Toxicity of ionic liquids: eco(cyto)activity as complicated, but unavoidable parameter for...

DOI: 10.1002/cssc.201300459

Toxicity of Ionic Liquids: Eco(cyto)activity as Complicated,but Unavoidable Parameter for Task-Specific OptimizationKsenia S. Egorova[a] and Valentine P. Ananikov*[a, b]

� 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemSusChem 2014, 7, 336 – 360 336

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1. Introduction

The development of sustainable chemical technologies hasclearly identified challenging problems concerning the com-plete life-cycle approach of chemical and industrial procedures,the renewable chemical supply chain, and the cost efficiencyof the production process including minimization of wastes.However, in spite of broadly discussed advantages of greenand sustainable chemical procedures, traditional production ofcompounds and materials based on toxic and non-renewablechemicals is still in use. Many old technologies are hard to re-place because newly developed “greener” and sustainable ana-logs are economically less competitive.

Technological cycle analysis revealed that a significant por-tion of the overall costs often is not associated with the prod-uct itself. Namely, these costs arise after the synthesis of a de-sired product and include treatment and recycling of the toxicchemicals (components of chemical systems and wastes). Ina typical procedure, the synthetic pathway is optimized withregard to high selectivity, yields, catalyst efficiency, and con-venient operation. However, environmental impact and utiliza-tion of the components of chemical systems left over after iso-lation of the final product are not considered in the initialdesign stage. Clearly, such an approach is hard to direct to-wards green and sustainable routes. To a great extent thismethodological drawback is responsible for the negativepublic perception of chemistry as an environmentally danger-ous activity that results in the accumulation of wastes.

The task-specific design of chemicals is one of the dominat-ing trends in modern research to develop a new generation ofchemical procedures. Rapid progress in the field led to the ap-plication of several breakthrough technologies: supercritical

fluids,[1] solid-state[2] and solvent-free syntheses,[3] reactions inwater,[4] smart functional materials,[5] microwave-promoted[6]

and ultrasound-promoted[7] processes, micro- and nanoreac-tors,[8] as well as many other advanced solutions. A remarkabletask-specific optimization has been achieved by refining theanionic and cationic parameters of ionic liquids (ILs) to meetthe requested target properties.[9]

ILs were designed as nonflammable, nonvolatile, and nonex-plosive reaction media with high thermal stability. ILs couldcontribute to the replacement of standard volatile and flamma-ble organic solvents with environmentally benign materials.Several chemical reactions performed in ILs were reported tobe more energy-efficient, considerably easier to carry out, andalso to produce less waste.[9m,q, 10] Numerous successful IL im-plementations include catalysis,[9i,t, 11] organic synthesis,[10i, 12]

biomolecular transformations,[13] electrochemistry,[14] and nano-particle research.[15] In addition to being advantageous mediafor traditional chemistry, ILs have demonstrated unique proper-ties in the application as lubricants[16] and battery electro-lytes[17] and for extraction,[18] biomass conversion,[19] solar andthermal energy conversion,[20] nuclear fuel processing,[18i, 21] bio-logical and medical procedures,[22] and others.[1k, 23] Such tre-mendous progress in diverse areas has been clearly confirmedby increasing interest and publication activity, which stillshows remarkable growth (Figure 1).

However, it should be pointed out that ILs are not intrinsical-ly “green”. Most of them are as toxic as other organic solvents,whereas some demonstrate extreme toxicity. Recently, theissues of biodegradability and toxicity of ILs have become ofprimary interest.[22c, 24] Quantification of the environmentalimpact of IL-based chemical technologies requires careful anal-ysis of possible ecological and biological activity. It is the majorlimitation that hampers practical application of many fascinat-ing chemical procedures developed in ILs. Environmentalimpact and ecological activity of ILs should be considered asprincipal parameters at the initial planning and optimization ofchemical processes. The overall impact of a chemical synthesisprocess should be taken into account because its eco- andbio-related properties are as important as chemical parameters(such as yield, activity, selectivity, etc.).

Rapid progress in the field of ionic liquids in recent decadesled to the development of many outstanding energy-conver-sion processes, catalytic systems, synthetic procedures, and im-portant practical applications. Task-specific optimizationemerged as a sharpening stone for the fine-tuning of structureof ionic liquids, which resulted in unprecedented efficiency atthe molecular level. Ionic-liquid systems showed promising op-portunities in the development of green and sustainable tech-nologies; however, the chemical nature of ionic liquids is notintrinsically green. Many ionic liquids were found to be toxic oreven highly toxic towards cells and living organisms. In thisReview, we show that biological activity and cytotoxicity ofionic liquids dramatically depend on the nature of a biological

system. An ionic liquid may be not toxic for particular cells ororganisms, but may demonstrate high toxicity towards anothertarget present in the environment. Thus, a careful selection ofbiological activity data is a must for the correct assessment ofchemical technologies involving ionic liquids. In addition tothe direct biological activity (immediate response), several indi-rect effects and aftereffects are of primary importance. The fol-lowing principal factors were revealed to modulate toxicity ofionic liquids: i) length of an alkyl chain in the cation; ii) degreeof functionalization in the side chain of the cation; iii) anionnature; iv) cation nature; and v) mutual influence of anion andcation.

[a] Dr. K. S. Egorova, Prof. V. P. AnanikovZelinsky Institute of Organic ChemistryRussian Academy of SciencesLeninsky Prospect 47, Moscow, 119991 (Russia)E-mail : [email protected]

[b] Prof. V. P. AnanikovDepartment of ChemistrySaint Petersburg State UniversityStary Petergof, 198504 (Russia)


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Recent environmental studies have demonstrated that indi-rect contaminant-induced changes can dramatically exceedthe immediate direct effect of a contaminant studied fora single target organism or cell culture.[25] For a reliable assess-

ment of the possible impact of a particular class of chemicals,an attempt should be made to consider the biological effecton a complex ecosystem through analysis of toxicity data mea-sured for several biological systems. Accumulation and multi-plication of toxic effects are especially dangerous in aquaticecosystems (Figure 2).[25] Because most ionic liquids are highlymiscible with water (as solutions, microemulsions, or stable bi-phasic systems), a risk assessment taking into account severalcontamination pathways should be performed.

In this Review, we discuss data on the toxicity of ILs withregard to different classes of biological systems, living organ-isms, and the environment, with special attention on possibledirections for the task-specific tuning of the IL properties. Thetoxicity of ILs has been considered for a broad range of objectsincluding cells, bacteria, algae, fungi, plants, invertebrates, andvertebrates. An important topic is concerned with understand-ing the molecular interactions of ILs with proteins and reveal-ing their possible influence on enzymatic activity.

2. Determination and Analysis of Eco- and Cy-totoxicity of Ionic Liquids

2.1. Characteristics of biological activity of ionic liquids

This Review covers a wide range of ionic liquids (ILs) activelyinvolved in chemical research and development of industrialapplications. The structures of the most common cations andanions used in ILs are shown in Scheme 1.

In this Review, we analyze the following main indices of ILtoxicity:

EC50—effective concentration resulting in 50 % reduction ofprocesses, such as growth or reproductive activity, of the ex-posed organisms relative to the control ;

LC50—lethal concentration/dose that kills half the membersof a population tested in a specified time;

IC50—inhibitory concentration resulting in 50% inhibition ofthe activity of biological or biochemical systems (in thisReview, it is applied to enzyme inhibition only) ;

LD50—median lethal dose;MIC—minimum inhibitory concentration, or the lowest con-

centration that inhibits visible growth of a microorganism afterovernight incubation.

Currently, there is no convention for scaling the hazard ofILs. Some authors compare the toxicity of ILs with that ofknown and widely used organic solvents. Thus, when studyingtoxicity of ILs towards freshwater organisms, Pretti and co-workers used the acute toxicity rating developed by Passinoand Smith.[26] According to this rating, substances could be di-vided into several groups: relatively harmless (no acute toxicityin concentrations higher than 1000 mg L

�1) ; practically harm-less (acutely toxic in concentrations of 100–1000 mg L

�1) ; mod-erately toxic (10–100 mg L

�1) ; slightly toxic (1–10 mg L�1) ;

highly toxic (0.1–10 mg L�1) ; extremely toxic (0.01–0.1 mg L

�1) ;and supertoxic (less than 0.01 mg L�1).[27] There is also a scaleproposed by Draize and colleagues to study the toxicity ofsubstances applied to skin and mucous membranes.[28] It wasused when studying acute IL toxicity in mammals.[29]

Valentine Ananikov received his Ph.D.

degree in 1999, habilitation in 2003,

and in 2005 he was appointed Profes-

sor and Laboratory Head of the ND Ze-

linsky Institute of Organic Chemistry,

Russian Academy of Sciences. In 2008,

he was elected as Member of the Rus-

sian Academy of Sciences. In 2012, he

became Professor at the Department

of Chemistry at the Moscow State Uni-

versity. He was recipient of the Russian

State Prize for Outstanding Achieve-

ments in Science and Technology (2004), an Award of the Science

Support Foundation (2005), a medal of the Russian Academy of

Sciences (2000), and the Balandin Prize for outstanding achieve-

ments in the field of catalysis (2010) and appointed Liebig Lecturer

by the German Chemical Society (2010). His scientific interests

focus on catalysis, development of new sustainable methods, and

mechanistic studies.

Ksenia Egorova graduated from Lomo-

nosov Moscow State University with

a Master of Science in Biochemistry in

2006 (2005–2006 research assistant at

the University of Medicine and Dentist-

ry of New Jersey, USA); between 2006

and 2011, she worked at the Institute

of Molecular Genetics RAS (Ph.D. in

Molecular Biology, 2010). Since 2012,

she has been a research fellow at the

Zelinsky Institute of Organic Chemistry.

Her major scientific interests are the

biological activity of ionic liquids, carbohydrate databases, and

cancer proteomics.

Figure 1. Number of publications dealing with ILs in the past decade (fromthe Web of Science database, http://www.webofknowledge.com/).



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Most studies of biological activity of ILs conducted in recentyears aimed at revealing correlations between IL structure andits toxicity. A large number of IL groups with different anionsand cations bearing side chains of variable length were tested.

