Journal of Environmental Protection, 2011, 2, 298-303
doi:10.4236/jep.2011.23033 Published Online May 2011 (
Copyright © 2011 SciRes. JEP
Toxicity and Antimicrobial Activities of Ionic
Liquids with Halogen Anion
You Yu, Yi Nie
College of Chemistry and Chemical Engineering, Qufu Normal University, Qufu, China.
Received January 2nd, 2011; revised February 18th, 2011; accepted March 24th, 2011.
To investigate the eco-toxicity of ionic liquids (ILs), experiments on growth of three kinds of bacteria were carried out
for six common ILs with halogen anion b y a micro-calorimetric method at 310 K. The results indicate that the growth of
all the bacteria was inh ibited in the presence o f ILs. In addition, all ILs at definite concentrations show some toxicity to
Escherichia coli, Staphylococcus aureus and Bacillus subtilis. Anti-microbial activities of the ILs with halogen anion
are strongly related to structures of the ILs. An increase in alkyl group chain length corresponds with an increase in
toxicity, and the ILs with pyridinium cation exhibit stro ng er restraining effect than the same series ILs with imida zolium
Keywords: Toxicity, Ionic Liquids, Inhibition
1. Introduction
Ionic liquids (ILs) were applied in many fields such as
biological and chemical reactions for they are non-
flammable, sparsely volatile and thermally stable. For
these properties, air pollution was prevented greatly.
However, release of ILs into aquatic environments may
lead to water pollution because of their high solubilities.
In recent years, some reports that risks of ionic liquids
have been available in environment and organisms. Li
Xiaoy et al. [1-5] studied the toxicity of ionic liquids to
Daphnia magna. The results showed that the toxicity to
Daphnia magna increased with the increasing of n-alkyl
chain length. Zhang Feng et al. [6] carried out standard
test methods for evaluating the toxicity of chemicals to
three aquatic organisms. The results indicated that the
inhibiting effects increased significantly with increasing
concentrations of ILs. In addition, large-scale application,
ILs released into the environments will be inevitable.
These cause environmental and agricultural pollution.
Some reports [7,8] on the germination rate of seeds and
growth of seedlings have indicated different seeds have
different reaction to ILs which shown eco-toxicity.
Moreover, it is dangerous that poisoning organisms can
be increased by increasing the food chain. The food
chain of primary producers, primary consumers and
predators cause acute and chronic toxicity or cancer. So
studying the toxicity of ILs is important.
Microcalorimetry was confirmed to be valid as an al-
ternative method in the study of metabolism of the cell.
[9]. It is a useful tool for investigating the biological
processes because it permits the continuous monitoring
of the activity of a living process in situ without disturb-
ing the system, and the heat evolved or adsorbed is
strictly proportional to the rate of the biological proc-
esses [10,11]. With its abundant thermodynamic and
kinetic information, micro-calorimetry has been widely
applied in clinical analysis, pharmacology, ecology, bio-
technology and agriculture [12,13].
In this paper, the effects of common ILs with Halogen
anion (as shown in Scheme 1) on Escherichia coli (E.
coli), Staphylococcus aureus (S. aureus) and Bacillus
subtilis (B. sutilis) growth were investigated by micro-
calorimetry, and the bacterial growth rate constants
different concentrations (c) of ILs aqueous solutions
were calculated via power-time curves along with the
generalized Logistic equation. The
-c correlation equa-
tions were formulated. The results indicate antimicrobial
activities are related to the structures of ILs, indicating a
potential eco-toxicity of the ILs to the micro-organisms
in the water.
2. Theory
The growth of bacteria is often limited by some external
constraints, including substrate, product concentration,
temperature, pH-values and so on. In the logarithmic
growth phase, the number of bacteria and time are re-
lated according to [14,15]:
Toxicity and Antimicrobial Activities of Ionic Liquids with Halogen Anion 299
Scheme 1. Chemical structures of prepared Ils.
where Nt represents the number of bacteria at time (t),
is the growth rate constant,
is the deceleration rate
constant, and t is the experimental time.
