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Short communication
Abstract
Several bioassays have been carried out to analyze the toxicity and biodegradability of several imidazolium ionic liquids (ILs) in aqueous phase.
The synthetized compounds consist of an imidazolium cation with two alkyl substituents in positions 3 (R1 ) and 1 (R2 ) and a counter-ion. The
alkyl substituent R1 has been fixed as a methyl group and the effect of the alkyl chain length (C1 C8 ) of the other substituent (R2 ) has been tested.
Moreover, the influence of diverse counter-ions A (Cl , PF6 , XSO4 ) has been analyzed. Acute toxicity and EC50 values of each compound in
the aqueous solution have been determined by using the Microtox standard procedure. Biodegradability of IL has been determined by measuring
BOD5 of aqueous samples containing IL and/or d-glucose and the IL residual content and/or d-glucose concentration after this assay. The viability
of the microorganisms used in the BOD5 has been related to the ATP in the samples, measured by a bioluminescence assay. All the ILs tested were
not biodegradable in the considered conditions. Besides, it was found that the shorter the chain length of side chain R2 , the lower the toxic effect
is. On the contrary, the anion has a little effect on the IL toxicity.
2007 Elsevier B.V. All rights reserved.
Keywords: Ionic liquid; Biodegradability; Microtox; Imidazolium
1. Introduction
Ionic Liquids, ILs, are low-melting-point salts that have
become increasingly attractive as green solvents for industrial
applications [110]. This green adjective is mainly attributed to
their negligible vapor pressure, which avoids the loss of solvent
to the atmosphere and decreases the worker exposure risk. Thus,
room-temperature ionic liquids could provide environmentally
friendly solvents for the chemical and pharmaceutical industries
[1,5,6,11]. Typical ILs consist of an organic cation with delocalized charges and a small inorganic anion, most often halogen
anions weakly coordinating such as Cl, BF4 or PF6 [12]. An
ionic liquid can be thought of as designer solvent [13] so it
should be possible to design, or tailor, a solvent for a certain
reaction. Therefore, many cation and anion combinations are
possible, changing properties as polarity, hydrophobicity and
solvent miscibility behavior. Among these possibilities, the 1alkyl-3-methylimidazolium is one of the most used because it
is non-volatile, non-flammable, presents high thermal stability
Corresponding author.
E-mail address: aromeros@quim.ucm.es (A. Romero).
0304-3894/$ see front matter 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.jhazmat.2007.10.079
269
Acronym
R2
CH3
CH2 CH3
(CH2 )3 CH3
(CH2 )5 CH3
(CH2 )7 CH3
(CH2 )5 CH3
(CH2 )7 CH3
CH3 SO4
CH3 CH2 SO4
Cl
Cl
Cl
PF6
PF6
2. Experimental
ILs were prepared according to slightly modified literature procedures [2529] in our laboratory. The progress of the
reaction was monitored by thin layer chromatography using aluminium sheets silica gel 60 GF-254, CH2 Cl2 10% MeOH as
eluent. H NMR analysis was made at 400 MHz and in D2 O. The
maximum water contents of the liquids were determined using
a 756 Karl Fisher coulometer.
2.1. Luminiscent bacteria acute toxicity test
100
IC50
(1)
100Ci
EC50
(2)
270
(3)
271
Fig. 1. Example of an inhibition plot of the luminescence vs. the concentration of the ionic liquid. The IL used was ClC6 min at Ci = 430 mg/L. (a) Gamma vs.
concentration, as (VS /VF ) 100 and (b) % effect vs. concentration.
Table 2
EC50 values and confidence intervals for the tested ionic liquids
IL
C1 mimMSO4
C2 mimESO4
C4 mimCl
C4 mimPF6
C4 mimBr
C4 mimBF4
C6 mimCl
C6 mimPF6
C6 mimBr
C6 mimBF4
C8 mimCl
C8 mimPF6
C8 mimBr
C8 mimBF4
>4.76
4.02 0.14
3.39 0.15
3.71 0.13
4.01 0.05
2.18 0.09
2.11 0.11
0.94 0.14
0.70 0.16
3.34
3.07
3.27
3.10
2.32
2.17
1.42 0.10
0.63 0.06
Common VOCs
3.07 0.03
3.55 0.04
Methanol
Ethanol
2-Propanol
Acetonitrile
Acetone
Dicloromethane
Chloroform
Benzene
Phenol
Toluene
5.557.00
5.706.08
5.775.84
5.77
5.355.70
4.074.53
3.554.32
1.413.12
2.35
2.292.64
3.18 0.03
1.41 0.07
(4)
0.13
0.29
0.09
0.17
0.16
0.06
1.19 0.11
0.95 0.12
out and the following equation has been obtained in this work:
log EC50 = 5.33 0.549nCR2
(5)
Fig. 2. Effect of the anion and alkyl chain length on the acute toxicity
(Microtox ) for 1-alkyl-3-methylimidazolium ionic liquids.
