water research 44 (2010) 352–372

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Review

Environmental fate and toxicity of ionic liquids: A review

Thi Phuong Thuy Pham a, Chul-Woong Cho a, Yeoung-Sang Yun a,b,*

a
Department of Bioprocess Engineering, Chonbuk National University, Jeonju, Chonbuk 561-756, Republic of Korea
b
Division of Semiconductor and Chemical Engineering and Research Institute of Industrial Technology, Chonbuk National University,
Chonbuk 561-756, Republic of Korea

article info abstract

Article history: Ionic liquids (ILs) are organic salts with low melting point that are being considered as
Received 31 May 2009 green replacements for industrial volatile organic compounds. The reputation of these
Received in revised form solvents as ‘‘environmental friendly’’ chemicals is based primarily on their negligible vapor
27 August 2009 pressure. Nonetheless, the solubility of ILs in water and a number of literature
Accepted 12 September 2009 documenting toxicity of ILs to aquatic organisms highlight a real cause for concern. The
Available online 24 September 2009 knowledge of ILs behavior in the terrestrial environment, which includes microbial
degradation, sorption and desorption, is equally important since both soil and aquatic
Keywords: milieu are possible recipients of IL contamination. This article reviews the achievements
Ionic liquids and current status of environmental risk assessment of ILs, and hopefully provides
Toxicity insights into this research frontier.
Degradation ª 2009 Elsevier Ltd. All rights reserved.
Biodegradation
Environmental fate
Sorption

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353
2. Toxicological aspect of ILs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354
2.1. Effects of ILs in an enzyme level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354
2.2. Antibacterial activity of ILs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356
2.3. Toxicity of ILs to algae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361
2.4. Cytotoxicity of ILs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361
2.5. Phytotoxicity of ILs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
2.6. Toxicity of ILs to invertebrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
2.7. Inhibitory effects of ILs on vertebrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364
3. Environmental fate of ILs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364
3.1. Chemical degradation of ILs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364
3.2. Biodegradability of ILs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
3.3. Sorption of ILs in environmental systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367

* Corresponding author. Division of Semiconductor and Chemical Engineering, Chonbuk National University, Jeonju, Chonbuk 561-756,
Republic of Korea. Tel.: þ82 63 270 2308; fax: þ82 63 270 2306.
E-mail address: ysyun@chonbuk.ac.kr (Y.-S. Yun).
0043-1354/$ – see front matter ª 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.watres.2009.09.030
 water research 44 (2010) 352–372
4. Concluding remarks and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 368
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369
1. Introduction
Most of volatile organic compounds (VOCs) commonly used in
industrial applications cause a major concern in the current
chemical processing industry. The main problems are the
toxicity of the organic solvents to both the process operators
and the environment as well as the volatile and flammable
nature of these solvents which make them a potential explo-
sion hazard (Schmid et al., 1998). Recently, the deleterious
effects of many solvents combined with serious
environmental issues, such as atmospheric emissions and
contamination of aqueous effluents are making their use
prohibitive. Thus, many researchers have focused on the
development of ‘‘green engineering’’ which represents
research aimed at finding environmentally benign alternatives
to harmful chemicals. Among the neoteric solvents applicable
in ‘‘green technologies’’ ionic liquids (ILs) have garnered
increasing attention over the others such as supercritical CO2
(Blanchard et al., 1999; Blanchard and Brennecke, 2001;
Kazarian et al., 2000) and aqueous biphasic systems (Myasoe-
dov et al., 1995; Rogers et al., 1995; Willauer et al., 1999).
Ionic liquids, formerly known as molten salts, constitute
one of the hottest areas in chemistry these days. Basically,
they have melting points below 100  C, which can be achieved
by incorporating a bulky asymmetric cation into the structure
together with a weakly-coordinating anion (Ranke et al., 2004).
The unique, highly solvating, yet non-coordinating environ-
ment of ILs provides an attractive medium for various types of
chemical processes. Also, the physical properties of ILs can be
tailored by a judicious variation in the length and branching of
the alkyl chain and the anionic precursor (Fuler et al., 1997;
Huddleston et al., 2001). In this way, ILs can be made task-
specific for a certain application. The almost limitless struc-
tural possibilities of ILs, as opposed to limited structural
variations within molecular solvents, make them ‘‘designer
Fig. 1 – Applications of ionic liquids.
353
solvents’’ (Marsh et al., 2004; McFarlane et al., 2005; Sheldon,
2005). Some independent reports (Hagiwara and Ito, 2000;
Olivier, 1999; Welton, 1999) and many reviews (Earle and
Seddon, 2000; Rooney and Seddon, 2001) have highlighted ILs
as representing a state-of-the-art, innovative approach to
sustainable chemistry, with the argument that their vapor
pressure is immeasurably low and they are not flammable.
Recently, the application of these liquids as reaction media for
organic synthesis, catalysis, or biocatalysis has been well
documented (Earle and Seddon, 2000; Wasserscheid and
Keim, 2000; Welton, 1999) (Fig. 1). Gordon (2001) pointed out
that there is an obvious advantage in performing many reac-
tions in ILs due to the improvement in process economics,
reaction activity, selectivity and yield.
Although ILs can lessen the risk of air pollution due to their
insignificant vapor pressure, they do have significant solu-
bility in water (Anthony et al., 2001; McFarlane et al., 2005;
Wong et al., 2002). As a result, this is the most likely medium
through which ILs will be released into the environment. Ionic
liquids currently are not widely used in industrial applica-
tions; nonetheless, continued development and further use of
these solvents may lead to accidental discharge and
contamination. The properties that make them be the target
of industrial interest (i.e. high chemical, thermal stability and
non-volatility) suggest potential problems with degradation or
persistence in the environment. In general, the deficiency of
information and uncertainty surrounding the environmental
impact of ILs is a major barrier to the utilization of these
compounds by industry. Initial efforts have been made to
overcome this drawback and offer a preliminary insight into
the behavior of ILs in the aqueous environments. These
studies provided extensive data sets, e.g. on (eco)toxicity,
biodegradability, bioaccumulation and distribution of ILs in
different environmental compartments. Therefore, it is
necessary to consolidate all the available data in a single
354 water research 44 (2010) 352–372

review to lay the groundwork for more comprehensive
community and ecosystem investigations. The overall objec-
tive of this review is to systematically gather and interpret
existing information about the fate, removal options and
(eco)toxicological assessment strategies of ILs.
2. Toxicological aspect of ILs
The current literature represents a number of studies address-
ing the biological effects of ILs evaluated on the basis of
toxicological test systems. The ILs toxicities towards these
systems of different levels of biological complexity as well as
several environmental compartments (Fig. 2) are successively
discussed in the following subsections. All the structures of
IL compounds discussed in this review were listed in Table 1.
The acronyms used for these substances were adapted
from Ranke et al. (2007a). In this way, the cation head groups
were abbreviated as ‘‘IM’’ for imidazolium, ‘‘Py’’ for pyridinium,
‘‘Pyr’’ for pyrrolidinium, ‘‘Mor’’ for morpholinium, ‘‘Pip’’ for
piperidinium, ‘‘Quin’’ for quinolinium, ‘‘N’’ for quaternary
ammonium and ‘‘P’’ for quaternary phosphonium. The alkyl
chains attached to the head group were given as numbers
corresponding to the number of carbon in the alkyl residues. For
example, the 1-butyl-3-methylimidazolium moiety was deno-
ted as IM14. In case the carbon chain length equals or exceeds 10,
the numbers were separated by a hyphen (e.g. IM1-10 indicated
1-decyl-3-methylimidazolium). Particularly, for pyridinium
entities, the carbon-bound alkyl chains were appended to the
head group at different positions and the abbreviation was
made by noting the position of attachment and a symbol for the
attached group (e.g. Py4-2Me for 1-butyl-2-methylpyridinium).
The anionic components were shortened as they are in the
periodic table for the halides. For tetrafluoroborate, hexa-
fluorophosphate, bis(trifluoromethylsulfonyl)imide, dicyana-
mide and hydrogen sulfate the abbreviations were BF4, PF6,
(CF3SO2)2N, CN(N)2 and HSO4 in respective to their structural
formula.
2.1. Effects of ILs in an enzyme level
Enzyme inhibition data by ILs include those of the acetyl-
cholinesterase from electric eel (Electrophorus electricus)
(Arning et al., 2008; Jastorff et al., 2005; Matzke et al., 2007;
Ranke et al., 2007b; Stasiewicz et al., 2008; Stock et al., 2004;
Torrecilla et al., 2009; Zhang and Malhotra, 2005), the AMP
deaminase (Sk1adanowski et al., 2005) and the antioxidant
enzyme system of mouse liver (Yu et al., 2009a). The enzyme
acetylcholinesterase plays the most important role in nerve
response and function. Also, acetylcholinesterase catalyzes
the hydrolysis of acetylcholinesters with a relative specificity
for acetylcholine, which is a neurotransmitter common to
many synapses throughout mammalian nervous systems
(Fulton and Key, 2001; Massoulié et al., 1993). Thus, an inhi-
bition of acetylcholinesterase leads to various adverse effects
in neuronal processes, such as heart diseases or myasthenia

Fig. 2 – The flexible (eco)toxicological test battery considering aquatic and terrestrial compartments as well as different
trophic levels including enzymes, luminescent marine bacteria, freshwater green algae, mammalian cells, duckweed,
freshwater crustacean and zebrafish (Adapted from Matzke et al. (2007) by permission of the Royal Society of Chemistry).
 water research 44 (2010) 352–372 355

Table 1 – Selection of cationic and anionic structures of commonly used ionic liquids.
Head group Side chain

R2 R1 ¼ -C2H5, -C3H7, -C4H9,

+ -C5H11, -C6H13, -C7H15,
N N R1
-C8H17, -C9H19, -C10H21,
Imidazolium (IM) -C14H29, -C16H33, -C18H37,
-C19H39
R2 ¼ -CH3, -C2H5
CH3

CH3
R1
+
N R1 ¼ -C2H5, -C3H7, -C4H9,
-C5H11, -C6H13, -C8H17
CH3
Pyridinium (Py)