By integrating Equation (1) with respect to time (t),
the following equation was obtained:
(1 )
NK e
 (2)
in Equation (2) represents the maxi-
mum bacterial number during the whole bacterial growth,
is the final multiple of the initial bacterial number
(being the integral constant).
Under the assumption that the heat production rate Pt
is proportional to the bacterial number, and P0 represents
the heat production rate by one bacterium, then,
Inserting it into Equation (2), the following equation
was obtained:
(1 )
Pm in Equation (3) is the maximum heat production
rate during the whole bacterial growth.
Using the experimental data Pt and t obtained from the
power-time curves, the growth rate constant (
) can be
calculated by Equation (3).
3. Experimental and Material
3.1. Instrument
A thermometric eight channel Thermal Activity Monitor
(3114/3236TAM Air, Sweden) in conjunction with the
operating and analytical software was used in this ex-
periment. With this instrument, reactions can be studied
at any given temperature in the temperature range of 5˚C
- 60˚C within ± 2 × 102˚C uncertainty. The system is
very sensitive, its detection limit is 1 × 105 W, and the
baseline stability (over a period of 24 h) is 2 × 105 W.
The measuring range contains between 60 mW and 600
mW. The maximum sample volume is 24 mL.
3.2. Materials
Standard strains of E. coli, S. aureus and B. subtilis were
used as the test organism.
The beef culture medium was used, containing 1 g
NaCl, 2 g peptone and 1 g beef extract in every 200 mL.
The pH of medium was adjusted to 7.2 - 7.4 before auto-
claving. The culture medium was sterilized in high pres-
sure steam at 121 for 30 min before experiments.
Ionic liquids with halide anion used in this experiment
were synthesized according to the published method
[16-17]. Aqueous solutions of ILs at different concentra-
tions were prepared using doubly distilled water.
3.3. Experimental Method
Ampoule mode was used in this experiment. The bacte-
rial suspension with a volume of 8 mL was poured into
each 24 mL glass ampoule in sterile conditions. After
adding ILs aqueous solution at different concentrations
into the ampoule, the ampoules were then sealed with
caps and placed into channels. Power-time curves of
continuous bacterial growth were recorded by computer.
When the baseline was re-established and became stabi-
lized, the process of bacterial growth was complete.
The micro-calorimeter was controlled at 310 K by
thermostat in the whole process, which is the optimum
growth temperature.
4. Results and Discussion
4.1. Power-Time Curves of Bacterial Growth
All ILs with halogen anion were tested for antimicrobial
activities against E. coli, S. aureus and B. subtilis. The
power-time curves of bacterial growth at 310 K in the
absence and presence of ILs have been determined, and
parts of the fit curves in logarithmic growth phase are
plotted in Figure 1. It can be seen that the slopes of ex-
ponential growth phase at different IL concentrations are
different. It can be concluded that the bacterial growth
phase changes with adding the ILs aqueous solution into
the culture medium.
4.2. Bacterial Growth Rate Constants
The data of Pt and t were obtained from Figure 1. Ac-
cording to Equation (3), the bacterial growth rate con-
stants (
) were calculated, and shown in Tables 1-3. The
growth rate constants (
) of the E. coli, S. aureus and B.
subtilis gradually decrease with the increase of the IL
concentration (c). This is mainly attributed to the inhibi-
tory effect of the halide ILs to some cells in the bacteria
suspension. The survivors remain metabolizing continu-
ously at a lower level of heat production rate, depending
on the concentration of IL in the solution.