However, the results of sucharray investigations have givenno simple and uniform picture.As will be discussed below, ILsexhibit rather different toxic be-havior : some inhibit biologicalsystems drastically, whereasother display weak or even noapparent effect. It is also evidentthat both ions, as well as thebiological environment used forthe measurements, can influencethe observed toxicity of ILs.[30]

The first apparent observationis that not only internal but alsoexternal factors such as the typeof a biological system govern ILtoxicity (Table 1, Figure 3). Asa representative example, thetoxicity of the widely used IL 1-

butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imidevaries from 30 [EC50 of the springtail Folsomia candida (F. candi-da) reproduction inhibition][30a] to 2000 mm and higher [24 hminimum inhibitory concentration for Escherichia coli (E. coli),

Staphylococcus aureus (S. aureus), and Candida sp.] .[30i]

Therefore, the nature of a biological system shouldbe considered before constructing any model for theevaluation of IL toxicity. Because different experimen-tal conditions and assays may result in different toxic-ity indices, even when using the same organism, theexact experimental details must be available for a reli-able comparison of toxicities. Unfortunately, somepublications lack necessary description, which ren-ders the data unreliable for comparison. Herein, weconsider experimental details when comparing toxici-ties of various ILs with regard to a single biologicalsystem or of a single IL towards various systems.Whenever available in original publications, the corre-sponding experimental data are given in the tables.

Figure 4 shows different models used for studyingthe (cyto)toxicity of ILs. Most scientists use model or-ganisms traditionally used for the determination oftoxicity of various chemicals and wastes. These or-ganisms usually have short life cycles and are easy tokeep and breed under laboratory conditions. IL toxici-ties are studied on different biological levels, fromseparate proteins (e.g. , acetylcholinesterase) to verte-brate animals (fish, mice, and rats). ILs show toxicitytowards cell cultures (cytotoxicity), inhibit growth ofbacteria, fungi and plants, and exhibit acute andchronic toxicity towards invertebrate and vertebrateanimals.

First, we will discuss the experimental data on ILtoxicities with regard to groups of organisms pub-lished in recent years and then highlight commontendencies that can be derived from these data. Wewill also summarize the four major internal factors in-

Figure 2. Ecosystem networks vulnerable to direct and indirect contaminant effects of chemical pollutants.

Scheme 1. Structures of cations and anions commonly used in ILs.



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fluencing toxic properties of ILs, as revealed by the existingdata: i) length of a cation side chain; ii) presence of a functional-ized cation side chain or degree of functionalization (that is,bearing a functional group, for example, hydroxy-, cyano-,phenyl-, etc.) ; iii) nature of an anion moiety; and iv) nature ofa cation moiety.

2.2. Cytotoxicity of ionic liquids

Cytotoxicity of ILs has beenstudied using cellular systemssuch as IPC-81 (rat promyeloticleukemia cell line),[24b, 30b,e,m,q, 33]

PC12 (rat pheochromocyto-ma),[34] V79 (lung fibroblasts ofChinese hamster),[35] NIH 3T3(murine fibroblasts),[36] J774(murine macrophage cellline),[30k] HT-29 and CaCo-2(human colon carcinoma),[30f, 37]

MCF-7 (human breast can-cer),[30g, 38] HeLa (human cervicalcancer),[32c, 39] U937 (human leu-kemic monocyte lymphoma cellline),[40] LoVo (colon adenocarci-noma), DLD-1 (colon adenocarci-noma), HepG2 (hepatocyte carci-noma), AGS (gastric adenocarci-noma), A549 (lung carcinoma),HaCaT (human immortalized ker-atinocytes),[41] S2 (Drosophilamelanogaster cell culture),[30n]

and CCO (channel catfish ovarycell line).[42]

The main method for estimating IL cytotoxicity is the MTTassay, which is based on measuring the activity of cellular en-zymes that reduce the tetrazolium dye MTT (3-(4,5-dimethylth-iazol-2-yl)-2,5-diphenyltetrazolium bromide) or the closely relat-ed XTT, MTS, and WST dyes.[43] These dyes can be reduced onlyby mitochondria in metabolically active cells ;[44] therefore, theintensity of an MTT reduction reflects the number of viablecells in the culture. Some scientists also use fluorescence mi-croscopy and visualization of DNA fragmentation for detectingapoptosis in cells[32c, 34b,c, 36, 40] or measure mitochondrial depola-rization, reactive oxygen species levels, and capsase-3 activi-ty.[32c, 34b,c]

It has been repeatedly demonstrated that the toxic effect ofILs depends on the alkyl side-chain length of the cation. Nu-merous evidences for this fact were obtained using cell cul-tures (Table 2). Thus, for 1-alkyl-3-methylimidazolium-based ILsthe increase of the side-chain length from four to 12 carbonatoms resulted in a 380-fold decrease of 24 h EC50 (that is, anincrease of toxicity) in PC12 cells : from >9123 mmol L�1 for 1-butyl-3-methylimidazolium bromide ([C4MIM][Br]) to24 mmol L�1 for 1-dodecyl-3-methylimidazolium bromide([C12MIM][Br])[34b] (Table 2, entries 1–5; the same effect was ob-served in HeLa cells) ;[32c] whereas in CaCo-2 cells, the increaseof the side-chain length from four to ten carbon atoms result-ed in a 956-fold decrease of 24 h EC50: from 28 690 mmol L�1

for 1-butyl-3-methylimidazolium chloride ([C4MIM][Cl]) to30 mmol L�1 for 1-decyl-3-methylimidazolium chloride ([C10MIM][Cl])[37b] (Table 2, entries 7–10; the same effect was observed inIPC-81 cells).[24b, 33a, 45] The effect was confirmed for 1-alkyl-3-methylimidazolium-based ILs containing different anions and

Table 1. Toxicity dependence of the IL 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide on bio-logical systems.

Biological system EC50

[mmol L�1]LC50

[mmol L�1]MIC[mmol L�1]

IC50

[mmol L�1]Ref.

cell cultures HeLa 1170[a] – – – [32c]IPC-81 500[a] – – – [30b]

invertebrates D. magna – 45.1[b] – – [26]F. candida 30[c] – – – [30a]

algae S. capricornutum 63.2[d] – – – [26]S. vacuolatus 50[e] – – – [30a]

higher plants L. minor 380[f] – – – [30a]T. aestivum 110[g] – – – [30a]

gram-negative bacteria V. fischeri 300–339[h] – – – [30a, 109]E. coli – – 2000[i] – [30i]

gram-positive bacteria S. aureus – – >2000[i] – [30i]Enterococcus sp. – – 500

(E. faecium,E. hirae)[i]

– [30i]

fungi Candida sp. – – >2000(C. albicans,C. glabrata,

C. tropicalis)[i]

– [30i]

S. cerevisiae – – 1300–2000[i] – [30i]enzyme acetylcholinesterase – – – 90[j] [30a]

[a] 48 h EC50. [b] 48 h LC50. [c] 96 h EC50 ; mmol kg�1 dry weight soil, reproduction inhibition. [d] 72 h EC50,growth inhibition. [e] 24 h EC50, reproduction inhibition. [f] EC50, growth inhibition. [g] EC50, mmol kg�1 dryweight soil, growth inhibition. [h] 15 min EC50, luminescence inhibition. [i] 24 h MIC. [j] IC50.

Figure 3. Toxicity of ionic liquids depends on the biological system. Toler-ance of different biological systems to 1-octyl-3-methylimidazolium bromideis shown in per cent of the maximum tolerance (in this case, demonstratedby U. lactuca ; see Table 2, entry 3, Table 5, entry 3, and Table 7, entry 3).(Images from [22c] , [31] are used in this figure).



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in different cell cultures: for tet-rafluoroborates in HeLa, CaCo-2,HT-29, and IPC-81 cells ;[32c, 33a, 37a]

for hexafluorophosphates inCaCo-2 cells ;[37b] and for bis(tri-fluoromethylsulfonyl)imides inHeLa[32c] and CCO cells.[42] A simi-lar but less pronounced effectwas demonstrated for quinolini-um-based ILs (Table 2, en-tries 14–19): the increase of theside-chain length from eight to18 carbon atoms resulted ina 46-fold decrease of 24 h EC50

in NIH 3T3 cells, from137 mmol L�1 for 1-octylquinolini-um bromide to 3 mmol L�1 for 1-octadecylquinolinium bromide[36]

(the same was observed for qui-nolinium-based tetrafluorobo-rates in IPC-81 cells).[33a] Interest-ingly, in case of the quinolinium-based ILs, the toxicity leveled offon reaching a threshold side-chain length (14 carbon atoms;Table 2, entries 18 and 19). The

Table 2. Influence of alkyl side-chain length on IL toxicity (EC50) on cell cultures.

Entry Alkyl chain EC50 [mmol L�1] for cell culturesPC12[a] CaCo-2 IPC-81[b] HeLa[b] NIH 3T3[a]

1-alkyl-3-methylimidazolium bromides[34b]

1 butyl >91232 hexyl 68743 octyl 5204 decyl 505 dodecyl 24

1-alkyl-3-methylimidazolium chlorides6 ethyl 7244[45]

7 butyl 28 690 (24 h)/3800 (48 h)[37b] 3548–3600[30b, 33a]

8 hexyl 5240 (24 h)/240 (48 h)[37b] 708[33a]

9 octyl 540 (24 h)/30 (48 h)[37b] 102[24b]

10 decyl 1910 (4 h)/30 (24 h)/10 (48 h)[30f, 37b] 22[33a]

1-alkyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imides[32c]

11 ethyl 182012 butyl 117013 octyl 190

1-alkylquinolinium bromides[36]

14 octyl 1280–65 (1–72 h)15 decyl 1416 dodecyl 618 tetradecyl 3 (15–2 for 1–72 h)19 octadecyl 3

[a] 24 h EC50, if not specified otherwise. [b] 48 h EC50.