CH3
+
N
R1 ¼ -C4H9, -C6H13, -C8H17
R1
Pyrrolidinium (Pyr)

CH3
+
O N
R1 R1 ¼ -C4H9
Morpholinium (Mor)

Cation
R1
+
N
CH3 R1 ¼ -C4H9
Piperidinium (Pip)

+
N
R1 ¼ -C4H9, -C6H13, -C8H17
R1
Quinolinium (Quin)

R1 R2
+
N
R1-4 ¼ -CH3, -C2H5, -C3H7,
R4 R3 -C4H9, -C6H13
Quaternary ammonium (N)

R1 R2
+
P
R4 R3 R1-4 ¼ -C4H9, -C6H13, -C14H29
Quaternary phosphonium (P (

(continued on next page)

356 water research 44 (2010) 352–372
Table 1 (continued)
Head group
Anion Chloride
Bromide
Tetrafluoroborate [BF4]  B
Hexafluorophosphate [PF6]
Bis(trifluoromethylsulfonyl)imide [(CF3SO2)2N]
Dicyanamide [(CN)2N]
in humans (Chemnitius et al., 1999; Pope et al., 2005). Ranke
et al. (2007b) published a comprehensive collection of acetyl-
cholinesterase inhibition values for 292 compounds covering
a large variety of ILs and closely related salts. Among these
data, only those of the commonly tested ILs are summarized
in Table 2 for the ease of comparison of ILs toxicity from
molecular up to organism levels of biological complexity.
It was found that all observed inhibitory effects on the enzyme
could be exclusively accounted for the cationic moiety (Arning
et al., 2008). In particular, the IL with pyridinium as cationic
core structure inhibited the enzyme slightly stronger than the
imidazolium analogue whereas the compounds based on
phosphonium was less inhibitory. All anion species exerted
no effect on the enzyme activity with only exception of the
fluoride anion and the fluoride containing [SbF6] and [PF6]
species. Both species are known to readily undergo hydrolysis
in contact with moisture and thus the fluoride seems to be the
active compound. The non-inhibiting effects of anion might
be explained by their limited interactions with the active site
of this enzyme (Matzke et al., 2007). In addition, a correlation
between an increasing chain length of the side chains
connected to the cationic head groups and an enhanced
inhibitory potential of the ILs was found. It is believed that the
mechanism involves the similarity of the positively charged
imidazolium or pyridinium to the choline part that binds to
the anionic site of the enzyme, such that the longer alkyl
chain results in an improved fit (Stock et al., 2004).
Sk1adanowski et al. (2005) discussed the usefulness of in
vitro AMP deaminase inhibition assay as a potential molecular
method in prospective risk analysis of imidazolium-based ILs.
The results revealed that IM14 salts associated with [PF6],
[BF4], p-tosylate and [Cl] demonstrated a dose-dependent
inhibition of AMP deaminase activity. The IC50 values
(concentration of ILs inhibiting 50% of enzyme activity) for
those containing a fluorine compartment [PF6] and [BF4] are
lower (5 mM) than those for [Cl] and p-tosylate (10 mM), which
indicated the adverse effect of these fluoride-containing
anions. The other study on enzyme inhibition assay dealt with
Side chain
Cl
Br
F
-
F
F F
F
F - F
P
F F
F
O O
F3 C CF3
S S
- O
O N
-
N N N
the effects of acute exposure of intraperitoneal injection of
aqueous IM18 Br on the antioxidant enzymes of the treated
mouse liver (Yu et al., 2009a). The antioxidant enzymes tested
included superoxide dismutase, catalase, glutathione peroxi-
dase and glutathione-S-transferase. The results showed that
administration of IM18 Br modified activities of these defense
enzymes in mouse liver, and caused damage to livers of
treated mice at median lethal dose (LD50) of 35.7 mg/kg.
Though data published by these authors did not cover
a large variety of ILs, the enzyme inhibition assays suggest the
trend in which cationic moiety is the dominating factor
influencing the toxicity of ILs, especially when substituted
with a long alkyl side chain. Regarding the anion types,
perfluoronated ions are of toxicological interest due to
hydrolysis resulting in HF formation, while the others cause
less prominent effect.
2.2. Antibacterial activity of ILs
Bacteria serve as an ideal starting point for ILs toxicity
estimations as they have short generation times. Preliminary
toxicological investigations have shown quaternary ammo-
nium and pyridinium compounds have critical inhibitory
effects on a variety of bacteria and fungi (Babalola, 1998;
Kelman et al., 2001; Li et al., 1998). In the studies of Pernak’s
group (Cieniecka-Ros1onkiewicz et al., 2005; Pernak et al.,
2001a; Pernak et al., 2001b; Pernak and Chwa1a, 2003; Pernak
et al., 2003; Pernak et al., 2004a), they observed a trend of
increasing toxicity with an increase in the alkyl chain length
substituent in the pyridinium, imidazolium and quaternary
ammonium salts to various bacteria including rods, cocci
and fungi. As a measure of microbial activity of imidazolium
and pyridinium ILs with varying alkyl chain lengths, Docherty
and Kulpa (2005) also used a group of microorganisms
possessing a variety of physiological and respiratory activities.
It was found that imidazolium and pyridinium bromides
incorporated hexyl- and octyl-chain had considerable anti-
microbial effect to pure cultures of Escherichia coli,
Table 2 – Toxicity of ILs to different levels of biological complexity including enzyme, bacteria, algae, rat cell line, human cell lines, duckweed and invertebrate.
Compound Log10EC50 (mM)a

Acetylcholin esterase Vibrio Escherichia Pseudo kirchneriella Scenedesmus IPC-81 HeLa MCF7b Lemna Daphnia
fischeri coli subcapitata vacuolatus minor magnac

IM12 Cl 2.0614 4.5510 N.A. N.A. 2.78  0.0619 N.A. N.A. N.A. N.A. N.A.
4.33  0.1119
IM12 BF4 2.0514 N.A. 5.25  0.069 N.A. N.A. 3.4414 4.00  0.0420 N.A. N.A. N.A.
IM12 PF6 2.0514 N.A. N.A. N.A. N.A. 3.9214 N.A. N.A. N.A. N.A.
IM12 (CF3SO2)2N 2.0314 N.A. N.A. N.A. N.A. N.A. 3.26  0.0420 N.A. N.A. N.A.
IM13 Cl 2.2714 N.A. N.A. N.A. N.A. >4.3014 N.A. N.A. N.A. N.A.
IM13 BF4 2.28  0.0318 3.94  0.0613 N.A. N.A. N.A. 3.4714 N.A. N.A. N.A. N.A.
IM13 PF6 2.2214 N.A. N.A. N.A. N.A. >3.0014 N.A. N.A. N.A. N.A.
IM14 Cl 1.91  0.0411 3.71  0.144 N.A. 2.34  0.0121 2.26  0.0811 3.5514 N.A. N.A. 2.8211 1.934
2.955 1.93  0.061
3.34  0.136
3.47  0.0419

water research 44 (2010) 352–372

IM14 Br 1.90  0.0218 4.01  0.054 N.A. 3.46  0.0623 N.A. 3.4314 3.44  0.1120 N.A. N.A. 1.574
3.07  0.0313 1.56  0.071
3.355 1.85  0.0622
3.27  0.096
IM14 BF4 1.98  0.01811 3.55  0.0413 4.60  0.029 N.A. 2.1111 3.1214 3.72  0.0517 N.A. 2.497 1.684
3.10  0.176 3.66  0.0820 1.67  0.111
3.12  0.3516
IM14 PF6 2.15  0.0518 3.07  0.296 4.15  0.069 2.20  0.0421 N.A. 3.1014 4.14  0.2217 N.A. N.A. 1.854
1.85  0.101
IM14 (CF3SO2)2N 1.96  0.02111 3.39  0.084 2.55  0.159 1.80  0.0712 1.81  0.1511 2.6814 3.07  0.0820 N.A. 2.45  0.0811 N.A.
IM14 (CN)2N 1.95  0.0718 3.67  0.104 N.A. N.A. N.A. 3.1514 N.A. N.A. N.A. N.A.
2.995
IM15 Cl 1.9614 N.A. N.A. N.A. N.A. >3.0014 N.A. N.A. N.A. N.A.
IM15 BF4 1.8614 3.14  0.0213 N.A. N.A. N.A. >3.0014 N.A. N.A. N.A. N.A.
IM15 PF6 1.8514 N.A. N.A. N.A. N.A. >3.0014 N.A. N.A. N.A. N.A.
IM16 Cl 1.9214 1.9410 N.A. 1.92  0.0921 0.0819 2.8514 N.A. N.A. N.A. N.A.
2.32  0.166
2.91  0.0913
IM16 Br N.A. 1.42  0.124 N.A. 2.57  0.152 N.A. N.A. N.A. N.A. N.A. 0.784
0.815 1.06  0.0422
IM16 BF4 1.8814 3.18  0.0313 N.A. N.A. N.A. 2.9814 N.A. N.A. N.A. N.A.
IM16 PF6 1.8814 2.17  0.066 3.25  0.679 N.A. N.A. 2.9114 N.A. N.A. N.A. N.A.
IM16 (CF3SO2)2N 2.1514 N.A. 2.53  0.159 N.A. N.A. 2.2414 N.A. 2.818 N.A. N.A.
IM17 Cl 2.0714 N.A. N.A. N.A. N.A. 2.5314 N.A. N.A. N.A. N.A.
IM17 BF4 2.1214 2.44  0.0613 N.A. N.A. N.A. 2.5814 N.A. N.A. N.A. N.A.
IM17 PF6 1.9114 N.A. N.A. N.A. N.A. 2.3014 N.A. N.A. N.A. N.A.
IM18 Cl 1.6014 1.19  0.116 N.A. 1.4621 2.67  0.3719 2.0114 N.A. N.A. N.A. N.A.
1.01  0.0619
IM18 Br N.A. 0.63  0.074 N.A. 1.65  0.252 N.A. N.A. 2.48  0.0420 N.A. N.A. 1.334
0.075 0.54  0.1222