272
Table 3
Biodegradability and microorganism inhibition for ionic liquid-D-glucose aqueous solutions (bioluminescence assay used for ATP measurements)
Sample
t = 0 days
D-Glucose (mg/L)
1
2
3
4
5
6
100
100
100
100
100
100
t = 5 days
IL type
C2 mimC2 H5 SO4
C4 mimCl
C6 mimCl
C8 mimCl
IL (mg/L)
100
100
100
100
t = 10 days
containing 100 mg/L of D-glucose as carbon source was calculated as 106 mg O2 /L. Therefore, a higher value of BOD5 than
100 mg O2 /L would be expected from samples containing IL as
additional carbon source.
Final concentration of D-glucose and IL in the samples
were analyzed, using an enzymatic kit or by UV-spectrometry,
respectively. Values obtained are also given in Table 3. DGlucose has been almost totally consumed in all the samples
while the ionic liquid remains always in a concentration close
to the initial one. Thus, all the ionic liquids tested were poorly
biodegradable in presence of glucose. As the living organisms
inoculated in this assay consumed only this monosacharide
and not the ionic liquid, the almost constant BOD5 (100 mg/L)
obtained is consistent.
Since this preference for the glucose was unsurprising, it
was analyzed if the microorganisms would consume the IL in
absence of another source of carbon. For this, the aqueous samples after the BOD5 measurements, free of glucose, containing
only the microorganism and the ionic liquids, were stirred in aerobic atmosphere for 5 days more at 20 C in the dark. Remaining
concentration of the ionic liquid at this final time of 10 days in
contact with the bacteria was determined and values obtained are
summarized in Table 3. The amounts of ionic liquid in the solutions after 10 days were almost the same as the initial ones and
those obtained after 5 days. In conclusion, the poor biodegradability of ILs in presence and absence of other carbon source is
confirmed.
3.3. Inuence on the biological activity
To examine if the presence of an ionic liquid produce cellular
stress on the microorganism in the aqueous phase, a bioluminescence assay have been used to measure the ATP in the samples
after BOD5 experience. The light emitted (proportional to the
ATP in the samples) is given in Table 3. Control samples are set
like those containing only glucose in the BOD5 measurements.
In Table 3, the light emission values obtained in the samples
that contained ILs were lower than those obtained in the control samples. Therefore, the aqueous samples with ionic liquid
added yield a lower living cells concentration than the control
samples. Because all the samples were inoculated with the same
type and amount of microorganisms, it can be deduced that a
lower microorganism growth takes place if the microorganism
is inoculated in a medium containing an ionic liquid. Again, the
shorter the chain length of side R2 , the lower the toxic effect.
D-Glucose (mg/L)
0
0
0
0
0
0
IL (mg/L)
Light emission
IL (mg/L)
89
98
99
95
2.8 107
90
97
98
96
2.7 107
2.1 107
2.2 107
1.3 107
1.2 107
Acknowledgement
The authors acknowledge financial support for this research
from the Spanish MCYT (contract no. CTM2006-00317).
References
[1] J.D. Holbrey, K.R. Seddon, Room-temperature ionic liquids as replacements for organic solvents in multiphase bioprocess operations, Clean Prod.
Process. 1 (1999) 223236.
[2] T. Welton, Room-temperature ionic liquids. Solvents for synthesis and
catalysis, Chem. Rev. 99 (1999) 20712084.
[3] M. Freemantle, Ionic liquids prove increasingly versatile, Chem. Eng. News
77 (1999) 2324.
[4] M.J. Earle, P.B. McCormac, K.R. Seddon, DielsAlder reactions in ionic
liquids, Green Chem. 1 (1999) 2325.
[5] D.W. Rooney, K.R. Seddon, in: G. Wypych (Ed.), Handbook of Solvents,
ChemTech Publishing, Toronto, 2001, pp. 14591484.
[6] C.M. Gordon, New developments in catalysis using ionic liquids, Appl.
Catal. A 222 (2001) 101117.
[7] R. Sheldon, Catalytic reactions in ionic liquids, Chem. Commun. 23 (2001)
23992407.
[8] J. Dupont, R.F. de Souza, P.A. Suarez, Ionic liquid (molten salt) phase
organometallic catalysis, Chem. Rev. 102 (2002) 36673692.
[9] F. Rantwijk, R.M. Lau, R.A. Sheldon, Biocatalytic transformations in ionic
liquids, Trends Biotechnol. 21 (2003) 131138.