Figure 4. Eco(cyto)toxicity of ILs. ILs display toxicity towards different biological systems, including enzymes, cells, bacteria, fungi, plants, and animals. [Imagesfrom [22c] , [30a] , [31a] , [32] are used in this figure; the structure of the electric eel (Electrophorus electricus) acetylcholinesterase is from PDBe (Protein DataBank Europe, http://www.ebi.ac.uk, entry 1c2b)] .



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cytotoxic effect of other ILs also seemed to depend on thealkyl side-chain length of the cation, as was demonstrated forammonium-,[32c] pyrrolidinium-,[30g, 33a, 38a] piperidinium-,[30g, 38a]

and pyridinium-based[32c, 33a] ILs.The second observation concerns the presence of a function-

al group on the side chain of the cation. Important data havebeen accumulated on the effect of oxygen-containing func-tional groups (Table 3). The introduction of oxygen atoms (inparticular the terminal hydroxyl group or ethoxy moieties) intothe side chain of imidazolium cations resulted in lower IL cyto-toxicities in IPC-81 and PC12 cells,[30b, 34a] whereas ILs with oxy-genated cations in cytotoxicity assays in PC12 cells demon-strated weaker toxicity in comparison to 1-butyl-3-methylimi-dazolium, independent of anion and number of ethoxy unitsin the side chain.[34a] Thus, in IPC-81 cells, the 48 h EC50 values of1-(3-hydroxypropyl)-3-methylimi-dazolium and 1-(3-methoxyprop-yl)-3-methylimidazolium chlor-ides were >20 000 mmol L�1, ascompared to 3600 mmol L�1 for1-butyl-3-methylimidazoliumchloride (Table 3, entries 2, 3,and 6).[30b] A similar effect wasobserved in IPC-81 cells for mor-pholinium-based ILs [48 h EC50

of 4-(3-methoxypropyl)-4-methyl-morpholinium bis(trifluorome-thylsulfonyl)imide was5900 mmol L�1 compared to2700 mmol L�1 for 4-butyl-4-methylmorpholinium bis(trifluor-omethylsulfonyl)imide] and pyr-rolidinium-based ILs [48 h EC50 of1-(2-ethoxyethyl)-1-methylpyrro-lidinium bromide was>20 000 mmol L�1, as comparedto 5888 mmol L�1 for 1-butyl-1-methylpyrrolidiniumbromide] .[30b, 33a] Data on otherfunctional groups in the cationside chain confirm the effect, asdemonstrated for 1-cyanometh-yl-3-methylimidazolium chloride

(IPC-81, 48 h EC50>20 000 mmol L�1) ;[30b] 1-benzyl-3-methylimi-dazolium chloride and 1-p-fluorobenzyl-3-methylimidazoliumchloride (CaCo-2, 24 h EC50 30 760 and 20 370 mmol L�1, respec-tively) ;[37b] and 1,3-dibenzylimidazoilium chloride (CaCo-2, 4 h,not toxic).[30f]

The third observation concerns the effect of an anionmoiety on the IL toxicity, which was observed in many biologi-cal systems (Table 4). For 1-butyl-3-methylimidazolium-basedILs, chlorides seemed to be the least toxic, whereas bis(trifluor-omethylsulfonyl)imides and bis(trifluoromethyl)imides were themost toxic: in IPC-81 cells, 48 h EC50 of 1-butyl-3-methylimida-zolium chloride, bis(trifluoromethylsulfonyl)imide and bis(tri-fluoromethyl)imide were 3600,[33a] 500,[30b] and 155 mmol L�1,[24b]

respectively (Table 4, entries 2, 5, and 6). However, 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide, as well as1-butyl-3-methylimidazolium hexafluorophosphate, wereshown to be nontoxic in CaCo-2 cells (Table 4, entries 4 and5).[37a] 1-Butyl-3-methylimidazolium chloride also demonstratedlow toxicity in HeLa[39] and CaCo-2 cells : its 48 h EC50 was3800 mmol L�1 compared to 23.3 mmol L�1 for 1-butyl-3-methyli-midazolium O-methyl sulfate[37b] (Table 4, entries 2 and 8). Itshould be noted that the effect was time dependent: for 1-butyl-3-methylimidazolium chloride, 24 h and 48 h EC50 inCaCo-2 cell were 28 690 and 3800 mmol L�1, respectively.[37b] 1-Butyl-3-methylimidazolium hexafluorophosphate showed lowtoxicity in channel catfish ovary cell lines (72 h EC50>

10 000 mmol L�1), whereas 1-butyl-3-methylimidazolium tetra-

Table 3. Influence of functionalized side chain on the toxicity of 1-alkyl-3-methylimidazolium chloride (48 h EC50) on the IPC-81 cell culture.

Entry Side chain EC50 [mmol L�]

1 ethyl 7244[45]

2 butyl 3548–3600[30b, 33a]

3 3-hydroxypropyl >20 000[30b]

4 ethoxymethyl 4000[30b]

5 2-methoxyethyl >20 000[30b]

6 3-methoxypropyl >20 000[30b]

7 cyanomethyl >20 000[30b]

Table 4. Influence of ionic moiety on IL toxicity (EC50) on cell cultures.

Entry Ionic moiety EC50 [mmol L�1] for cell culturesCaCo-2 IPC-81[a] HeLa[a]

anions1-butyl-3-methylimidazolium ILs1 bromide 2692[33a] 2750[32c]

2 chloride 28 690 (24 h)/3800 (48 h)[37b] 3548–3600[30b, 33a] 12 300 (24 h)[39]

3 tetrafluoroborate >6026 (4 h)[37a] 1318[33a] 4550[32c]

4 hexafluorophosphate NT[b][37a] 1259[33a] 13 900 (24 h)[39]

5 bis(trifluoromethylsulfonyl)imide NT[b][37a] 500[30b] 1170[32c]

6 bis(trifluoromethyl)imide 155[24b]

7 dicyanamide >6026 (4 h)[37a] 1413[24b]

8 O-methyl sulfate 20.5 (24 h)/23.3 (48 h)[37b] 1622[24b]

9 O-octyl sulfate 1698[24b]

10 trifluoromethanesulfonate 1047[24b]

1-butyl-1-methylpyrrolidinium-based Ils11 bromide 5888[33a]

12 chloride >20 000[30b, 33a]

13 bis(trifluoromethylsulfonyl)imide 1000[30b]

14 O-methyl sulfate >3947[30q]

cations (bromide as anion)15 1-butyl-3-methylimidazolium 2692[33a]

16 1-butylquinolinium 209[33a]

17 4-butyl-4-methylmorpholinium >20 000[30b]

18 1-butyl-1-methylpyrrolidinium 5888[33a]

19 1-butyl-1-methylpiperidinium 11 000[30b]

20 1-butylpyridinium 8000[30b, 33a]

21 tetrabutylammonium 178[33a]

22 tetrabutylphosphonium 46[33a]

[a] 48 h EC50, if not specified otherwise. [b] Not toxic.



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fluoroborate, bromide and bis(trifluoromethylsulfonyl)imidewere more toxic, but also demonstrated relatively low toxicity(72 h EC50 4460, 3810, and 2880 mmol L�1, respectively).[42] Incase of 1-octyl-3-methylimidazolium-based ILs, bromide andtetrafluoroborate exhibited similar toxicity (48 h EC50

300 mmol L�1 in HeLa cells), whereas bis(trifluoromethylsulfony-l)imide was more toxic (48 h EC50 190 mmol L�1 in HeLacells).[32c] In the case of 1-butyl-1-methylpyrrolidinium-basedILs, chloride also seemed to be the least toxic, whereas bis(tri-fluoromethylsulfonyl)imide was the most toxic: in IPC-81 cells,48 h EC50 of 1-butyl-1-methylpyrrolidinium chloride and bis(tri-fluoromethylsulfonyl)imide were >20 000 and 1000 mmol L�1,respectively (Table 4, entries 12 and 13).[30b, 33a] Among the mostrecent works concerning the anion impact on IL toxicity is thestudy of Frade and colleagues, who investigated the cytotoxici-ty of imidazolium- and cholinium-based ILs containing mag-netic anions ([FeCl4] , [GdCl6] , [CoCl4] , and [MnCl4]). Accordingto their data, the [CoCl4] and [MnCl4] anions increased the tox-icity of the investigated ILs in CaCo-2 cells and normal humanskin fibroblasts (CRL-1502).[46]

The afore-mentioned observations on the anion impactseem to depend on the nature of the cation. Thus, the strongimpact of the anion was demonstrated for phosphonium-based cations, but in contrast to imidazolium- and pyrrolidini-um-based ILs trihexyl(tetradecyl)phosphonium chloride([P6,6,6,14][Cl]) showed higher cytotoxic effects in S2 cells (to-gether with the highest water solubility) in comparison to tri-hexyl(tetradecyl)phosphonium bis(trifluoromethylsulfonyl)imide([P6,6,6,14][NTf2]), which showed the least cytotoxic effect ; trihex-yl(tetradecyl)phosphonium methanesulfonate, on the otherhand, showed extremely high cytotoxicity, possibly because ofthe high solubility of methanesulfonic acid and its derived IL inwater.[30n] The anion was a significant determinant of cytotoxic-ity also for 4-benzyl-4-methylmorpholinium ILs.[30m]

The fourth observation concerns the cation effect itself.Thus, comparison of different ILs demonstrate that morpholini-um-based ILs seem to be the least toxic (48 h EC50 of 4-butyl-4-methylmorpholinium bromide >20 000 mmol L�1), whereasphosphonium-based ILs are the most toxic (48 h EC50 of tetra-butylphosphonium bromide 46 mmol L�1) in IPC-81 cells(Table 4, entries 17 and 22).[30b, 33a]