357
(continued on next page)
 358
Table 2 (continued)
Compound Log10EC50 (mM)a

Acetylcholin esterase Vibrio Escherichia Pseudo kirchneriella Scenedesmus IPC-81 HeLa MCF7b Lemna Daphnia
fischeri coli subcapitata vacuolatus minor magnac

IM18 BF4 1.53  0.02511 1.41  0.0713 N.A. N.A. 2.3011 1.5914 2.48  0.0220 2.848 0.907 N.A.
IM18 PF6 2.0314 0.95  0.126 2.64  0.159 N.A. N.A. 1.9614 N.A. N.A. N.A. N.A.
IM18 (CF3SO2)2N 2.0314 N.A. N.A. N.A. N.A. 1.6414 2.28  0.0220 N.A. N.A. N.A.
IM19 BF4 N.A. 0.72  0.0413 N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A.
IM1-10 Cl 1.0914 0.50  0.0713 N.A. N.A. 3.57  0.0619 1.3414 N.A. N.A. N.A. N.A.
0.23  0.0619
IM1-10 BF4 1.10  0.0418 0.18  0.0613 N.A. N.A. N.A. 0.7714 N.A. N.A. N.A. N.A.
IM1-10 PF6 1.6814 N.A. N.A. N.A. N.A. 1.5014 N.A. N.A. N.A. N.A.
IM1-14 Cl 0.5414 0.15  0.0719 N.A. N.A. 2.48  0.219 0.4214 N.A. N.A. N.A. N.A.
IM1-16 Cl 0.6814 0.23  0.0819 N.A. N.A. >2.0019 0.1914 N.A. N.A. N.A. N.A.
IM1-18 Cl 0.9614 1.45  0.0519 N.A. N.A. >2.0019 0.0114 N.A. N.A. N.A. N.A.
IM1-19 Cl 1.3614 N.A. N.A. N.A. N.A. 1.4014 N.A. N.A. N.A. N.A.

water research 44 (2010) 352–372

IM1-19 BF4 1.4314 N.A. N.A. N.A. N.A. 1.6514 N.A. N.A. N.A. N.A.
IM1-19 PF6 1.6214 N.A. N.A. N.A. N.A. 1.8514 N.A. N.A. N.A. N.A.
IM22 Br 2.0814 N.A. N.A. N.A. N.A. >3.0014 N.A. N.A. N.A. N.A.
IM23 Br 2.2114 N.A. N.A. N.A. N.A. >3.3014 N.A. N.A. N.A. N.A.
IM24 BF4 2.03  0.0118 2.8  0.0413 N.A. N.A. N.A. 3.2614 4.36  0.0917 N.A. N.A. N.A.
IM25 BF4 N.A. 3.1413 N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A.
IM26 Br 1.7714 N.A. N.A. N.A. N.A. 2.0114 N.A. N.A. N.A. N.A.
IM26 BF4 1.8414 2.15  0.0513 N.A. N.A. N.A. 2.2614 N.A. N.A. N.A. N.A.
IM2-10 Br 0.9214 N.A. N.A. N.A. N.A. 0.5314 N.A. N.A. N.A. N.A.
Py Cl >3.0014 N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A.
Py2 Cl 2.1014 N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A.
Py3 Br 2.2214 N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A.
Py3 (CF3SO2)2N 2.2114 N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A.
Py4 Cl 1.7014 3.41  0.084 N.A. 2.57  0.0621 2.59  0.1119 N.A. N.A. N.A. 2.32  0.1819 N.A.
2.645
3.18  0.0619
Py4 Br 1.7714 3.40  0.014 N.A. N.A. N.A. 3.9014 3.50  0.0720 N.A. N.A. N.A.
2.735
Py4 BF4 1.8014 N.A. N.A. N.A. N.A. 3.1814 N.A. N.A. N.A. N.A.
Py4 PF6 1.8414 N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A.
Py4 (CN)2N N.A. 3.31  0.104 N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A.
2.615
Py5 Br 1.5214 N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A.
Py5 (CF3SO2)2N 1.5514 N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A.
Py6 Cl 1.7214 N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A.
Py6 Br N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. 1.074
Py6 PF6 1.7614 N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A.
Py6 (CF3SO2)2N 1.8514 N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A.
Py8 Cl 1.6014 N.A. N.A. N.A. N.A. 1.2714 N.A. N.A. N.A. N.A.
Py8 (CF3SO2)2N 1.4014 N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A.
Py4-2Me Cl 0.7014 N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A.
Py4-2Me BF4 0.8214 N.A. N.A. N.A. N.A. 3.2514 N.A. N.A. N.A. N.A.
Py4-3Me Cl 1.1514 N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A.
Py4-3Me Br N.A. 2.75  0.134 N.A. 3.46  0.0623 N.A. N.A. N.A. N.A. N.A. 1.764
2.125
Py4-3Me BF4 1.53  0.0218 N.A. N.A. N.A. N.A. 3.3014 N.A. N.A. N.A. N.A.
Py4-3Me PF6 1.45  0.0218 N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A.
Py4-3Me (CN)2N 1.2214 2.66  0.054 N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A.
1.995
Py6-3Me Cl 1.0614 1.4410 N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A.
Py6-3Me Br N.A. 2.06  0.164 N.A. N.A. N.A. N.A. N.A. N.A. N.A. 0.594
1.485
Py6-4Me Cl 1.4414 N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A.
Py6-4Me BF4 1.4814 N.A. N.A. N.A. N.A. 2.1714 N.A. N.A. N.A. N.A.
Py8-3Me Cl 0.6414 N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A.
Py8-3Me Br N.A. 0.79  0.054 N.A. N.A. N.A. N.A. N.A. 1.008 N.A. 0.404
0.255
Py8-4Me Cl 1.1114 N.A. N.A. N.A. N.A. 1.6314 N.A. N.A. N.A. N.A.
Py8-4Me BF4 1.2214 N.A. N.A. N.A. N.A. 1.4914 N.A. N.A. N.A. N.A.
Pyr14 Cl 1.9214 >4.3019 N.A. N.A. 3.37  0.1019 >4.3014 N.A. N.A. 2.16  0.2519 N.A.

water research 44 (2010) 352–372

Pyr14 Br 1.9314 N.A. N.A. 3.67  0.283 N.A. 3.7714 N.A. 4.64  0.0215 N.A. N.A.
4.258
Pyr14 BF4 1.9114 N.A. N.A. N.A. N.A. 2.9014 N.A. N.A. N.A. N.A.
Pyr14 (CF3SO2)2N 2.1314 N.A. N.A. >2.3812 2.5319 3.0114 N.A. 3.148 2.98  0.3219 N.A.
Pyr14 (CN)2N 1.9814 N.A. N.A. N.A. N.A. 4.2314 N.A. N.A. N.A. N.A.
Pyr16 Cl 2.4814 2.9910 N.A. N.A. N.A. 2.9114 N.A. N.A. N.A. N.A.
Pyr16 (CF3SO2)2N 2.6014 N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A.
Pyr18 Cl 2.3614 N.A. N.A. N.A. N.A. 2.5914 N.A. N.A. N.A. N.A.
Pyr18 BF4 2.0214 N.A. N.A. N.A. N.A. 1.8214 N.A. N.A. N.A. N.A.
Pyr66 2.0814 N.A. N.A. N.A. N.A. 1.2314 N.A. N.A. N.A. N.A.
Mor14 Cl N.A. >4.3019 N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A.
Mor14 Br 2.7114 N.A. N.A. N.A. >4.0019 >4.3014 N.A. N.A. 3.11  0.1319 N.A.
Mor14 (CF3SO2)2N 2.7814 N.A. N.A. N.A. 2.0019 3.4314 N.A. N.A. 3.15  0.1319 N.A.
Pip14 Br 1.8314 4.27  0.0919 N.A. N.A. 3.27  0.1219 4.0314 N.A. 4.15  0.0415 0.4719 N.A.
4.158
Pip14 (CF3SO2)2N 1.7814 N.A. N.A. N.A. 2.0819 3.4114 N.A. 2.938 2.85  0.0719 N.A.
Quin4 Br 0.7914 N.A. N.A. N.A. N.A. 2.3214 N.A. N.A. N.A. N.A.
Quin4 BF4 0.6214 N.A. N.A. N.A. N.A. 2.1614 N.A. N.A. N.A. N.A.
Quin6 BF4 0.4814 N.A. N.A. N.A. N.A. 1.0714 N.A. N.A. N.A. N.A.
Quin8 Br N.A N.A. N.A. N.A. N.A. 0.0314 N.A. N.A. N.A. N.A.
Quin8 BF4 0.3014 N.A. N.A. N.A. N.A. 0.1714 N.A. N.A. N.A. N.A.
N1111 Br N.A. >5.004 N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A.
N1114 (CF3SO2)2N 2.6014 N.A. N.A. N.A. N.A. 3.6114 N.A. N.A. N.A. N.A.
N1123 (CF3SO2)2N 2.3414 N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A.
N1124 Cl 2.0614 N.A. N.A. N.A. >4.0019 >4.3014 N.A. N.A. 0.83  0.6719 N.A.
N1124 (CF3SO2)2N 2.0314 N.A. N.A. N.A. 1.78  0.1719 3.4313 N.A. N.A. N.A. N.A.
N2222 Cl 2.8014 N.A. N.A. N.A. N.A. >3.4814 N.A. N.A. N.A. N.A.
N2222 Br N.A. >5.004 N.A. N.A. N.A. N.A. 4.26  0.0420 N.A. N.A. N.A.
N2226 Br N.A. 2.46  0.164 N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A.
N4444 Br 2.3014 3.27  0.074 N.A. N.A. N.A. 2.2514 N.A. N.A. N.A. 1.474
2.6114 2.714 1.6614 0.954

359
P4444 Br N.A. N.A. N.A. N.A. N.A. N.A.
P666-14 Br 2.8514 3.41  0.024 N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A.
(continued on next page)
360 water research 44 (2010) 352–372