4.3. Growth Rate Constants VS Concentrations
and Structure of ILs
The growth rate constants decrease with the increase of
L concentrations. The results indicate that the ILs with I
Copyright © 2011 SciRes. JEP
Toxicity and Antimicrobial Activities of Ionic Liquids with Halogen Anion
Copyright © 2011 SciRes. JEP
Table 1. Growth rate constants μ/min1 of E. Coli at different concentrations of ILs at 310 K.
c(mmol/L) 4.985 7.431 16.924 25.123 30.001 39.617
(min1) 0.06029 0.05459 0.045 0.04277 0.03509 0.02689
c(mmol/L) 1.44 9.132 11.133 13.784 18.084
(min1) 0.06456 0.05515 0.04788 0.04211 0.03701
c(mmol/L) 0.537 0.892 1.422 1.773 2.122 2.643
(min1) 0.05583 0.04861 0.04152 0.03558 0.02063 0.01481
c(mmol/L) 0 0.646 1.288 1.928 3.197
(min1) 0.09126 0.0768 0.04322 0.03798 0.01876
c(mmol/L) 0 0.235 0.352 0.584 0.699
(min1) 0.09126 0.07036 0.04481 0.0341 0.02268
c(mmol/L) 0 3.491 6.947 13.757 20.435 26.985
(min1) 0.09126 0.08043 0.07743 0.06148 0.0582 0.03734
Table 2. Growth rate constants μ/min1 of S. aureus at different concentrations of ILs at 310 K.
c(mmol/L) 0 1.508 2.508 3.998 4.985 5.967 8.885
(min1) 0.10048 0.07313 0.06869 0.05495 0.04731 0.03729 0.0259
c(mmol/L) 0 1.44 2.155 2.866 3.574 4.278
(min1) 0.10048 0.07859 0.05113 0.04211 0.04367 0.03254
c(mmol/L) 0 0.178 0.529 0.701 0.87 1.038
(min1) 0.10048 0.09172 0.07211 0.06802 0.03621 0.02643
c(mmol/L) 0 0.0646 0.129 0.256 0.32 0.477
(min1) 0.10048 0.09299 0.0827 0.0677 0.05613 0.03075
c(mmol/L) 0 0.0235 0.0352 0.0468 0.0584
(min1) 0.10048 0.05 0.03573 0.03095 0.0271
c(mmol/L) 0 4.358 6.947 8.662 12.913
(min1) 0.10048 0.09307 0.07162 0.06862 0.05074
Table 3. Growth rate constants μ/min1 of B. subtilis at different concentrations of ILs at 310 K.
c(mmol/L) 0 2.508 4.985 9.848 14.594
(min1) 0.11248 0.09984 0.0799 0.06637 0.0472
c(mmol/L) 0 1.798 3.574 5.327 7.06 8.771
(min1) 0.11248 0.10884 0.09289 0.07786 0.06444 0.04914
c(mmol/L) 0 0.716 2.844 5.286 7.005 8.703
(min1) 0.11248 0.09693 0.0766 0.04609 0.03397 0.01186
c(mmol/L) 0 0.646 1.288 1.928 2.564 3.197
(min1) 0.11248 0.10393 0.0887 0.07373 0.06781 0.05921
c(mmol/L) 0 0.59 1.177 1.693 2.342
(min1) 0.11248 0.09185 0.08533 0.0607 0.03813
c(mmol/L) 0 1.625 3.229 4.814 6.379 9.454
(min1) 0.112 48 0.091 81 0.079 63 0.0712 0.064 19 0.039 72
Toxicity and Antimicrobial Activities of Ionic Liquids with Halogen Anion 301
Figure 1. The power-time curves of E. coli, S. aureus and B.
subtilis at different ionic liquid concentrations in logarith-
mic growth phase at 310 K.
halogen anion show significant inhibition to E. coli, S.
aureus and B. subtilis growth. Moreover, their activities
are greatly affected by the alkyl chain length of the
cation ring, and variety of the cation.
For example, the growth rate constants of E. coli in the
presence of ILs follow the order: [BMIM]Br >
[EMIM]Cl > [BMIM]Cl > [HMIM]Cl. With the same
anion Cl-, the longer the alkyl chain length of the imida-
zolium ring is, the lower the bacterial growth rate con-
stant is. And with the same cation [BMIM]+, the growth
rate constant in the presence of [BMIM]Br is higher than
[BMIM]Cl. The growth rate constants of B. subtilis in
the presence of ILs follow the order: [HMIM]Cl >
[HPy]Cl >[OPy]Cl. With the same anion, the toxicity of
pyridinium ring is stronger than that of the imidazolium
ring. The inhibitory activity of [OPy]Cl is the highest
among all ILs.