It is also apparent that, apart from the structure, IL toxicitiesdepend on the cell type (Tables 1,2, and 4). However, themechanism of the IL cytotoxicity has not been studied thor-oughly yet. It has been proposed that long alkyl side chainsdisturb the cellular membrane. For example, 1-dodecyl-3-meth-ylimidazolium bromide is highly cytotoxic in U937 cells and, aswell as other ILs, shows strong attachment to the lipid bilayersurface, likely giving rise to a considerable disruption of themembrane (Figure 5 f–h).[40] 1-Octyl-3-methylimidazolium bro-mide caused a marked increase in lactate dehydrogenase re-lease, whereas the affected PC12 cells lost adhesion ability. Thepercentage of apoptotic cells was higher than in control, sug-gesting that the IL exposure induced apoptosis in PC12 cells(Figure 5 a and b).[34b] 1-Octyl-3-methylimidazolium chloride in-hibited PC12 cell growth and decreased their viability depend-ing on the dose, inducing DNA damage and overproduction of

reactive oxygen species, which gradually exhausted cellularATP and caused mitochondrial permeability transition andapoptosis,[34c] whereas 1-ethyl-3-methylimidazolium tetrafluoro-borate induced apoptosis in HeLa cells (Figure 5 c–e).[32c]

2.3. Toxicity of ionic liquids towards invertebrates

The main invertebrate ‘sensor’ for studying (eco)toxicity of dif-ferent substances, including ILs, is the crustacean Daphniamagna (D. magna),[26, 30q, 34a, 47] which serves as a link betweenbacteria and higher organisms.[24a] Substance toxicity towardsD. magna is assayed using acute immobilization tests, whichreveal the degree of toxicity on swimming capability, or viabili-ty, of D. magna.[48] In general, imidazolium-based ILs showedhigh toxicity towards D. magna and exhibited a dependence ofthe side-chain length of the cation: the increase of the side-chain length from four to 12 carbon atoms resulted in a 470-fold decrease of 48 h LC50 (from 70 mmol L�1 for 1-butyl-3-methylimidazolium bromide to 0.15 mmol L�1 for 1-dodecyl-3-methylimidazolium bromide) (Table 5, entries 1–5).[47d]

Introduction of an oxygenated side chain into the imidazoli-um cation reduced the IL toxicity towards D. magna : 48 h LC50

for 1-butyl-3-methylimidazolium and 1-(2-methoxyethyl)-3-methylimidazolium tetrafluoroborates were 50 and80 mmol L�1, respectively.[34a, 47b] There was no direct correlationbetween the acute toxicity of IL towards D. magna and the

Figure 5. IL effects on cell morphology and membrane integrity. a, b) nuclearmorphological alteration of PC12 cells induced by 1-octyl-3-methylimidazoli-um bromide (a: control ; b: 1-octyl-3-methylimidazolium bromide; cells werestained with acridine orange and visualized under fluorescent microscope).c–e) scanning electron microphotographs of untreated HeLa cells (c) andcells exposed to 1-ethyl-3-methylimidazolium tetrafluoroborate (d, e). f–h) confocal fluorescence microscopy images of rhodamine-phosphoethanol-amine-phosphatidylcholine-phosphatidylglycerol giant vesicles following in-cubation with ILs (f : control ; g: benzyltributylammonium chloride; h: 1-butyl-3-methylimidazolium chloride). (a, b from [34b]; c–e from [32c] ; f–hfrom [40]).



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number of ethoxy units, but the crustacean seemed somewhatsensitive to the anion type: the 48 h LC50 varied from 23–53 mmol L�1 (for 1-butyl-3-methylimidazolium tetrafluorobora-te)[34a, 47b] to 85 mmol L�1 (for 1-butyl-3-methylimidazolium chlo-ride)[49] (Table 6, entries 1–6). On the other hand, in case of pyr-rolidinium-based ILs, D. magna showed a significantly highersensitivity to the anion type: 48 h LC50 varied from 395 to88 mmol L�1 for 1-butyl-1-methyl-pyrrolidinium O-methyl sulfa-te[30q] and bis(trifluoromethylsulfonyl)imide,[26] respectively(Table 6, entries 7 and 8).

1-Butyl-3-methylimidazolium tetrafluoroborate destabilizedthe model membranes,[34a] whereas 1-methyl-3-octylimidazoli-um bromide significantly decreased the body length and inhib-ited the reproductive ability of D. magna.[47c] Exposure ofD. magna to higher IL concentrations and to ILs with longeralkyl chains generally increased the activities of antioxidant-de-fense enzymes (superoxide dismutase, catalase, glutathioneperoxidase, and glutathione S-transferase) and biomarkerlevels (the antioxidant glutathione and the lipid peroxidationby-product malondialdehyde, a marker of oxidative stress).[47d]

It was demonstrated that biodegradation products of 1-butyl-3-methylpyridinium, 1-hexyl-3-methylpyridinium and 1-octyl-3-methylpyridinium bromides were less toxic for D. magna thanthe initial compounds.[47e]

Swatloski and colleagues proposed a well-known nematodeCaenorhabditis elegans to be used as a model organism for in-vestigating the IL effect on the viability of worms by addingILs to the growth media.[50] By using 1-alkyl-3-methylimidazoli-um chlorides, they demonstrated a positive correlation be-tween the alkyl chain length and IL toxicity. Moreover, animalsexposed to 1-octyl-3-methylimidazolium showed an averse re-sponse. Still, the survivors showed no signs of injury and repro-duced normally.

The side-chain effect was also pronounced in case of 1-alkyl-3-methylimidazolium bromides exhibiting toxicity towards thezebra mussel Dreissena polymorpha (D. polymorpha)[51] andfreshwater snail Physa acuta (P. acuta).[52] For D. polymorpha,the increase of the side-chain length from four to eight carbonatoms resulted in a 74-fold decrease of 96 h LC50 (from5887 mmol L�1 for 1-butyl-3-methylimidazolium bromide to79 mmol L�1 for 1-octyl-3-methylimidazolium bromide; Table 5,entries 1–3),[51] whereas for P. acuta, the increase from six to 12carbon atoms resulted in a 350-fold decrease of 96 h LC50

(from 1455 mmol L�1 for 1-hexyl-3-methylimidazolium bromideto 4 mmol L�1 for 1-dodecyl-3-methylimidazolium bromide;Table 5, entries 2–5).[52] A similar effect was observed for 1-alkyl-3-methylpyridinium bromides in D. polymorpha : the in-crease of the side-chain length from four to eight carbonatoms resulted in a 52-fold decrease of 96 h LC50 (from3915 mmol L�1 for 1-butyl-3-methylpyridinium bromide to75 mmol L�1 for 1-octyl-3-methylpyridinium bromide).[51] Thelatter observation also demonstrates some sensitivity of D. pol-ymorpha to the IL cation (compare the 96 h LC50 5887 and3915 mmol L�1 for 1-butyl-3-methylimidazolium and 1-butyl-3-methylpyridinium bromides, respectively), but it apparently de-clines with elongation of the cation alkyl side chain (96 h LC50

79 and 75 mmol L�1 for 1-octyl-3-methylimidazolium and 1-octyl-3-methylpyridinium bromides, respectively).[51] Short-termexposure to 1-alkyl-3-methylimidazolium and 1-alkyl-3-methyl-pyridinium bromides reduced zebra mussel feeding;[51] 1-butyl-3-methylimidazolium bromide inhibited growth rates of P.acuta,[53] whereas 1-octyl-3-methylimidazolium bromide inhibit-ed the hatching rate of snail embryos; all of the treated em-bryos died when the exposure concentration was higher than15 mmol L�1.[52]

The alkyl-chain length–toxicity correlation could also be ob-served in toxicities of 1-methyl-3-alkylimidazolium bromides to-wards the earthworm Eisenia foetida. In filter paper contacttests, the 48 h LC50 decreased from 0.33 mmol cm�2 for 1-methyl-3-butylimidazolium bromide to 0.06 nmol cm�2 for 1-methyl-3-dodecylimidazolium bromide.[54] The 7-d LC50 of 1-methyl-3-octylimidazolium bromide was 750 mmol kg�1 artificialsoil (dry weight). 1-Methyl-3-octylimidazolium bromide mightinterfere with the nervous function of the earthworm, whereasin high concentrations it significantly inhibited growth.[54, 55] Re-productive ability of E. foetida was significantly repressed after42 days of subchronic exposure to 1-methyl-3-octylimidazoli-

Table 5. Influence of alkyl side-chain length on IL toxicity (LC50) on inver-tebrates.

Entry Alkyl chain LC50 [mmol L] for invertebratesD. magna[a] D. polymorpha[b] P. acuta[b]

1-alkyl-3-methylimidazolium bromides1 butyl 70[47d] 5887[51] 1045[110]

2 hexyl 11.5[47d] 429[51] 1455[52]

3 octyl 10.3[47d] 79[51] 1307[52]

4 decyl 0.5[47d] 30[52]

5 dodecyl 0.15[47d] 4[52]

1-alkyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imides6 ethyl 230[33b]

7 butyl 45.1[26]

[a] 48 h LC50. [b] 96 h LC50.

Table 6. Influence of ionic moiety on IL toxicity (LC50) on invertebrates.

Entry Ionic moiety LC50 [mmol L�1] for invertebratesD. magna[a] P. acuta[b]

anion1-butyl-3-methylimidazolium ILs1 bromide 70[47d]

2 chloride 85[49]

3 tetrafluoroborate 23–53[34a, 47b]

4 hexafluorophosphate 70.8[49]

5 bis(trifluoromethylsulfonyl)imide 45.1[26]

6 dicyanamide 78[34a]

1-butyl-1-methylpyrrolidinium Ils7 bis(trifluoromethylsulfonyl)imide 88[26]

8 O-methyl sulfate 395[30q]

cation (bromide as anion)[110]

9 1-butyl-3-methylimidazolium 104510 tetrabutylammonium 180011 tetrabutylphosphonium 613

[a] 48 h LC50. [b] 96 h LC50.



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um bromide at concentrations �18 mmol kg�1 artificial soil (dryweight), whereas during acute exposure, concentrations�145 mmol kg�1 artificial soil (dry weight) inhibited the activi-ties of Na+-K+- and Mg2+-adenylpyrophosphatase after 3 and7 days of exposure.[55]

2.4. Toxicity of ionic liquids towards vertebrates

Studying IL toxicity towards aquatic vertebrate organisms as-sists evaluation of the IL effect on the environment. However,experiments with vertebrate animals are rather time and costconsuming and the existing separate data on IL toxicity in ver-tebrates do not allow drawing general conclusions with regardto dependence of IL toxicity on IL structure and organismnature.