Staphylococcus aureus, Bacillus subtilis, Pseudomonas fluorescens

References: 1Bernot et al. (2005a); 2Cho et al. (2007); 3Cho et al. (2008a,b); 4Couling et al. (2006); 5Docherty and Kulpa (2005); 6Garcia et al. (2005); 7Jastorff et al. (2005); 8Kumar et al. (2009); 9Lee et al. (2005);
Luis et al. (2007); 11Matzke et al. (2007); 12Pretti et al. (2008); 13Ranke et al. (2004); 14Ranke et al. (2007b); 15Salminen et al. (2007); 16Samorı̀ et al. (2007); 17Stepnowski et al. (2004); 18Stock et al. (2004);
Daphnia and Saccharomyces cerevisiae. The anion performed nearly no
magnac effect on antimicrobial activity in the case of imidazolium

N.A.
N.A.
N.A.
N.A.
analogues (Docherty and Kulpa, 2005; Garcia et al., 2005; Lee
et al., 2005; Pernak et al., 2003; Pernak et al., 2004a) whereas
this was not the case for phosphonium salts. Within the group
Lemna

of alkyltrihexylphosphonium ILs in the study of Cieniecka-

minor

N.A.
N.A.
N.A.
N.A.
Ros1onkiewicz et al. (2005), both cation structure and the type
of anion had effects on the biological activity.
The antibacterial activity of ILs not only involves in
hampering the growth rate of microbes but also interferes
MCF7b

with their productivity. Matsumoto et al. (2004a) tested the

N.A.
N.A.
N.A.
N.A.

toxicity of imidazolium-based ILs to lactic acid producing

bacterium Lactobacillus rhamnosus to examine whether these
compounds can replace conventional organic solvents in the
1.90  0.0520

extractive fermentation of lactate. The results showed that

HeLa

the bacterium L. rhamnosus grew, consumed glucose, and

N.A.
N.A.

N.A.

produced lactate in the presence of imidazolium-based ILs.

A change of alkyl length in the imidazolium cation had little
difference on the survival of the cells. In a similar study
IPC-81

0.4814

0.2414
N.A.

N.A.

(Matsumoto et al., 2004b), they focused on hiochii bacteria,

Lactobacillus homochiochii and Lactobacillus fructivorans and also
found that the bacteria could produce lactic acid in the pres-
Scenedesmus
vacuolatus

ence of ILs. Nonetheless, the lactic acid producing activities of

Log10EC50 (mM)a

these bacteria generally decreased with the extension of alkyl

chain length in the imidazolium cation moiety.
N.A.
N.A.
N.A.
N.A.

Water miscible ILs had various effects on the physiology of

Clostridium sporogenes when tested as additives in culture
Pseudo kirchneriella

media or reaction media for reduction of nitrobenzene

(Dipeolu et al., 2008). In their study, 2-hydroxyethyltrimethyl-
subcapitata

ammonium dimethylphosphate and N,N-dimethylethano-

lammonium acetate increased the growth rate of C. spor-
ogenes; by contrast, IM14 BF4 and IM12 EtSO4 inhibited growth.
N.A.
N.A.
N.A.
N.A.

Stolte et al. (2007a); 20Wang et al. (2007); 21Wells and Coombe (2006); 22Yu et al. (2009).

Although IM12 EtSO4 inhibited growth, it was sufficiently

non-toxic to allow efficient reduction of nitrobenzene using
harvested cells. Thus, it is recommended that both non-
Escherichia

inhibitory and partially inhibitory ILs should be screened for

coli

use in biotransformation. Nonetheless, Ganske and Born-

N.A.
N.A.
N.A.
N.A.

b Toxicity of ILs is expressed as log10IC50 (mM) in case of MCF7 cell line.

scheuer (2006) referred that ILs could have substantial inhib-

itory effects on the growth of microorganisms when they
c Toxicity of ILs is expressed as log10LC50 (mM) in case of D. magna.

explored the effects of the two most commonly used ILs IM14
fischeri
Vibrio

BF4 and IM14 PF6 on the growth of E. coli, Pichia pastoris and
Bacillus cereus.
N.A.
N.A.
N.A.
N.A.

Regarding inhibition assays used in assessment of envi-

ronmental potential risk of a compound in aquatic milieu, the
Acetylcholin esterase

bioluminescence assay using Vibrio fischeri (formerly known as

a N.A. means not available (not determined).

Photobacterium phosphoreum) is one of the most applied (Kaiser

and Palabrica, 1991; Steinberg et al., 1995). This is a rapid, cost-
effective, and well-established method for toxicity determi-
3.47  0.0818

3.40  0.218

nation focusing on environmental issues, and also a standard

ecotoxicological bioassay in Europe (DIN EN ISO 11348). The
>3.3017
>3.4814

published data on ILs toxicity towards V. fischeri were listed on

Table 2 and were comprehensively interpreted in the study of
Table 2 (continued)