[10] R.A. Sheldom, Green solvents for sustainable organic synthesis: state of
the art, Green Chem. 7 (2005) 267278.
[11] C.F. Poole, Chromatographic and spectroscopic methods for the determination of solvent properties of room temperature ionic liquids, J. Chromatogr.
A 1037 (2004) 4982.
[12] P.A.Z. Suarez, S. Einloft, J.E.L. Dullius, R.F. de Souza, J. Dupont, Synthesis
and physicalchemical properties of ionic liquids based on 1-n-butyl-3methylimidazolium cation, J. Chim. Phys. 95 (1998) 16261639.
[13] M. Freemantle, Designer solvents ionic liquids may boost clean technology development, Chem. Eng. News 76 (March) (1998) 3237.
[14] K.L.E. Kaiser, V.S. Palabrica, Photobactrium phosporeum, toxicity data
index, Water Poll. Res. J. Can. 26 (1991) 361431.
[15] S.M. Steinberg, E.J. Poziomek, W.H. Engelmann, K.R. Rogers, A review
of environmental applications of bioluminescence measurements, Chemosphere 30 (1995) 21552197.
273
[16] T. Garca, N. Gathergood, N.J. Scammells, Biodegradable ionic liquids. Part II. Effect of the anion and toxicology, Green Chem. 7 (2005)
914.
[17] J. Ranke, K. Molter, F. Stock, U. Bottin-Weber, J. Poczobutt, J. Hoffman, B.
Ondruschka, J. Filser, B. Jastorff, Biological effects of imidazolium ionic
liquids with varying chain lengths in acute Vibrio scheri and WST-1 cell
viability assays, Ecotoxicol. Environ. Saf. 58 (2004) 396404.
[18] K.M. Docherty, C.F. Kulpa, Toxicity and antimicrobial activity of imidazolium and pyridinium ionic liquids, Green Chem. 7 (2005) 185189.
[19] J. Pernak, J. Kalewska, H. Ksycinska, J. Cybulski, Synthesis and
anti-microbial activities of some pyridinium salts with alkoxymethyl
hydrophobic group, Eur. J. Med. Chem. 36 (2001) 899907.
[20] J. Pernak, J. Rogoza, I. Mirska, Synthesis and antimicrobial activities of
new pyridinium and benzimidazolium chlorides, Eur. J. Med. Chem. 36
(2001) 313320.
[21] J. Pernak, P. Chwala, Synthesis and anti-microbial activities of cholinelike quaternary ammonium chlorides, Eur. J. Med. Chem. 38 (2003) 1035
1042.
[22] J. Pernak, I. Goc, I. Mirska, Anti-microbial activities of protic ionic liquids
with lactate anion, Green Chem. 6 (2004) 323329.
[23] R.P. Swatloski, J.D. Holbrey, S.B. Memon, G.A. Caldwell, K.A. Caldwell,
R.D. Rogers, Using Caenorhabditis elegans to probe toxicity of 1-alkyl-3methylimidazolium chloride based ionic liquids, Chem. Commun. 6 (2004)
668699.
[24] R.J. Bernot, M.A. Brueseke, M.A. Evans-White, G.A. Lamberti, Acute and
chronic toxicity of imidazolium-based ionic liquids on Daphnia magna,
Environ. Toxicol. Chem. 24 (2005) 8792.
[25] J.D. Holbrey, W.M. Reichert, R.P. Swatloski, G.A. Broker, W.R. Pitner,
K.R. Seddon, R. Rogers, Green Chem. 4 (2002) 407413.
[26] A.B. Pereiro, E. Tojo, A. Rodrguez, J. Canosa, J. Tojo, Properties of ionic
liquid HMIMPF6 with carbonates, ketones and alkyl acetates, J. Chem.
Thermodyn. 38 (2006) 651661.
[27] A.B. Pereiro, E. Tojo, A. Rodrguez, J. Canosa, J. Tojo, HMImPF(6) ionic
liquid that separates the azeotropic mixture ethanol plus heptane, Green
Chem. 8 (2006) 307310.
[28] A.B. Pereiro, F. Santamarta, E. Tojo, A. Rodrguez, J. Tojo, Temperature
dependence of physical properties of ionic liquid 1,3-dimethylimidazolium
methyl sulphate, J. Chem. Eng. Data 51 (2006) 952954.
[29] E. Gomez, B. Gonzalez, A. Domnguez, E. Tojo, J. Tojo, Physical properties
of pure 1-ethyl-3-methylimidazolium ethylsulfate and its binary mixtures
with ethanol and water at several temperatures, J. Chem. Eng. Data 51
(2006) 20962102.