1-Octyl-3-methylimidazolium hexafluorophosphate displayeda toxic effect on zebrafish (Danio rerio): it inhibited the activi-ties of antioxidant enzymes and caused the accumulation ofreactive oxygen species and DNA damage depending on con-centration and time.[56] Similar results were obtained with 1-decyl-3-methylimidazolium bromide, which caused DNAdamage in zebrafish liver and inhibited the anti-oxidative-stress enzymes catalase (CAT) and superoxide dismutase(SOD).[57]

1-Methyl-3-octylimidazolium bromide proved to be toxic to-wards the goldfish Carassius auratus,[58] the brocarded carpCyprinus carpio (C. carpio),[59] and the frog Rana nigromaculata(R. nigromaculata),[60] affecting both the early embryonic devel-opment and adult organisms. An exposure to 1-methyl-3-octy-limidazolium bromide prolonged the duration of embryo de-chorionation and decreased the hatching rates, causing in-creases in the number of embryonic malformation and themortality ratio.[58a] For adult goldfish, 24 h LC50 of 1-methyl-3-octylimidazolium bromide was 887 mmol L�1; acute exposure tothis IL induced superficial damage to skin, gill filaments, andintestinal villi of the fish, whereas histological examination alsoindicated damage to hepatopancreas and kidney.[58b] In addi-tion, 1-methyl-3-octylimidazolium bromide caused a significantincrease of the malondialdehyde level in the hepatopan-creas[58b] and induced changes in the activities of SOD, CAT,glutathione peroxidase, and glutathione.[58c] 1-Methyl-3-octyli-midazolium bromide had an immunotoxic effect on the bro-carded carp C. carpio : 1090 mmol L�1 of IL inhibited the specificand nonspecific immune systems, as demonstrated by IgMlevel, lysozyme activity, and complement C3 content, whereas363 mmol L�1 activated the immune system of the fish duringthe early periods of exposure (2–5 days). Exposure to1090 mmol L�1 of IL led to remarkable damages in hepatopan-creas, kidney, and spleen after 7 days of treatment.[59] 1-Methyl-3-octylimidazolium bromide also affected the early embryonicdevelopment of the frog R. nigromaculata, prolonging the du-ration of embryo dechorionation at the early cleavage andneural plate developmental stages.[60]

As for mammals, toxicity of ILs towards mice, rats, and rab-bits was studied. Oral toxicity, dermal and eye irritation, andlocal lymph node assay, as well as maternal toxicity, were in-vestigated. Landry and colleagues drew an acute toxicity pro-

file of 1-butyl-3-methylimidazolium chloride.[29] In female rats,the acute oral LD50 was estimated as 3150 mmol kg�1 of bodyweight. According to an acute-irritation study, 1-butyl-3-meth-ylimidazolium chloride was classified as slightly irritating to theskin (Draize score[28] 1.5) and mildly irritating to the eye.

1-Alkyl-3-methylimidazolium chlorides displayed toxic effectsin mice. 1-Butyl-3-methylimidazolium chloride showed devel-opmental toxicity at maternally toxic dosages (968 and1288 mmol per kg and day, maternal oral doses) leading toa decrease of fetal weight and displaying possible teratogenicactivity.[61] A teratogenic effect was also associated with 1-decyl-3-methylimidazolium chloride, whereas an exposure to1-ethyl-3-methylimidazolium chloride resulted in no morpho-logical defects despite maternal morbidity at the highestdosage.[62] Studies of the route of toxicokinetics of 1-butyl-3-methylimidazolium chloride[63] and 1-butyl-3-methylpyrrolidini-um chloride[64] in rats and mice showed that these ILs wereeliminated with the urine and did not accumulate heavily intissues.

The toxicity of didecyldimethylammonium saccharinate wasstudied in rats. In acute experiments, didecyldimethylammoni-um saccharinate induced exfoliation of the surface layer of thecolon and alveolar septa in lung parenchyma. However, theobserved changes in clinical chemistry parameters did notoccur in both sexes and were not related to the dose.[41]

Recent data propose a possible mechanism of the cytotoxicaction of ILs at the level of a complex organism. It was foundthat ILs inhibited organic cation transporters (OCTs) and multi-drug and toxic extrusion transporters (MATEs) in Chinese ham-ster ovary cells transfected with the corresponding rat orhuman gene constructs.[65] OCTs are polyspecific organic cationtransporters found in the kidney, liver, and other organs; theseproteins take part in elimination of small organic cations,drugs and toxins. MATEs apparently function as proton-drivencation efflux transporters in the liver and kidney.[66] Cheng andcolleagues studied the inhibitory activity of N-alkylpyridiniumchlorides as well as of 1-butyl-3-methylimidazolium chloride,N-butyl-N-methylpyrrolidinium chloride, and pyridine hydro-chloride.[65] N-alkylpyridinium chlorides exhibited strong inhibi-tion of OCT2, as demonstrated by the intracellular uptake oftetraethylammonium trifluoroacetate (IC50 ranging from36.7 mmol L�1 for N-ethylpyridinium chloride to 0.35 mmol L�1

for N-hexylpyridinium chloride) ; 1-butyl-3-methylimidazoliumchloride and N-butyl-N-methylpyrrolidinium chloride were alsopotent inhibitors of OCT2 (IC50 of 1.5 and 0.48 mmol L�1, respec-tively), whereas pyridine hydrochloride was significantly lesstoxic (IC50 : 790 mmol L�1). Similar kinetics was obtained for in-tracellular uptake of metformin (antidiabetic drug). The studyalso included the OCT1 transporter as well as MATE1 andMATE2K. Moreover, it was demonstrated that N-butylpyridini-um chloride reduced renal clearance of metformin in rats.These data suggest that ILs can shift drug pharmacokineticsin vivo.



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2.5. Phytotoxicity of ionic liquids

In many studies concerned with ecotoxicity of ILs, algae, whichare good indicators of environmental changes, are used as testobjects.[24a] The main toxic effect considered is growth inhibi-tion or, in some cases [e.g. , Scenedesmus vacuolatus (S. vacuo-latus)] , reproduction inhibition.[30a, c, 33b] So far, IL toxicities wereassessed towards microalga Selenastrum capricornutum (S. cap-ricornutum, Pseudokirchneriella subcapitata) ;[26, 30d, 67] green algaeChlamydomonas reinhardtii,[68] Scenedesmus quadricauda,[68a]

Scenedesmus obliquus (S. obliquus),[69] S. vacuolatus,[30a, c, 33b, 70]

Chlorella vulgaris (C. vulgaris),[30h, 71] Oocystis submarina[30h, 71] andChlorella ellipsoidea (C. ellipsoidea) ;[69] marine macroalga Ulvalactuca (U. lactuca) ;[72] diatoms Bacillaria paxillifer (B. paxillif-er),[73] Cyclotella meneghiniana, Skeletonema marinoi (S. mari-noi),[30h, 74] and Phaeodactylum tricornutum (P. tricornutum) ;[74]

and cyanobacterium Geitlerinema amphibium (G. amphibium ;belongs to bacteria but is considered together with algaeherein).[71, 73]

An impact of the cation side-chain length was observedwhen studying the toxicity of 1-alkyl-3-methylimidazolium-based ILs towards the freshwater green algae S. obliquus, C. el-lipsoidea,[69] and C. vulgaris[30h] and the diatom B. paxillifer.[73]

Thus, the increase of the side-chain length from four to 12carbon atoms resulted in 1690-fold and 460-fold decreases ofthe 96 h EC50 (from 102 and 110 mmol L�1 for 1-butyl-3-methyli-midazolium bromide to 0.06 and 0.24 mmol L�1 for 1-dodecyl-3-methylimidazolium bromide) for S. obliquus and C. ellipsoidea,respectively (Table 7, entries 1–5),[69] whereas the increase fromtwo to ten carbon atoms led to 35-fold and 1710-fold decreas-es of 72 h EC50 (from 34.4 and 6330.51 mmol L�1 for 1-ethyl-3-methylimidazolium chloride to 0.99 and 3.68 mmol L�1 for 1-

decyl-3-methylimidazolium chloride) for B. paxillifer and C. vul-garis, respectively (Table 7, entries 8–12).[30h, 73] Analogous ef-fects were observed when studying toxicity of 1-alkyl-3-methyl-imidazolium chlorides towards G. amphibium,[73] Oocystis sub-marina, Cyclotella meneghiniana, S. marinoi,[30h] and S. vacuola-tus.[33b] 1-Alkyl-3-methylimidazolium-based bis(trifluoromethyl-sulfonyl)imides exhibited similar effects towards S. vacuolatus(Table 7, entries 13–15)[30a, 33b, 70]

An influence of functionalized groups in the side chain waspronounced for 1-alkyl-3-methylimidazolium-based ILs, as dem-onstrated by reproduction inhibition of S. vacuolatus : 24 h EC50

of 1-(3-hydroxypropyl)-3-methylimidazolium chloride, 1-(ethox-ymethyl)-3-methylimidazolium chloride, 1-(2-methoxyethyl)-3-methylimidazolium chloride, and 1-(3-methoxypropyl)-3-meth-ylimidazolium chloride were >1000, 631–890, 1820, and>1000 mmol L�1, respectively,[30c, 70] as compared to 140–178 mmol L�1 for 1-butyl-3-methylimidazolium chloride[30a, 70] or600 mmol L�1 for 1-ethyl-3-methylimidazolium chloride[33b]

(Table 8). 1-Cyanomethyl-3-methylimidazolium chloride also

showed moderate toxicity to-wards S. vacuolatus (24 h EC50>

1000 mmol L�1).[30c] Similar effectswere observed with the marinediatoms S. marinoi and P. tricor-nutum : monoethoxy and dieth-oxy cations demonstratedweaker inhibition of growth andphotosynthesis.[74]

An influence of the anionmoiety on IL toxic propertieswas demonstrated for 1-butyl-3-methylimidazolium-based ILswhen studying their toxicity to-wards S. capricornutum[26, 30d, 67]

and B. paxillifer.[73] In case ofS. capricornutum, 1-butyl-3-meth-ylimidazolium bis(trifluorome-thylsulfonyl)imide exhibited thehighest toxicity (72 h EC50

63.2 mmol L�1),[26] whereas chlo-ride was the least toxic (96 hEC50 2884 mmol L�1)[30d] (Table 9,entries 2 and 5). In case of

Table 8. Influence of functionalized side chain on the toxicity of 1-alkyl-3-methylimidazolium chloride (24 h EC50, reproduction inhibition) on S. va-cuolatus.