Peraccini et al. (2007). Although it has been claimed that

(CF3SO2)2N

modifications of the anion lead to changes in chemical and

(CN)2N

physical properties of ILs (Sheldon, 2001), no clear increase in

Compound

BF4
PF6

toxicity caused by the anion could be observed, and toxicity

P666-14
P666-14
P666-14
P666-14

seemed to be determined mainly by the cationic component

(Ranke et al., 2004). This is likely explained by the fact that
10

19

lipophilic part of the molecules can be intercalated into the

 water research 44 (2010) 352–372
membrane, whereas their ionic head group is at least partially
solvated in the aqueous solution, as suggested by Austin et al.
(1998). The ILs toxicity was also observed to correlate directly
with the length of the n-alkyl residues in the methyl-
imidazolium cation (Romero et al., 2008). Interestingly, Ranke
et al. (2004) noted a slight hormetic effect at concentrations
below inhibitory concentrations. Concerning the anionic
influence, compounds with [PF6] were found to be slightly
more toxic than compounds with other anions in their study
(Ranke et al., 2004). The anion [(CF3SO2)2N] showed no
intrinsic toxicity to V. fischeri in the report of Matzke et al.
(2007); in contrast, an increased in toxicity was found for all
tested compounds combined with [(CF3SO2)2N] for V. fischeri
(Stolte et al., 2007a). Couling et al. (2006) extended the biolu-
minescence inhibition assay to pyridinium derivatives and it
was noted that the quaternary ammonium compounds
seemed to be less toxic to V. fischeri than the pyridinium
and imidazolium analogues. Also, the quantitative structure-
property relationship (QSPR) modeling suggested that imida-
zolium cations, with two nitrogen atoms, are predicted to
be more toxic than pyridinium moieties, which only have
one nitrogen atom in the structure. In addition, the QSPR
correlation predicted that quaternary ammonium cations are
less toxic than those with cations containing nitrogen-bearing
rings, which was in agreement with the experimental results
(Couling et al., 2006). However, in contrast to the cases of
aromatic ILs and ammonium compounds, the authors were
unsuccessful in modeling the behavior of phosphonium salts
using the developed correlation.
2.3. Toxicity of ILs to algae
As algae are primary producers, either directly or indirectly, of
organic matter required by animals in freshwater food chains,
their ecology is crucial in providing the energy for sustaining
other higher trophic levels. The ubiquity of algae makes these
organisms ideal for toxicological studies and, because they
have a short life cycle they can respond quickly to environ-
mental change (Blaise, 1993; Lewis, 1995). To date, several
groups have focused their attention on the use of algal
primary producers to assess the effects of ILs to aquatic
environments (Cho et al., 2007; Cho et al., 2008a,b,c;
Grabinska-Sota and Kalka, 2006; Kulacki and Lamberti, 2008;
Lata1a et al., 2005; Matzke et al., 2007; Matzke et al., 2008; Pham
et al., 2008a,b; Pretti et al., 2009; Stolte et al., 2007a; Wells and
Coombe, 2006). Cho and co-workers used Pseudokirchneriella
subcapitata (formerly known as Selenastrum capricornutum) to
study the effect of different head groups, side chains and
anions of ILs on algal growth rate and photosynthetic activity.
The data revealed that the toxic influence of ILs on growth
rates were more significant than those of photosynthetic
performance (Pham et al., 2008b). Once again, the trend of
increasing toxicity with increasing alkyl chain length was
observed in their reports (Cho et al., 2007; Pham et al., 2008b).
Regarding the anionic effects, P. subcapitata was sensitive
to the anion moieties in the order: [SbF6] > [PF6] >
[BF4] > [CF3SO3] > [C8H17OSO3] > [Br] z [Cl]. In particu-
larly, it was found that with respect to IL incorporating per-
fluorinated anion (i.e. IM14 BF4), EC50 values (concentrations
which lead to a 50% reduction of the exposed organisms
361
relative to control) of the previously prepared stock solution
(6 months prior to experiment) were significantly lower
compared to those of the freshly made one (Pham et al.,
2008a). This might be due to hydrolytic effects of IM14 BF4
leading to fluoride formation, as confirmed by ion chroma-
tography analysis. This implies that after ILs are released into
the aqueous system; they can become more hazardous than
expected by laboratory data with fresh ILs. In a detailed study
on hydrolysis of fluoride-containing anions, Cho et al. (2008a)
showed that IM14 SbF6 generated a greater amount of fluoride
compared to IM14 BF4, but no fluoride formation occurred
with the hexafluorophosphate. When only small amounts of
fluoride ions were formed from IM14 SbF6 and IM14 BF4 within
96 h, the formed fluoride ion did not affect the algal growth
rate. Nevertheless, the fluoride ion formation from IM14 BF4
increased with incubating time of the stock solution; thus, the
toxicity might significantly increase according to the further
formed fluoride ions. In view of cationic effect, Pyr14 Br was
found to be the least toxic of all the ILs tested to P. subcapitata
(Cho et al., 2008b). For the limnic green alga Scenedesmus
vacuolatus, a severe toxicity was found for 1-butyl-4-(dime-
thylamino)pyridinium, whereas the quaternary ammonium
and morpholinium compounds exhibited no toxicity (Stolte
et al., 2007a). Despite the extensive studies on the toxicolog-
ical impact of ILs towards freshwater phytoplankton, inhibi-
tion mechanism of both the growth rate and photosynthetic
activity by ILs has not been described by the authors.
Lata1a et al. (2005), who selected two marine algae Oocystis
submarina (green algae) and Cyclotella meneghiniana (diatom) as
testing organisms, found that the two species differed
dramatically in their ability to recover from IL exposure.
Additionally, it was discovered that IL toxicity declined with
increasing salinity. The lower toxicity of IL in this case is
probably due to the reduced permeability of IL cations through
the algal cell walls. High amounts of chloride provide a good
ion-pairing environment for imidazolium cations, which
consequently compete with hydroxyl or silanol functional
groups in the cell-wall structure of green alga and diatom,
respectively. Though no information on EC50 values was
described, the facts emerged from this work provide useful
information in the further fate assessment of ILs in marine
environments.
2.4. Cytotoxicity of ILs
As a cellular test system, promyelotic leukemia rat cell line
IPC-81 has been frequently used in cytotoxicity assays of ILs,
with the reduction of the WST-1 dye as an indicator of cell
viability (Matzke et al., 2007; Ranke et al., 2004; Ranke et al.,
2007a; Stasiewicz et al., 2008; Stolte et al., 2006; Stolte et al.,
2007b; Torrecilla et al., 2009). It was observed that ILs with
polar ether, hydroxyl and nitrile functional groups within the
side chains exhibited low cytotoxicity compared to those
incorporated with ‘‘simple’’ alkyl side chains (Kumar et al.,
2009; Stasiewicz et al., 2008; Stolte et al., 2007b). Those func-
tional groups were thought to impede cellular uptake by
membrane diffusion and reduce lipophilicity based interac-
tions with the cell membrane (Stolte et al., 2007b). Taking
a closer look at the effects of sub-structural elements of ILs,
[(CF3SO2)2N] anion and 4-(dimethylamino)pyridinium cation
362 water research 44 (2010) 352–372
were described to have intrinsic effects of anion and head
group on cytotoxicity, respectively (Stolte et al., 2007b). The
well known side chain length effect (decrease in EC50 values
with elongation of the alkyl side chain) could also be
confirmed in these studies.
To date many studies have analyzed the toxicity of ILs on
human cell lines (Frade et al., 2007; Garcı́a-Lorenzo et al.,
2008; Hassoun et al., 2002; Kumar et al., 2009; Salminen et al.,
2007; Stepnowski et al., 2004; Wang et al., 2007). These in vitro
systems have been extremely beneficial in studying the
molecular basis of chemical’s biological activity, including its
toxic mode of action (Blaauboer et al., 1998) and could facil-
itate extrapolation of in vitro data with regard to possible
effects on humans (Malich et al., 1997). Most of studies dealt
with HeLa cells exemplifying prototypical cells of the human
epithelium which is normally the site of first contact of an
organism with toxicants. According to Stepnowski et al.
(2004), the cytotoxicity data implied that effects of IM14
cation coupled with chloride, tetrafluoroborate or hexa-
fluorophosphate were probably dependent on the anionic
moieties. The lowest effect concentrations for tetra-
fluoroborate species were found to be 0.63 mM, whereas
hexafluorophosphate and chloride inhibited HeLa cell growth
at comparably high concentrations of >10 mM. Surprisingly,
when the anion effect was compared, the strongest inhibition
was found for [PF6]. This might be due to hydrolysis
affecting fluoride formation, thus causing serious toxicolog-
ical consequences through the decomposition product.
A similar phenomenon was observed by Ranke et al. (2004) in
IPC-81 leukemia cells, where the lower toxicity of 1-n-butyl-3-
methylimidazolium hexafluorophosphate in comparison to
the hexafluorophosphate anion alone was explained by
reduced anion uptake due to the formation of an ion pair.
The anion in this ion pair can, however, also be partially
decomposed. This was shown in recent work by Swatloski
et al. (2003), who identified traces of 1-n-butyl-3-methyl-
imidazolium fluoride hydrate as a decomposition product
formed during the purification of the 1-n-butyl-3-methyl-
imidazolium hexafluorophosphate.
As shown by Wang et al. (2007) the phosphonium
bis(trifluoromethylsulfonyl)imide salts performed the highest
inhibitory to HeLa cells, followed by alkylimidazolium,
alkylpyridinium, alkyltriethylammonium and N-alkyl-N,N-
dimethyl-N-(2-hydroxylethyl)ammonium salts, in decreasing
order. For each cation class the toxicity increased with
increasing chain length of the alkyl substituent for a given anion:
1-ethyl-3-methylimidazolium bromide yielded an EC50 of
8.4 mM, substituting the ethyl moiety for a butyl group led to an
EC50 of 2.8 mM, and for an octyl moiety an EC50 of 0.3 mM. This
result was consistent with what has been observed in other
studies. Salts containing the tetrafluoroborate anion showed the
highest EC50, followed closely by bromide and chloride. Bis(tri-
fluoromethylsulfonyl)imide salts were significantly more toxic
than their halide counterparts. However, the effect of changing
the anion was smaller than that of changing the alkyl substit-
uent, e.g. while 1-ethyl-3-methylimidazolium tetrafluoroborate
was observed to have an EC50 of 9.9 mM, the corresponding
bromide and bis(trifluoromethylsulfonyl)imide salts had EC50 of
8.4 and 1.8 mM, respectively – these all considerably less
toxic than 1-octyl-3-methylimidazolium bromide.
The CaCo-2 cells were used in the study of Garcı́a-Lorenzo
et al. (2008) with the aim of a convenient screening method
for obtaining first rough estimates for the toxic potential of
ILs. The obtained data showed that in general, ILs with longer
alkyl chains were more lipophilic than those with shorter
alkyl chains. The former can be presumed to have a tendency
to be incorporated into the phospholipid bilayers of biological
membranes. In this respect, some authors have indicated
that the increased toxicity of longer ILs can be accounted for
enhanced membrane permeability altering the physical
properties of the lipid bilayer (Lata1a et al., 2005; Ranke et al.,
2004; Stepnowski et al., 2004). Additionally, it has been
proposed that the mode of toxic action for ILs takes place
through membrane disruption because of the structural
similarity of imidazolium-based ILs to detergent, pesticides
and antibiotics able to cause membrane-bound protein
disturbance (Docherty and Kulpa, 2005). Recently, Ranke et al.
(2007a,b) have demonstrated that lipophilicity of ILs domi-
nates their in vitro cytotoxicity over a wide range of structural
variations. The contribution of the anionic part of the ILs to
the observed biological effect was evaluated by comparing
the EC50 values obtained for the cations IM16 and IM18,
combined with two different anions [Cl] and [PF6]. For both
cations, a stronger toxic effect was found for chloride deriv-
atives, but not for fluoride containing hexafluorophosphate.
A similar result was reported by Stock et al. (2004) where the
inhibitory effects of IM14 Cl and IM14 PF6 on the acetylcho-
linesterase activity were compared. In addition, slightly
higher cytotoxicity for the chloride derivative has also been
observed when the cytotoxicity of IM14 Cl and IM14 PF6 on
HeLa cells was tested (Stepnowski et al., 2004). This implies
the effect of perfluorinated ions is not drastic to all but vary
according to species of organisms tested. Several authors
have pointed out that altering the anion has only minimal
effects on the toxicity of several imidazolium compounds
(Bernot et al., 2005a; Garcia et al., 2005; Ranke et al., 2004).
This indicates that ILs toxicity seems to be related to the alkyl
chain branching and to the hydrophobicity of the imidazo-
lium cation but not to the various anions. In this respect,
a recent study using the IPC-81 rat leukemia cell line with
a large pool of anions demonstrated that most of the
commercially available anions showed no or only marginal
cytotoxic effects. However, anionic compartments with
lipophilic and hydrolysable structural elements are likely to
be of considerable relevance with respect to the toxicity of ILs
(Stolte et al., 2006).
In a recent study (Frade et al., 2007), the human cell lines
such as HT-29 and CaCo-2 cells were utilized to estimate the
inhibitory effect of ILs with several types of cations and
anions. In both cells, IM14, IM12OH (1-(2-hydroxyethyl)-3-
methylimidazolium), IM12O2O1 (1-(2-(2-methoxyethox-
y)ethyl)-3-methylimidazolium) and cholines were the least
toxic cations independently of the anion. Within the studied
combinations, it can be noted that IM14 PF6, IM14 acesulfame,
IM12OH BF4/PF6, IM12O2O1 BF4/PF6, IM12OH acesulfame and
IM12OH saccharine are not toxic and present good alterna-
tives to organic solvents. Meanwhile, increasing the length of
the substituent chain may contribute to a significant
increasing of imidazolium toxicity. It was also noted that
[(CF3SO2)2N] anion decreased the toxicity to a large extent,
 water research 44 (2010) 352–372
independently of the cation and for both cell types, which was
in accordance with Salminen et al. (2007).
2.5. Phytotoxicity of ILs
The studies on phytotoxic activity of ILs were conducted
mostly on the duckweed, Lemna minor, a common aquatic
vascular plant (Jastorff et al., 2005; Larson et al., 2008; Matzke
et al., 2007; Stolte et al., 2007a). In general, 1-alkyl-3-
methylimidazolium compounds with longer alkyl chains were
more toxic to L. minor than those with short alkyl chain
lengths. Imidazolium and pyridinium cations with butyl
groups had similar EC50s (the concentrations that produced
a 50% reduction in root growth) (39.07 and 32.54 mM, respec-
tively); while the equivalent ammonium cation had a much
higher EC50 (101.48 mM; i.e., less toxic) (Larson et al., 2008). In
consideration of anionic effect, [(CF3SO2)2N] was found to
cause moderate toxicity to this duckweed (EC50 ¼ 6300 mM)
(Matzke et al., 2007). On the other hand, this anion had no or
even a positive influence on the observed effects on L. minor
(Stolte et al., 2007a).
Focusing on the terrestrial environment, Matzke et al.
(2009a) investigated the influence of differently composed
soils, with varying contents of the clay minerals smectite and
kaolinite, on the toxicity of different anion species of imida-
zolium-based ILs towards the wheat Triticum aestivum. The
data showed that IM14 (CF3SO2)2N appeared the most toxic,
independently of the type and concentration of added clay.
This is totally in contrast to the findings of Stolte et al. (2007a),
who reported that [(CF3SO2)2N] caused no harm to L. minor,
indicating the toxic effect of this anion is different between
certain plants. The toxicity of 1-butyl-3-methylimidazolium
incorporated chloride, tetrafluoroborate and hydrogen sulfate
was mainly controlled by the cationic moiety. The observed
effects varied according to the added clay type and clay
concentration. An increase of clay content resulted in less
inhibitory effects of these substances. On the contrary, for
IM14 combined with bis(trifluoromethylsulfonyl)imide the
addition of clay minerals led to higher toxicity compared to
the reference soil. Since results are contradictious further
study is necessary to unravel the underlying mechanism.
Moreover, a detailed study on the effect of IM14 BF4 on the
wheat T. aestivum seedlings (Wang et al., 2009) showed that
IM14 BF4 was hazardous to the early development of wheat
and had varying effects on different organs. At low concen-
trations, IM14 BF4 did not inhibit, and even promoted, wheat
seedling growth. Nonetheless, at high concentrations, this IL
inhibited wheat seedling growth significantly and decreased
chlorophyll content, thereby reducing photosynthesis and
plant growth. Therefore, the authors suggested that dilution
could decrease the toxicity of IM14 BF4 to plants and would be
a good method for remediating IL-polluted environments.
In another research, the phytotoxicity tests of chiral ILs
