100 mM of alkyltributylphosphonium chlorides, [P4
n]Cl
4
4
(n = 1, 8 or 12), during two hours of incubation. Micrographs (A–D)
present conidia at 5000ꢂ magnification, and (A0–D0) show indivi-
dualised conidium at 15000ꢂ. (A, A0) Saline solution control; (B, B0)
n = 1; (C, C0) n = 8; D, D0: n = 12. Scale bars (D, D0): 1 mm.
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
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 [P4 4 4 1]Cl treatment presented conidia with morphology
similar to the control. [P4 4 4 8]Cl and [P4 4 4 12]Cl caused major
alterations resulting in an irregular shape (Fig. 5). The conidia
[P4 4 4 4] ]
+ and [P4 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.
of the control sample and treated with [P4
1]Cl had a
4
4
Conclusions
characteristic flat-ball shape (Fig. 5A and B) which was not
observed in the case of [P4 4 4 8]Cl and [P4 4 4 12]Cl (Fig. 5C
and D). Conidia with intact membranes collapse during
dehydration under high-vacuum conditions (sample prepara-
tion), due to a loss of turgor pressure.59 Membrane damage,
on the contrary, circumvents this effect.
Toxicity of alkyltributylphosphonium chlorides [P4
n]Cl
4
4
(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 [P4 4 4 n]Cl,
where n Z 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
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 [P4 4 4 n]Cl (n = 1, 3–8, 10, 12 or 14) was
c
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New J. Chem., 2012, 36, 56–63 61

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in B20% of the conidia, inhibition of growth and death are
more separated events. [P4 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.
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Acknowledgements
M. P. and D. O. H. are grateful to Fundac¸a˜o para a Cieˆncia 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 Partner-
ship Scheme, grant number EP/D029538/1) for their continu-
ing 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
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