Entry Alkyl chain EC50 [mmol L�1]

1 ethyl 600[33b]

2 butyl 140–178[30a, 70]

3 3-hydroxypropyl >1000[30c]

4 ethoxymethyl 631–890[30c, 70]

5 2-methoxyethyl 1820[30c, 70]

6 3-methoxypropyl >1000[30c]

7 cyanomethyl >1000[30c]

Table 7. Influence of alkyl side-chain length on IL toxicity (EC50, LC50) on algae.

Entry Alkyl chain EC50 [mmol L�1] LC50 [mmol L�1]S. obliquus[a] C. ellipsoidea[a] B. paxillifer[b] C. vulgaris[b] S. vacuolatus[c] U. lactuca[d]

1-alkyl-3-methylimidazolium bromides1 butyl 102[69] 110[69] 9800[72]

2 hexyl 24[69] 44[69] 3710[72]

3 octyl 1.2[69] 23[69] 1420[72]

4 decyl 0.3[69] 1.1[69] 150[72]

5 dodecyl 0.06[69] 0.24[69] 70[72]

6 tetradecyl 40[72]

7 hexadecyl 20[72]

1-alkyl-3-methylimidazolium chlorides8 ethyl 34.4[73] 6330.51[30h] 600[33b]

9 butyl 6.48[73] 1026.2[30h] 140–178[30a, 70]

10 hexyl 2.01[73] 64.5[30h]

11 octyl 1.52[73] 15.1[30h]

12 decyl 0.99[73] 3.68[30h]


14 butyl 50–63[30a, 70]

15 hexyl 1.1[70]

[a] 96 h EC50, growth inhibition. [b] 72 h EC50, growth inhibition. [c] 24 h EC50 reproduction inhibition. [d] 4-dLC50.



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B. paxillifer, all anions tested displayed high toxicity: the 96 hEC50 ranged from 5.16 mmol L�1 for 1-butyl-3-methylimidazoli-um dicyanamide to 16.05 mmol L�1 for 1-butyl-3-methylimida-zolium O-methyl sulfate (Table 9, entries 2, 7, 8, and 10).[73] Thecorrelation between the anion moiety and toxic effect was alsoobserved for other diatoms and green algae.[30h, 73] A similareffect was detected for 1-butyl-1-methylpyrrolidinium-basedILs in S. capricornutum[26, 30q, 67a] and S. vacuolatus[30c] (Table 9, en-tries 11–14). An estimation of the hydrolysis of fluoride-contain-ing ILs showed that hexafluoroantimonate produced more po-tentially harmful fluoride ions than tetrafluoroborate, whereasno fluoride formation occurred with hexafluorophosphate.Small amounts of fluoride ions did not affect the growth rateof algae; however, the fluoride ion formation from 1-butyl-3-methylimidazolium tetrafluoroborate increased with incubationtime of the stock solution,[30d] possibly resulting in significantlylower EC50 values.[67b]

Apparently, the cation structure also influences IL toxicity.Thus, in case of S. capricornutum, tetrabutylphosphonium bro-mide was the most toxic (96 h EC50 224 mmol L�1), whereas 1-butyl-1-methylpyrrolidinium bromide demonstrated the lowesttoxicity (96 h EC50 12 303 mmol L�1; Table 9, entries 16 and

18).[67a] The effect of ILs on S. capricornutum was also stronglyrelated to the incubation time: the toxicities of tetrabutylphos-phonium, tetrabutylammonium, 1-butyl-3-methylpyridinium,and 1-butyl-1-methylpyrrolidinium bromides decreased, where-as the toxicity of 1-butyl-3-methylimidazolium bromide in-creased when the incubation time was increased from 48 to96 h.[67a] Interestingly, 1-butyl-3-methylimidazolium bromidehad a prominent hormetic effect on S. capricornutum ;[67a] itshould be also mentioned that the toxic effect of the IL ongrowth rates was more pronounced than that on photosyn-thetic activity (96 h EC50 23 998 and 2138 mmol L�1 for photo-synthetic activity and growth rate, respectively).[67b] It was dem-onstrated that the cell wall was involved in susceptibility ofChlamydomonas reinhardtii to such ILs as 1-butyl-2-methylpyri-dinium bromide and tetrabutylammonium bromide, whereas ithad no influence on toxicity of 1-alkyl-3-methylimidazoliumbromides.[68b]

The mentioned effects were also shown for the marine mul-ticellular green alga U. lactuca[72] and higher plants, particularlythe freshwater plant Lemna minor (L. minor), or common duck-weed[30a, c, 33b] (Tables 7 and 10). For L. minor, the effects weremore pronounced for growth inhibition of roots than that offronds.[75] The butyl-substituted 3-methylpyridinium and 3-methylimidazolium cations expressed similar toxicity towardsL. minor, whereas the tetrabutylammonium cation was consid-erably less toxic; still, toxicity of ILs seemed to be similar tothat of other organic solvents, for example, phenol.[75] An expo-sure to 1-dodecyl-3-methylimidazolium bromide triggered thereactive oxygen species damage of the membrane and DNAand inhibition of antioxidant systems in U. lactuca.[72] 1-Butyl-3-methylimidazolium tetrafluoroborate reduced Triticum aestivum(T. aestivum) germination and led to a decrease of the root andshoot lengths, which was accompanied by a decrease of thechlorophyll content, at concentrations �900 mmol L�1.[76] 1-Octyl-3-methylimidazolium chloride inhibited root and stemgrowth (5-d EC50: 2.6 and 3.0 mmol L�1, respectively) and de-creased activity of antioxidant enzymes in rice (Oryza sativasativa) seedlings. Increased root length and weight were ob-

Table 10. Influence of alkyl side-chain length, of functionalized sidechain, and anion on the toxicity of ILs (EC50, growth inhibition) onL. minor.

Entry Modification of IL EC50 [mmol L�1]

alkyl chain (1-alkyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imides)1 ethyl 290[33b]

2 butyl 380[30a]

functionalization (1-alkyl-3-methylimidazolium chlorides)3 ethyl 700[33b]

4 butyl 660[30a]

5 3-hydroxypropyl 3350[30c]

6 ethoxymethyl >5000[30c]

7 2-methoxyethyl 420[30c]

8 3-methoxypropyl 988[30c]

anions (1-butyl-1-methylpyrrolidinium Ils)[30c]

9 chloride 14510 bis(trifluoromethylsulfonyl)imide 955

Table 9. Influence of ionic moiety on IL toxicity (EC50) on algae.

Entry Ionic moiety EC50 [mmol L�1] for algaeS. vacuola-tus[a]

B. paxilli-fer[b]

S. capri-cor-nutum[c]

anions1-butyl-3-methylimidazolium ILs1 bromide 1047–

2138[30d, 67]

2 chloride 140–178[30a, 70]

6.48[73] 2884 [30d]

3 tetrafluoroborate 2512[30d]

4 hexafluorophosphate 1318[30d]

5 bis(trifluoromethylsulfonyl)imide 63.2(72 h)[26]

7 dicyanamide 5.16[73]

8 O-methyl sulfate 16.05[73]

9 O-octyl sulfate 2239[30d]

10 trifluoromethanesulfonate 6.99[73] 2188[30d]

1-butyl-1-methylpyrrolidinium Ils11 bromide 9333

(72 h)/12 303(96 h)[67a]

12 chloride 2344[30c]

13 bis(trifluoromethylsulfonyl)imide 340[30c] >237(72 h) [26]

14 O-methyl sulfate >395(72 h) [30q]

cations (bromide as anion)15 1-butyl-3-methylimidazolium 1047–

2138[30d, 67]

16 1-butyl-1-methylpyrrolidinium 12 303[67a]

17 tetrabutylammonium 2240[67a]

18 tetrabutylphosphonium 224[67a]

[a] 24 h EC50, reproduction inhibition. [b] 72 h EC50, growth inhibition.[c] 96 h EC50, growth inhibition, if not specified otherwise.



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served at a lower concentration of IL (0.43 mmol L�1).[77] Shortaliphatic protic ionic liquids (2-hydroxyethanolamine formate,2-hydroxydiethanolamine propionate, and 2-hydroxytriethanol-amine pentanoate) showed no significant toxicity towards soilmicrobiota and plants, although the toxicity increased with in-crease of complexity of ILs (branch and length of the aliphaticchain).[30l]

The above-listed data emphasize the dependence of thetoxic effects of ILs on the nature of a targeted organism. Whencomparing effects of the 1-alkyl-3-methylimidazolium-based ILson the characteristic benthic species B. paxillifer and G. am-phibium, Latała and co-workers observed that the EC50 valuesfor the exposure of the two organisms to 1-ethyl-3-methylimi-dazolium chloride were quite similar (30.9 and 34.4 mmol L�1,respectively) ;[73] however, in case of B. paxillifer, elongation ofthe alkyl chain in the cationic moiety of the ionic liquids bytwo, four, six, or eight carbon atoms did not cause any sharpdecrease in the EC50 values (growth inhibition of the algae ex-posed to 1-decyl-3-methylimidazolium chloride occurred at0.99 mmol L�1), whereas in case of G. amphibium, for every twocarbon atoms elongation, the EC50 value fell by nearly oneorder of magnitude (the longest IL inhibited growth of the cya-nobacterium at 0.02 mmol L�1). A significant difference in sensi-tivities was found between the two diatoms S. marinoi andP. tricornutum (72 h E50 of 1-butyl-3-methylimidazolium chloridewas 120 and 1260 mmol L�1 for S. marinoi and P. tricornutum, re-spectively), possibly attributable to different silica uptake andorganization of the outer cell walls.[74] In general, diatomsshowed significantly higher sensitivity to ILs than green algae(Tables 7 and 9).[30h] Interestingly, the IL toxicity decreased withincreasing salinity, probably because of the reduced penetra-tion of the IL cations through the algal cell walls.[71] Low con-centrations of natural dissolved organic matter also reducedthe toxicity of the imidazolium-based ILs.[68a, 75] Matzke and co-workers observed that an increase in concentration of organicmatter in soil decreased the toxicity of 1-alkyl-3-methyl-imida-zolium tetrafluoroborates towards wheat (T. aestivum) andcress (Lepidium sativum),[78] whereas the presence of cadmiumreduced the toxicity of the mixture of 3-methyl-imidazolium-based ILs towards T. aestivum.[79] Notably, the imidazolium-based ILs tested were found to be more toxic than their corre-sponding alkali salts.[30d]