containing (-)-nopyl derivatives were carried out in a plant
house using spring barley (Hordeum vulgare) which is a mono-
cotyledonous plant, and a common radish (Raphanus sativus L.
subvar. radicula Pers.) which is a dicotyledonous plant (Ba1c-
zewski et al., 2007). According to the data obtained, increasing
the concentration of ILs resulted in a systematic decrease in
the crop fresh weight of total sprouts and the crop fresh
363
weight per plant, both for spring barley and for common
radish. It could also be noted that common barley was a more
resistant plant which fairly well tolerates test IL concentra-
tions up to 200 mg kg1 of soil; whereas, for radish, the growth
and development inhibiting concentration is 100 mg kg1 of
soil. Using the same target plant (H. vulgare), Pernak et al.
(2004b) reported that the 1,3-dialkoxymethylimidazolium
tetrafluoroborate salts introduced to the soil at concentration
of 1,000 mg kg1, or 100 mg kg1 dry mass of soil, were found
to exert a phytotoxic effect on monocotyledonous plants.
On the other hand, at a concentration of 10 mg kg1 no such
effect on the growth of the roots was notified. Concerning
phytotoxicity of ILs to garden cress (Lepidium sativum L.) in soil
environment, Studzińska and Buszewski (2009) have proved
that hazardous effects of imidazolium ILs are closely
connected with organic matter content in soil. Soil with more
organic carbon was observed to sorb IL cations more exten-
sively than soil with little or no organic matter; hence, the
more fertile in soil, the lower probability of hazardous effect of
ILs to plants. On the other hand, the hazardous character of
analyzed ILs was strongly connected with their hydropho-
bicity, indicating that the more hydrophobic IL, the higher
decrease of seed germination.
Although intensive work has not been conducted on
phytotoxic influence of ILs, the available data offer initial
hints for environmental scientists dealing with the potential
impact of ILs towards aqueous and terrestrial plants.
2.6. Toxicity of ILs to invertebrates
Ecotoxicological literature of ILs to invertebrates mainly focus
on the use of Daphnia magna as a test organism (Bernot et al.,
2005a; Couling et al., 2006; Garcia et al., 2005; Grabinska-Sota
and Kalka, 2006; Luo et al., 2008; Nockemann et al., 2007; Pretti
et al., 2009; Samorı̀ et al., 2007; Wells and Coombe, 2006; Yu
et al., 2009b). Daphnia is an important link between microbial
and higher trophic levels (McQueen et al., 1986), and has been
the subject of hundreds of intensive ecological studies. The
results of all studies again observed the well-established link
between toxicity and alkyl chain length of the tested ILs
containing imidazolium, pyridinium or quaternary ammo-
nium as counter cations. The most toxic compound towards
D. magna was found to be IM18 Br whereas the least toxic one
was IM14 Cl with log10EC50 values of 1.33 and 1.93, respec-
tively (Table 2). Also, the nature of the anion was suggested to
have smaller effects compared to those of the cation. In a
recent study, Luo et al. (2008) investigated the developmental
toxicity of IM18 Br on D. magna. It was found that this
compound exhibited toxicity on the development of three
generation of D. magna with the decrease of number of
offspring and average brood size correlated to increasing IM18
Br concentrations. This indicated that IM18 Br could cause
deleterious effect to the population of Daphnia and indirectly
disturb freshwater food webs. Couling et al. (2006) used
experimental data to determine which part of the IL molecule
is responsible for the observed toxic effects through a quanti-
tative structure-property relationship (QSPR) modeling. In this
respect, correlative and predictive equations were generated
and proved that there was a distinct influence of the length of
alkyl residues attached to the aromatic nitrogen atoms
364 water research 44 (2010) 352–372
towards D. magna. Moreover, the models predicted that the
toxicity increased slightly with increasing number of aromatic
nitrogen atoms in cation ring. This implies that ammonium
salts are less toxic than pyridinium salts, which in turn are
less toxic than imidazolium moieties. Interestingly, it was
noted that methylating the aromatic carbons could be
effective in reducing toxicity to D. magna, indicating that 1-n-
butylpyridinium bromide can be more toxic than 1-n-butyl-3-
methylpyridinium bromide, which is more toxic than 1-n-
butyl-3,5-dimethylpyridinium analogue. The QSPR, though is
still in its infancy, has contributed initial guidelines for
a rationale design of a new category of ILs with an acceptable
environmental profile.
Other studies include data on the snail Physa acuta (Bernot
et al., 2005b), the spring tail Folsomia candida (a soil inverte-
brate) (Matzke et al., 2007), Caenorhabditis elegans (a soil
roundworm) (Swatloski et al., 2004) and Dreissena polymorpha
(zebra mussel) (Costello et al., 2009). It was also demonstrated
a positive relationship between alkyl chain length and toxicity
in these reports. In the research of Bernot et al. (2005b), the
estimated LC50 (median lethal concentration) ranged from
3.50 to 1799.8 mM (0.54 to 3.26 in the logarithmic form), which
implied that P. acuta are less sensitive to ILs than are D. magna
(log10LC50 (mM) ranging from 1.33 to 1.93 (Table 2)). Also, the
authors observed that at low concentrations, the IL may
suppress snail movement, but concentrations above this
threshold level trigger an escape response, causing the
organism to move faster. Grazing patterns, nonetheless,
showed that snails grazed less at higher IL concentrations.
Physa spp. are key components of freshwater food webs,
because they graze algae and are themselves important prey
for fish and invertebrate predators (Bernot and Turner, 2001;
Osenberg and Mittelbach, 1989). Thus, nonlethal IL concen-
trations affected P. acuta behaviors, potentially influencing
individual fitness and good web interactions.
2.7. Inhibitory effects of ILs on vertebrates
Zebrafish (Danio rerio) plays an important role in ecotoxicology
as a prominent model vertebrate. Concerning toxicity of ILs to
the zebrafish, Pretti et al. (2006) revealed that ILs may cause
a completely different effect on fish according to their chem-
ical structures. As imidazolium, pyridinium and pyrrolidi-
nium showed a LC50 (lethal effect) >100 mg L1, they could be
regarded as non-highly lethal towards zebrafish. On the other
hand, the ammonium salts showed LC50 remarkably lower
than that reported for organic solvents and tertiary amines.
In general, these data referred that fish are less sensitive to ILs
toxicity compared to other species belonging to lower trophic
levels.
In a recent report, Li et al. (2009) used the frog Rana nigro-
maculata as an amphibian model for toxicity testing.
Amphibians are often the main vertebrate group prone to
contaminant exposure in aquatic systems mostly because
their larvae live in water (Lahr, 1997; Mann and Bidwell, 2000).
In their study, they evaluated the toxic effects of IM18 Br on
the early embryonic development of the frog R. nigromaculata.
The results demonstrated that the highest embryonic
mortality occurred in the neural plate stage, followed by the
early gastrula and early cleavage stages with the LC50 values
being 42.4, 43.4 and 85.1 mg/L, respectively, indicating that the
developmental toxicity of IM18 Br in the frog was stage-
sensitive. The number of dead embryos was also found to
increase with the increasing concentrations of the IL IM18 Br.
The developmental impact of IM18 Br was claimed not only in
this finding but also in the work of Luo et al. (2008), who
investigated on D. magna.
Other work in the literature has focused on the acute
toxicity of ILs on rats and mice (Bailey et al., 2008; Cheng et al.,
2009; Landry et al., 2005; Pernak and Czepukowicz, 2001; Sipes
et al., 2008). The values of acute toxicity of 3-hexyloxymethyl-
1-methylimidazolium tetrafluoroborate were found to be
LC50 ¼ 1400 and 1370 mg kg1 for female and male Wistar rats,
respectively (Pernak and Czepukowicz, 2001). Bailey et al.
(2008) studied the effects of prenatal exposure of mice to IM14
Cl due to the potential for human exposure as a result of water
or soil contamination from industrial effluent or accidental
spills. As shown in the experimental data, after being con-
tacted to the IL, fetal weight was considerably reduced at the
two highest concentrations (169 and 225 mg kg1 d1).
Malformations were also somewhat more numerous at the
highest dosage, suggesting that IM14 Cl may be teratogenic.
Maternal toxicity was also present, indicating that IM14 Cl
appeared to be developmentally toxic at maternally toxic
dosages. Also, IM14 Cl has been shown to cause thermal irri-
tation when applied topically to rats, but produced only
minimal contact sensitization when evaluated in the mouse
local lymph node assay (Landry et al., 2005). Additionally, in
this report, it is worth noting that the transdermal toxicity of
IM14 Cl was influenced by the vehicle of administration. Use
of the organic solvent, dimethylformamide, accentuated the
acute toxicity. Very high concentrations of IM14 Cl (up to 95%
IM14 Cl in water) applied to the rat skin were markedly less
acutely toxic. This result may have a practical guideline that to
reduce the acute toxicity, ILs can be handled in pure form with
water as a co-solvent.
3. Environmental fate of ILs
3.1. Chemical degradation of ILs
Ionic liquids possess excellent chemical and thermal stability,
which gives, unfortunately, a negative aspect for their treat-
ment after usage prior to disposal. To assess the persistence of
ILs in the environment as well as verify possibilities of their
cleanup by chemical methods, several groups have focused
their attention on oxidative and thermal degradation of ILs in
aqueous media (Awad et al., 2004; Baranyai et al., 2004;
Berthon et al., 2006; Itakura et al., 2008; Li et al., 2007; Mor-
awski et al., 2005; Siedlecka and Stepnowski, 2009; Siedlecka
et al., 2008a,b; Stepnowski and Zaleska, 2005). Pioneering work
in the field of oxidative degradation was done by Stepnowski
and Zaleska (2005) and Morawski et al. (2005) who showed that
the greatest degradation efficiency for imidazolium ILs was
achieved with a combination of UV light and a catalytic
oxidant such as hydrogen peroxide or titanium dioxide.
Subsequently, Li et al. (2007) studied the oxidative degradation
of 1,3-dialkylimidazolium ILs in hydrogen peroxide/acetic acid
medium assisted by ultrasonic chemical irradiation. It was
 water research 44 (2010) 352–372
observed that 99% of tested compounds was degraded after
72 h. In addition, advanced oxidative degradation in the
presence of reactive peroxides generated by Fenton reagent
has been applied for the removal of ILs from water (Siedlecka
and Stepnowski, 2009; Siedlecka et al., 2008a,b). According to
the results, in a Fenton system with 1 mM of Fe(III) and
100 mM of H2O2, more than 97% of IM14 Cl was observed to
degrade after 90 min. For Pyr14 Cl, IM16 Cl and IM18 Cl, the
levels of degradation were 92%, 88% and 68%, respectively.
Investigations of the degradation mechanisms indicated IM18
Cl was more resistant to oxidation by OH radicals cleaved
from H2O2, suggesting that the oxidation rates of imidazolium
ILs by OH are structure-dependent (Siedlecka and Stepnow-
ski, 2009). The level of degradation was dependent on the alkyl
chain length, consistent with Stepnowski and Zaleska (2005),
who indicated that lengthening the alkyl chain lowered the
rate of IL degradation. On contrast, the different length of the
side chains and the type of anions did not affect the degra-
dation process (Li et al., 2007). Regarding the thermal degra-
dation studies of alkylimidazolium salts (Awad et al., 2004),
extension of the alkyl chain enhanced the thermo-oxidative
degradation of imidazolium salts. Interestingly, methyl
substitution in the 2-position (i.e. between the two N atoms)
was observed to decrease the oxidative decomposition of
imidazolium ILs. The longer alkyl chain was also observed to
induce an enhancement in photocatalytic decomposition of
ILs (Morawski et al., 2005), which was not in the case of
oxidative degradation (Stepnowski and Zaleska, 2005; Sied-
lecka and Stepnowski, 2009). Nonetheless, detailed account on
the degradation of ILs by photocatalysis is required to verify
this phenomenon.
3.2. Biodegradability of ILs
In contrast to chemical degradation, which requires the
assistance of a certain oxidant for catalysis, biodegradation is
the microbial breakdown of chemical compounds. Biodegra-
dation seems to be more environmentally friendly compared
to chemical decomposition process. The initial attempt to
examine the degradation potential of different IM14 cations
combined with [Br], [BF4], [PF6], [N(CN)2], [(CF3SO2)2N]
and octylsulfate as the counter ion was done using the Sturm
and Closed-Bottle test protocols by the group of Scammells
(Garcia et al., 2005; Gathergood and Scammells, 2002; Gath-
ergood et al., 2004; Gathergood et al., 2006). Nonetheless, no
compound showed significant degree of biodegradation with
the exception of the octylsulfate-containing IL. The next step
study on the biodegradation of ILs involved the design of ILs
containing biodegradable side chains (Gathergood and
Scammells, 2002). The design was done according to the
principles of Boethling (Boethling, 1994, 1996; Howard et al.,
1991) who identified three important parameters including
the potential sites of enzymatic hydrolysis (for example,
esters and amides) and oxygen in form of hydroxyl, aldehyde
or carboxylic acid groups as well as unsubstituted linear alkyl
chains (especially  4 carbons) and phenyl rings, which
represent possible sites for attack by oxygenases. However,
for a balance between chemical properties and biodegrad-
ability, not all of these factors were suitable for ILs. The
365
addition of oxygen containing functional groups such as
alcohols, aldehyde and carboxylic acids was reported to
restrict the ILs performance as reaction media whereas the
incorporation of phenyl rings was known to increase the
melting points of IL solvents (McGuinness and Cavell, 2000).
Therefore, ester or amide group was selected to be coupled in
alkyl side chain of ILs. The introduction of ester groups
derived from a C2 acid and C4 or higher alcohol in the
3-N-substitutent was demonstrated to increase the biodegra-
dation of imidazolium-based ILs (Gathergood et al., 2004). This
can be explained by the fact that introduction of ester moiety
probably provides a site susceptible to enzymatic attack
(Gathergood et al., 2004; Gathergood et al., 2006) and hence,
improves the biodegradation level. Though the addition of
amide group is informed to improve the biodegradation of
organic compounds (Boethling, 1994, 1996; Howard et al.,
1991), no critical enhancement of biological degradation was
noted when this group was appended into the imidazolium-
based ILs (Gathergood et al., 2004). However, no compound
could be classified as ‘‘readily biodegradable’’ corresponding
to Organization for Economic Cooperation and Development
(OECD) standards (U.S. EPA, 1998), for which 60–70% or greater
biodegradation by activated sludge microbial inoculate is
required within a 10-day window in a 28-day period. Finally,
the combination of the octylsulfate anion and imidazolium
cation containing ester side chains resulted in readily biode-
gradable IL (Gathergood et al., 2006). Recently, Stolte et al.
(2008) also paid their attention on investigation of functional
groups incorporating alkyl chain ILs. Nonetheless, the intro-
ductions of terminal hydroxyl, carboxyl, ether and nitrile
groups did not improve the biological degradation as
expected.
Kumar et al. (2006) investigated the fate of IM14 BF4 when
in contact with soil-microorganisms, wastewater microor-
ganisms, Pseudomonas putida and E. coli. Although IM14 BF4
was indicated to be recalcitrant in Sturm and Closed-Bottle
test assays as mentioned above, it was observed in this study
that P. putida was able to break down IM14 BF4 after 15 days of
incubation. The breakdown products were monitored
using GC-MS and identified to be 1-H-methylimidazole and
1-H-butylimidazole, which were in consistent with the
theoretical metabolism scheme proposed by Jastorff et al.
(2003). In case of bacteria from soil and wastewater, the
metabolic intermediates appeared on the 12th day. It was
also noted that different intermediate peaks were observed at
different retention time with different microbes, indicating
that the degradation mechanism of IM14 BF4 may vary in
correspondence to certain microbes and metabolic pathways.
In another study, the biodegradation pathway of IM18 moiety
(Fig. 3) was proposed based on intermediate products via
HPLC-MS analysis after 24-day period of incubation with
activated sludge (Stolte et al., 2008). The metabolism of IM18
cation appeared to undergo oxidation reactions catalyzed
probably by mono-oxygenases, e.g. the cytochrome P450
system on the terminal methyl group (u-oxidation). The
alcohol formed was subsequently oxidized and converted
into aldehydes, and then into carboxylic acids by dehydro-
genases. The resulting carboxylic acids then might undergo
b-oxidation and finally generated two carbon fragments that
can enter the tricarboxylic acid cycle as acetyl Co-A (Fig. 3).
366 water research 44 (2010) 352–372