-c relationship can be well represented by equa-
tions, as listed in Table 4. The correlations between
and c of E. coli, S. aureus were formulated by quadratic
equations, and the correlations between
and c of B. sub-
tilis were formulated by linear equations. The results
suggest all ILs may have the same inhibition mechanism
on the E. coli, S. a ureus and B. subtilis growth .
4.4. Inhibition Mechanism
E. coli, S. aureus and B. subtilis are significantly inhib-
ited by the IL-treatments. The toxic effect of ILs may be
related to a common cellular structure or process. It is
assumed that the toxicity mechanism of ILs is through
interaction with the cell wall and membrane, leading to a
membrane disruption [18]. ILs consisting of cation-anion
pairs is similar to the structure of surfactants, pesticides
and antibiotics that attack lipid structure, and induce po-
lar narcosis due to their interfacial properties, and may
cause membrane-bound protein disruption [19]. More-
over, disruption to cell membrane is related to the alkyl
chain length of the cation ring and variety of the cation
of ILs.
4.5. Conclusions
The effects of six halide ionic liquids on the bacterial
growth were studied by microcalorimetry. The ionic liq-
uids studied show inhibition activities on the metabolism
of E. coli, S. aureus and B. subtilis, and may follow the
same inhibition mechanism on the bacteria growth. The
antimicrobial activities of ILs are associated with the
alkyl side chain length of cation and the variety of the
cation. With the same anion, the longer the alkyl side
chain length of cation is, the stronger the antibacterial
activitie of the IL is. The toxicity of pyridinium ring is
stronger than that of the imidazolium ring. The micro-
calorimetry can be effectively used to study the micro-
bial growth and toxic properties of ILs. The antimicro-
bial effects of ILs should be considered in their potential
Copyright © 2011 SciRes. JEP
Toxicity and Antimicrobial Activities of Ionic Liquids with Halogen Anion 302
Table 4. μ-c correlation equations.
bacteria Ionic liquids c
[EMIM]Cl 62 4
1.74 109.76 100.0633cc
  20.9738R
[BMIM]Cl 52 3
1.22 101.50 100.0672cc
 
   20.9708R
[HMIM]Cl 42
5.70 100.01790.0650cc
  20.9974R
[BMIM]Br 62 3
5.16 101.69 100.0890cc
  20.9662R
[HPy]Cl 32
5.30 100.04030.0936cc
  20.9679R
[OPy]Cl 22
4.70 100.1320.0924cc
  20.9684R
[EMIM]Cl 42
6.30100.01370.0979 cc
  20.9873R
[BMIM]Cl 32
2.24 100.02580.102cc
  20.9519R
[HMIM]Cl 22
4.78100.02260.0993 cc
  20.9702R
[BMIM]Br 523
6.52103.22100.102 cc
 
  20.9575R
[HPy]Cl 22
5.93100.1180.100 cc
  20.9986R
S. aureus
[OPy]Cl 2
24.92.690.100 cc
 20.9983R
[EMIM]Cl 5
2.99100.109 c
  20.973R
[BMIM]Cl 3
7.57100.118 c
  20.981R
[HMIM]Cl 2
1.11100.108 c
  20.9929R
[BMIM]Br 3
7.20100.107 c
  20.9769R
[HPy]Cl 2
1.73100.112 c
  20.9673R
B. subtilis
[OPy]Cl 2
3.06100.114 c
  20.9716R
industrial applications and overall risk assessment.
5. Acknowledgements
Shandong Province Natural Scientific Foundation of
China (ZR2009BM035), and Shandong Province Science
Research Reward Foundation for Excellent Young and
Middle-aged Scientists (2008BS02021).
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