2.6. Toxicity of ionic liquids towards fungi and bacteria

Antifungal and antibacterial properties of ILs are of significantimportance both in medicine and industry. For instance, it wasfound that the residual 1-ethyl-3-methylimidazolium ions inbiomass pretreated with 1-ethyl-3-methylimidazolium acetateinhibited microbial growth and ethanol production by Saccha-romyces cerevisiae (S. cerevisiae), thus imposing a negativeeffect on biofuel manufacture[80] and suggesting that (eco)toxicproperties of ILs should be considered before any industrialapplication. On the other hand, 1-alkyl-3-methylimidazoliumchlorides possess a broad-spectrum antibiofilm activity to-wards clinically significant microbial pathogens[81] and the am-picillin ILs display up to 43-times improved antibacterial activi-

ty towards E. coli, Klebsiella pneumoniae (K. pneumoniae), S.aureus, and Enterococcus faecium (E. faecium) compared tosodium ampicillin.[82] The presence of (1R,2S,5R)-(�)-menthol inthe cation moiety increased the antimicrobial activity of ILsand the optically active ILs were significantly more potent thantheir racemic counterparts, suggesting possible application inantisepsis.[83] Bacteria were also used for studying ecotoxicityof ILs.

One of the best-known bacteria used in (eco)toxicity tests isthe gram-negative marine bacterium Vibrio fischeri (V. fischeri ;Aliivibrio fischeri, or Photobacterium phosphoreum). It is widelyexploited in bioluminescence assays designed for assessmentof ecotoxicity of various substances.[84] Tests with V. fischeri re-peatedly confirmed the cation side-chain length effect ob-served for other organisms: the increase of the side-chainlength from two to ten carbon atoms resulted in a 6563-folddecrease of 15 min EC50 (from 21 000 mmol L�1 for 1-ethyl-3-methylimidazolium chloride to 3 mmol L�1 for 1-decyl-3-methyl-imidazolium chloride; Table 11, entries 6–10).[30a, 33b, 85] The effectwas also pronounced for 1-alkyl-3-methylimidazolium- and 1-alkylquinolinium-based ILs towards the freshwater luminescentbacterium Vibrio qinghaiensis (V. qinghaiensis) sp.-Q67,[30o, 86]

other gram-negative bacteria, such as E. coli, K. pneumoniae,Klebsiella aerogenes (K. aerogenes), Proteus mirabilis (P. mirabilis),and Pseudomonas aeruginosa (P. aeruginosa), and gram-positivebacteria, such as Staphylococcus epidermidis (S. epidermidis), S.aureus, Enterococcus faecium (E. faecium), Enterococcus hirae (E.hirae), and Bacillus cereus (B. cereus)[30i, 81, 82, 87] (Table 11; notethat some data on MIC obtained for E. coli by different groupsvary as greatly as 20-fold; partially—but not completely—itmay be explained by different strains of E. coli used in thetests). Similar effects were demonstrated for the fungi Candidatropicalis (C. tropicalis) and S. cerevisiae (Table 12).[30i] The imida-zolium-based ILs also showed a negative effect towards Penicil-lium sp., with the expected positive correlation between thealkyl side-chain length and toxicity.[88] As for other IL cations,systematic elongation of one of the alkyl substituents in alkyl-tributylphosphonium chlorides ([P4,4,4,n][Cl]) generally resultedin higher toxicities of ILs towards Aspergillus nidulans, likely be-cause of a stronger interaction with the conidia cellular boun-daries (MIC decreased from 32 500 mmol L�1 for [P4,4,4,1][Cl] to11 mmol L�1 for [P4,4,4,14][Cl]). It was demonstrated that the toxic-ity of [P4,4,4,n][Cl] with n�4 was realized through direct interac-tions with the conidia plasma membrane and cell wall, where-as [P4,4,4,1][Cl] had no evident effect (Figure 6).[89] At the sametime, Aspergillus was found to be generally tolerant to 1-ethyl-3-methylimidazolium acetate.[90]

Interesting data on the impact of chain length were ob-tained when studying toxicity of choline-based ILs towards var-ious bacterial species. Thus, toxicity increased from octoxy-methyl(2-hydroxyethyl)dimethylammonium acesulfamate tododecyloxymethyl(2-hydroxyethyl)dimethylammonium acesul-famate, whereas tetradecyloxymethyl(2-hydroxyethyl)dimethy-lammonium acesulfamate showed lesser toxicity ; the samewas observed for (2-acetoxyethyl)octoxymethyldimethylammo-nium–(2-acetoxyethyl)tetradecyloxymethyldimethylammoniumacesulfamate series ; however, the latter group was more toxic



www.chemsuschem.org

Tab

le11

.In

fluen

ceo

fal

kyl

sid

e-ch

ain

len

gth

on

ILto

xici

ty(E

C50

,MIC

;mm

olL�

1 )o

nb

acte

ria.

Entr

yA

lkyl

chai

nG

ram

-neg

ativ

eb

acte

ria

Gra

m-p

osi

tive

bac

teri

aE.

coli[a

]K.

pn

eum

o-n

iae[a

]

K.ae

roge

nes

[a]

V.fis

cher

i[b]

V.qi

ngh

aien

sis

sp.-Q

67[b

]

P.m

irab

i-lis

[a]

P.ae

rugi

nos

a[a

]P.

vulg

ar-

is[a

]

S.m

arce

s-ce

ns[a

]

Ente

roco

ccus

sp.[c

]

E.h

irae

[a]

S.au

reus

[a]

S.ep

ider

mi-

dis[a

]

B.ce

r-eu

s[a]

M.l

u-te

us[a

]

1-al

kyl-

3-m

eth

ylim

idaz

oliu

mbr

omid

es1

oct

yl93

0[87b

]

2d

ecyl

106[8

7b]

844[8

7b]

3d

od

ecyl

386[8

7b]

733[8

7b]

97[8

7b]

193[8

7b]

4te

trad

ecyl

356

[87b

]35

6[87b

]45

[87b

]6[8

7b]

5h

exad

ecyl

10[8

2]15

[82]

15[8

2]

1-al

kyl-

3-m

eth

ylim

idaz

oliu

mch

lori

des

6et

hyl

261

800

[30i

]21

000[3

3b]

9772

3/12

134

(12

h)

[30o

,86]

261

800[3

0i]

261

800[3

0i]

7b

uty

l20

750

0[3

0i]

2454

–25

00[3

0a,8

5a]

1513

6[86]

207

500[3

0i]

207

500[3

0i]

8h

exyl

1644

–31

500[3

0i,8

1]

>16

44[8

1]15

1[85a

]80

0[86]

3120

0[30i

]16

44–

1560

0[30i

,81]

9o

ctyl

>72

2[8

1]>

1444

[81]

9[85a

]22

[86]

722–

1444

[81]

10d

ecyl

321–

1000

[30i

,81]

643[8

1]3[8

5b]

500[3

0i]

40–

500[3

0i,8

1]

1-al

kyl-

3-m

eth

ylim

idaz

oliu

mbi

s(tr

ifluo

rom

eth

ylsu

lfon

yl)im

ides

11et

hyl

844[1

08]

12b

uty

l20

00[3

0i]

339[1

08]

13h

exyl

1000

[87c

]51

[108

]20

00[8

7c]

14o

ctyl

700

[87c

]14

00[8

7c]

1-al

kylq

uin

olin

ium

brom

ides

[87a

]

15o

ctyl

121.

260

242

484.

812

1.2

121.

248

4.8

16d

ecyl

55.7

55.7

111.

522

355

.755

.722

317

do

dec

yl25

.812

.925

.851

.612

.912

.956

.118

tetr

adec

yl11

.65.

85.

823

.95.

83

23.9

19h

exad

ecyl

35.1

35.1

70.2

70.2

35.1

35.1

140.

520

oct

adec

yl10

4.9

104.

910

4.9

419.

514

0.9

140.

941

9.5

cho

liniu

m-b

ased

aces

ulf

amat

esal

koxy

met

hyl

(2-h

ydro

xyet

hyl

)dim

etyl

amm

oniu

mac

esul

fam

ate

s[91]

21o

cto

xy12

6725

34>

2534

1267

2534

1267

317

634

317

22n

on

oxy

306

612

1224

612

1224

306

153

153

153

23d

ecyl

oxy

296

592

592

296

592

296

148

7474

24u

nd

ecyl

oxy

143

286

573

286

286

7272

7237

25d

od

ecyl

oxy

6913

927

713

927

736

2636

1826

tetr

adec

ylo

xy13

126

125

526

152

265

6565

33(2

-ace

toxy

eth

yl)a

lkox

ymet

hyl

dim

eth

ylam

mon

ium

aces

ulfa

mat

es[9

1]

27o

cto

xy57

311

4522

9011

4511

4528

614

371

7128

no

no

xy27

755

522

1955

511

1013

913

969

6929

dec

ylo

xy26

953

821

5226

910

7667

6767

6730

un

dec

ylo

xy13

126

152

213

152

265

6565

3331

do

dec

ylo

xy32

127

507

6350

732

3232

1632

tetr

adec

ylo

xy60

240

960

120

960

6060

6031

[a]

24h

MIC

.[b

]15

min

EC50

;lu

min

esce

nce

inh

ibit

ion

ifn

ot

spec

ified

oth

erw

ise.