retention time m/z+ intensity m/z+

in min.

8.9 195 4*105 N +

N

13.5 / 14.4 211 3*105 / 2*105 N N

+

OH

OH
12.2 / 12.7 209 1*106 / 2*106 +
N N N +
N
211
O

10.0 – 12.5 225 2*104 – 6*104 N + H

N
O
O

19.5 183 1*105 +

OH
N +
N N N
209
OH
5
16.2 197 3*10 N +
N
OH O
O +

OH
26.5 155 0.5*105 +
N N N +
N
OH
225

24.2 169 4*106 N +

N
OH
O

26.7 141 1*105 N +

N
O
OH

Fig. 3 – Biodegradation pathways of 1-octyl-3-methylimidazolium by activated sludge microbial community (Reproduced

from Stolte et al. (2008) by permission of the Royal Society of Chemistry).

The proposed pathways provide basic information to both
environmental scientists and chemical engineers; however,
no studies have sought to examine the toxicity of metabolic
products after degradation of ILs. This issue is of paramount
importance since metabolism might not always end in less
toxic products.
Subsequently, Wells and Coombe (2006) extended the
microbial degradation study with ammonium, imidazolium,
phosphonium and pyridinium compounds by measuring the
biological oxygen demand. The authors observed no biode-
gradability of cations incorporated short chains (C  4) within
this test series, which was in agreement with Docherty et al.
(2007) and Stolte et al. (2008). For longer alkyl chains (C12, C16
and C18) containing ILs, a strong inhibitory effect of these
compounds on the inoculum used was found, indicating the
active microbial consortium was significantly impacted by ILs
toxicity. In recent studies (Docherty et al., 2007; Grabinska-
Sota and Kalka, 2004; Harjani et al., 2008; Stasiewicz et al.,
2008), pyridinium-based ILs were reported to be fully catabo-
lized by microbial community in activated sludge. This can be
inferred from the fact that degradation pathways for pyridine –
the precursor of pyridinium-based compounds – under
aerobic and anaerobic conditions were intensively investi-
gated in the work of Kaiser et al. (1996). With respect to the
common 1,3-dialkylpyridinium ILs, Pham et al. (2009) reported
that after 21 days of incubation, microorganisms from acti-
vated sludge were able to break down Py4-3Me Br. Analyses of
HPLC and MS/MS demonstrated that this biodegradation
led to the formation of 1-hydroxybutyl-3-methylpyridinium,
1-(2-hydroxybutal)-3-methylpyridinium, 1-(2-hydroxyethyl)-
3-methylpyridinium and methylpyridine. Based on these
intermediate products, biodegradation pathways were also
suggested (Fig. 4), thereby providing the basic information
which might be useful for assessing the factors related to the
environmental fate and behavior of this commonly used
pyridinium IL. Although this is the first report on biodegra-
dation intermediates and pathway of pyridinium ILs, the
authors have failed to systematically screen a single micro-
organism or a microbial consortium responsible for biodeg-
radation of ILs. Therefore, it is needed to further investigate
which type of microorganism is adaptable to ILs and which is
responsible for degradation process. Also, the broken
 water research 44 (2010) 352–372 367

II
H3C + H3C +
N CH3 N CH3
OH

I
H3C + OH
N H3C + OH CH3
N
+ H3C

H3C + O
N H3 C H
+ H3C
OH N
+ OH

H3C + OH H3C
N +
O

H3 C +
H
N H3C
+ OH

Fig. 4 – Biodegradation pathways of 1-butyl-3-methylpyridinium entity by microorganisms from activated sludge (Reprinted
with permission from Pham et al. (2009). Copyright 2009 American Chemical Society).

structures much further than methylpyridinium were not
measured in the study. In particular, the possibility of
cleavage of heterocyclic ring in ILs molecular structure (both
pyridinium in this work and imidazolium in the study of Stolte
et al. (2008)) has not been indentified. This issue should be
clarified through further studies. Interestingly, Py4-3Me Br
was not found to be metabolized by the activated sludge
community (Docherty et al., 2007). This was attributed likely
to the high IL concentration used in the study of Docherty’s
group, which consequently inhibited the microbial consor-
tium. Nonetheless, it was demonstrated that the structural
manipulation of the pyridinium skeleton may lead to ILs with
greater biodegradable extent compared to imidazolium-based
compouds (Harjani et al., 2008).
Concerning the anionic effect, ILs with halide counter ions
were postulated to be more stable to degradation than
perfluoronated ions (Awad et al., 2004; Gathergood and
Scammells, 2002). In a preliminary study, Gathergood and
Scammells (2002) confirmed this assumption and showed that
the biodegradation efficiency decreased in the order
[PF6] > [BF4] > [Br] with 60%, 59% and 48% of CO2 evolution
values, respectively. In a later study (Gathergood et al., 2006),
it was found that the octylsulfate anion is considerably more
biodegradable than the other commonly used anions.
The alkyl chain with C4, C6 or C8 was found to increase the
rate of degradation (Docherty et al., 2007; Stolte et al., 2008);
nonetheless, further increasing the chain length to C12, C16 or
C18 was noted to cause toxic effect towards inoculum (Wells
and Coombe, 2006). However, it was stated that the long octyl
side chain was not a compulsory factor for biodegradation, but
more important is a certain overall lipophilicity of the
compound (Stolte et al., 2008).
3.3. Sorption of ILs in environmental systems
Because the aquatic and terrestrial environments are possible
recipients for contaminants, the distribution and behavior of
ILs in soil are also extremely important. The retention and
mobility of ILs in soils and sediments are strongly influenced by
its tendency to be sorbed onto the various components of the
soil matrix (Stepnowski, 2005). Since imidazolium-based ILs
possess high electron acceptor potential of delocalized
aromatic systems and hydrophobic components (e.g. the alkyl
chain) (Stepnowski, 2005), they could be sorbed onto soils and
sediments via several mechanisms. In a preliminary study,
Stepnowski (2005) proved that electrostatic interactions
contributed to the sorption of the imidazolium cations. More-
over, totally contrast trends were also observed demonstrating
an extremely strong and practically irreversible sorption onto
fine-textured marine sediments and a relatively weakly and
reversibly binding to peaty soil (with the highest organic
carbon content) (Stepnowski, 2005). This indicates the impor-
tance of the mineral component of the soil (sediment) in the
sorption mechanism of ILs. Also, in this work, the author
pointed out that compounds with longer alkyl chains were
irreversibly bound to the soil component, which was in
agreement with Stepnowski et al. (2007) and Matzke et al.
(2009b). Interestingly, Beaulieu et al. (2008) did not find a posi-
tive effect of alkyl chain length on the sorption of alkylimida-
zolium-based ILs to aquatic sediments and suggested that
hydrophobic interactions were not the most important sorp-
tion mechanism. The contrast results between these groups
imply that sorption mechanisms of ILs may vary according to
properties and composition of the environmental systems. The
studies suggest that ILs may be retained by aquatic sediments;
368 water research 44 (2010) 352–372
nonetheless, the toxic action of these sorbed materials towards
aquatic organisms has not been addressed. Also, further
efforts should be continued to elucidate the reversibility of IL
sorption on these sediments to give a better understanding of
the ILs fate after being released into aqueous environment.
Matzke et al. (2009b) investigated the influences of the two
different clay minerals kaolinite and smectite as well as of
organic matter on the cation sorption and desorption behav-
iors of three imidazolium based ILs including IM14 BF4, IM18
BF4 and IM14 (CF3SO2)2N in soil. The addition of organic matter
and clays was observed to increase the sorption/decrease the
desorption of all ILs tested, and in particular smectite had
striking effects on the sorption efficiency of all substances.
It is worth noting that not only the cationic moiety with
different alkyl side chain lengths but the anionic compart-
ments can also play an importance role in the sorption/
desorption processes. Imidazolium compounds with BF 4 as
a counter anion showed higher sorption capacity compared to
that of [(CF3SO2)2N], indicating the high potential of this type
of IL to form ionic pairs in the soil matrix (Matzke et al., 2009b).
However, further work with a variety of ILs incorporated
different cationic and anionic moieties should be carried out
to verify these phenomena.
Gorman-Lewis and Fein (2004) examined the sorption
behavior of IM14 Cl onto a range of surfaces which are
commonly found in the near-surface environment. The results
suggested that IM14 Cl could be minimally retarded by non-
interlayer clay system and might lead to unimpeded transport
through subsurface groundwater. Also, the adsorption
capacity of this IL onto bacterial surfaces was not high, which
might be due to the low hydrophobicity of IM14 Cl. Addition-
ally, investigations of the adsorption of IM14 Cl towards
different media carried out by our group (Vijayaraghavan et al.,
2009) have shown that retardation of this compound was
possible only by an ion-exchange resin and activated carbon,
which was in consistency with the work of Anthony et al.
(2001). However, no significant adsorption of IM14 Cl in the
media of a fermentation waste (Corynebacterium glutamicum)
and dried activated sludge was observed in our study.
Conclusively, the data currently available demonstrated
that ILs incorporated imidazolium cation can be sorbed to
organic matter, whether found in aquatic sediments or
terrestrial soils, and the presence of clays significantly
enhanced the sorption capacity.
4. Concluding remarks and future directions
Ionic liquids, of which the most often cited attribute is their
negligible vapor pressure, have been suggested as a green
alternative to traditional organic solvents with the desire to
minimize diffusion to the atmosphere. Low volatility, however,
does not completely eliminate potential environmental
hazards and might pose serious threats to aquatic and terres-
trial ecosystems. The studies of environmental fate and
toxicity of ILs have shown that the ILs commonly used to date
are toxic in nature and their toxicities vary considerably across
organisms and trophic levels. In general, the effect of anionic
moieties is not drastic as the alkyl length effect except for the
case of [(CF3SO2)2N], which shows a clear (eco)toxicological
hazard potential. The other perfluorinated anions have been
also proved to be hazardous due to hydrolytically unstable
properties. In addition, the introduction of functional polar
groups to the alkyl chain has been shown to reduce the toxicity
of ILs and increase the biodegradation efficiency to some
extent. This indicates the possibility of tailoring ILs by coupling
suitable functional groups to their structure, which in turn
leads to a more environmental friendly compound. The side
chain length effect has been found to be consistent in all levels
of biological complexity as well as different environmental
compartments. Also, an increase in alkyl-chain length, or lip-
ophilicity, was observed to be related to an increase in the rate
of degradation as well as an increase in toxicity. This indicates
a conflict of aims between minimizing the toxicity and maxi-
mizing the biodegradability of these neoteric solvents.
Regarding the cationic compartment, pyridinium has been
found to be more environmental friendly than imidazolium
from both viewpoints of toxicology and microbial degradation.
It can therefore be suggested that the structural manipulation
of the pyridinium skeleton should be considered in design of
a sustainable IL. From the currently available data, it is clear
that some commonly used ILs are very far away from the image
of green chemicals that are often cited in the literature. The
uncertainties in their sustainable development hinder the
applications of ILs under real conditions. Although some
attempts have been made to give important hints in the
prospective design and synthesis of inherently safer ILs,
comprehensive studies dealing with the behaviors of ILs in
aqueous media still await to be conducted. The important
features required for the thorough insight into environmental
fate of ILs include, but are not limited to:
- Providing more fundamental understanding into the
mechanism for IL-induced toxicity to different levels of
biological complexity. The underlying mechanisms of IL
toxicity have rarely been studied.
- Assessing the biodegradability of cationic and anionic
compartments and toxicity of their degradation interme-
diates. This may provide useful information in consciously
designing safer chemicals.
- Investigating the aerobic and anaerobic biodegradation of
ILs, which would suggest initial guidelines for the treatment
of ILs waste by using the existing aerobic and anaerobic
wastewater treatment facilities. Especially, anaerobic
degradation awaits to be investigated.
- Defining which organisms or enzymes may promote
degradation pathways and determining specific microbial
consortium or cultivatable communities capable of
biotransformation of ILs.
- Performing the ecotoxicity and biodegradation tests in real
environmental conditions instead of controlled conditions
of laboratory experiments, which would be advantageous in
understanding the fate and behavior of ILs under real
conditions. For this, the potential toxicological effects at
population level and community level should be addressed.
It must be encouraged to use tools such as experimental
mesocosms to study the effects of ILs at higher levels of
organization.
- Creating database of environmentally benign structure
moieties of ILs based upon their toxicological and
 water research 44 (2010) 352–372
biodegradation information, which would be practically
useful as a reference for manufacturers and regulators to
properly develop and regulate the use of ILs.
Acknowledgements
This work was supported by NRF Grant funded by the Korean
Government (KRF-2007-521-D00106, NRL 2009-0083194, and in
part WCU R31-2008-000-20029-0).
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