[c]

24h

MIC

;E.f

aeci

um,E

.hir

ae.



www.chemsuschem.org

[24 h MIC of octoxymethyl(2-hydroxyethyl)dimethylammonium,and (2-acetoxyethyl)octoxymethyldimethylammonium acesulfa-mates were 1267 and 573 mmol L�1, respectively, as shown forE. coli ; Table 11, entries 21–32].[91] The ILs displayed similar ef-fects towards the fungi Candida albicans (C. albicans) and Rho-dotorula rubra (R. rubra ; Table 12, entries 15–26).[91] At thesame time, cholinium chloride did not inhibit Penicilliumgrowth,[88] whereas cholinium alkanoates were less toxic thanthe corresponding sodium salts.[30j]

V. fischeri was studied with regard to the impact of function-alized groups in the side chain: 1-(3-hydroxypropyl)-3-methyli-midazolium, 1-(2-methoxyethyl)-3-methylimidazolium, and 1-(3-methoxypropyl)-3-methylimidazolium chlorides, as well as 1-cyanomethyl-3-methylimidazolium chloride, showed lower tox-icity than 1-butyl-3-methylimidazolium chloride, even at longertimes of exposure [30 min EC50 of 1-(3-hydroxypropyl)-3-meth-ylimidazolium chloride was >20 000 mmol L�1,[30c] compared to15 min EC50 of 1-butyl-3-methylimidazolium chloride being2500 mmol L�1;[30a, 85a] Table 13).

The anion was shown to impact both bacteria and fungi. Incase of V. fischeri, when using 1-butyl-3-methylimidazolium-based ILs, O-octyl sulfate had the most toxic effect (15 minEC50 70 mmol L�1),[30a] whereas dicyanamide was the least toxic(15 min EC50 4677 mmol L�1) ;[49] 1-butyl-3-methylimidazolium tri-fluoromethanesulfonate also demonstrated relatively low toxic-ity (15 min EC50 3981 mmol L�1;[92] Table 14, entries 7, 9, and 10).However, the impact of the anion appeared to depend strong-ly on the nature of the organism: thus, in case of E. coli, toxici-ties of 1-butyl-3-methylimidazolium O-octyl sulfate and tri-fluoromethanesulfonate did not differ so greatly (24 h MIC31 300 and 62 630 mmol L�1, respectively), whereas the highesttoxicity was observed for 1-butyl-3-methylimidazolium bis(tri-fluoromethylsulfonyl)imide (24 h MIC 2000 mmol L�1; Table 14,entries 5, 9, and 10).[30i] Fungi also seemed most sensitive tothe bis(trifluoromethylsulfonyl)imide anion: 24 h MIC was

Table 12. Influence of alkyl side-chain length on IL toxicity (MIC;mmol L�1) on fungi.

Entry Alkyl chain C. tropicalis[a] C. albicans[b] S. cerevi-siae[a]

R.rubra[b]

1-alkyl-3-methylimidazolium chlorides1 ethyl 26 1800[30i] 261 800[30i]

2 butyl 20 7500[30i] 207 500[30i]

3 hexyl 1644–15 600[30i, 81]

7800–15 600[30i]

4 octyl >1444[81]

5 decyl 321–500[30i, 81] 100–500[30i]

1-alkyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imides6 butyl >2000

(24 h)[30i]

7 hexyl >2000(24 h)[87c]

8 octyl 1400(24 h)[87c]

1-alkylquinolinium bromides[87a]

9 octyl 24210 decyl 55.711 dodecyl 12.912 tetradecyl 313 hexadecyl 35.114 octadecyl 140.9

cholinium-based acesulfamatesalkoxymethyl(2-hydroxyethyl)dimetylammonium acesulfamates[91]

15 octoxy 2534 26716 nonoxy 612 30617 decyloxy 592 14818 undecyloxy 286 7219 dodecyloxy 193 3620 tetradecyloxy 261 65

(2-acetoxyethyl)alkoxymethyldimethylammonium acesulfamates[91]

21 octoxy 573 57322 nonoxy 555 27723 decyloxy 538 26924 undecyloxy 261 13125 dodecyloxy 127 6326 tetradecyloxy 240 120

[a] 24 h MIC. [b] 48 h MIC.

Figure 6. IL effect on conidia of Aspergillus nidulans. Scanning electron mi-crophotographs of conidia treated with (a–a’) saline solution control, (b–b’)methyltributylphosphonium chloride, and (c–c’) octyltributylphosphoniumchloride. (From [89]).

Table 13. Influence of functionalized side chain on the toxicity of 1-alkyl-3-methylimidazolium chlorides (30 min EC50, luminescence inhibition ifnot specified otherwise) on the gram-negative bacterium V. fischeri.

Entry Alkyl chain EC50 [mmol L�1]

1 ethyl 21 000 (15 min)[33b]

2 butyl 2454–2500 (15 min)[30a, 85a]

3 3-hydroxypropyl >20 000[30c]

4 ethoxymethyl 12 000[30c]

5 2-methoxyethyl 15 000[30c]

6 3-methoxypropyl >20 000[30c]

7 cyanomethyl >10 000[30c]



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about 2000 mmol L�1 in case of C. tropicalis and S. cerevisiae,whereas chloride produced the weakest effect (24 h MIC207 500 mmol L�1; Table 14, entries 2 and 5).[30i] The anionimpact was also pronounced for 1-butyl-1-methylpyrrolidini-um-based ILs (Table 14, entries 11–14).[30c, q, 93] The Penicilliumfungal isolates grew in media containing relatively high con-centrations of cholinium alkanoates; nevertheless, MIC valuesvaried from 1000 to 1500 000 mmol L�1 and the length ofthe linear chain seemed to define the anion toxicity asfollows: ethanoate<propanoate<butanoate<pentanoate<hexanoate<octanoate<decanoate. However, the longer linearchain anions proved to be better biodegradable.[30j]

Imidazolium and ammonium saccharinates inhibited growthof E. coli and caused a drop in the pH value from pH 7.5 topH 5.0, which might influence the viability of microorga-nisms.[30p] The cations tested were also toxic when combinedwith the halide anions, implying that the IL toxicity could bedominated by the most toxic component, in this case, thecation. Similar observations were made for ethanoate ILs.[30p]

The cation influence was pronounced in case of V. fischeri. 4-Butyl-4-methylmorpholinium bromide had a low toxic effecteven at longer exposition times (30 min EC50>

20 000 mmol L�1),[30c] whereas tetrabutylphosphonium bromide

was the most toxic (15 min EC50 509 mmol L�1) ;[30s] pyrrolidini-um-based ILs also demonstrated low toxicity: in case of 1-butyl-1-methylpyrrolidinium bromide, 15 min EC50 was25 119 mmol L�1 [93] (Table 14, entries 16, 17, and 21).

A comparative study of a wide range of ILs clearly demon-strated complex interdependencies of IL toxicities and types ofanion, cation, and organism.[30r] Thus, in case of 1-butyl-3-meth-ylimidazolium ILs, the anion toxicity towards E. coli decreasedas follows: hydrogen sulfate>bis(trifluoromethylsulfonyl)i-mide> triflate> tetrafluoroborate>hexafluorophosphate>di-methylphosphate>chloride, whereas in case of tetradecyltri-hexylphosphonium ILs, the chloride anion showed the highesttoxicity: chloride>phosphinate>dicyanamide>bis(trifluoro-methylsulfonyl)imide. Toxicity towards C. albicans demonstrat-ed the following pattern: in case of 1-butyl-3-methylimidazoli-um ILs, it decreased as hydrogen sulfate> tetrafluoroborate>bis(trifluoromethylsulfonyl)imide� triflate>dimethylphos-phate>hexafluorophosphate�choride, whereas in case of tet-radecyltrihexylphosphonium ILs the highest toxicity belongedto the dicyanamide ion and chloride was nontoxic.[30r]

Attempts to investigate the mechanism of IL toxicity havebeen made. It was demonstrated that phosphonium- and am-monium-based ILs accumulated within E. coli cells, as detected

Table 14. Influence of ionic moiety on IL toxicity (MIC, EC50 ; mmol L�1) on bacteria and fungi.

Entry Ionic moiety Gram-negative bacteria Gram-positive bacteria FungiE. coli[a] V. fischeri[b] V. qinghaiensis sp.-

Q67[b]

Enterococcus sp.[c] S. aureu-s[a]

C. tropicali-s[a]

S. cerevisiae[a]

anions1-butyl-3-methylimidazolium ILs1 bromide 3359[30s]

2 chloride 207 500[30i] 2454–2500[30a, 85a] 15 136[86] 207 500[30i] 207 500[30i] 207 500[30i] 207 500[30i]

3 tetrafluoroborate 15 700[30i] 3500[30a, o] 5821/31 915 (12 h)[30o] 15 600[30i] 15 700[30i] 62 500[30i] 31 250[30i]

4 hexafluorophosphate 1175 (30 min)[47g]

5 bis(trifluoromethylsulfonyl)imide 2000[30i] 300–339[30a, 109] 500[30i] >2000[30i] >2000[30i] 1300–2000[30i]

6 bis(trifluoromethyl)imide 3000[30a]

7 dicyanamide 4677[49]

8 O-methyl sulfate 12 3500[30i] 4900 [86] 62 500[30i] 62 500[30i] >92 000[30i] 46 000[30i]

9 O-octyl sulfate 31 300[30i] 70[30a] 2950 [86] 15 700 [30i] 11 500[30i] 31 250[30i] 15 700–31 250[30i]

10 trifluoromethanesulfonate 62 630[30i] 3981[92] 62 500 (E. hirae),31 200 (E. faeciu-m)[30i]

62 500[30i] 125 000[30i] 62 500[30i]

1-butyl-1-methylpyrrolidinium Ils11 bromide 25 119[93]

12 chloride 20 000 (30 min)[30c]

13 bis(trifluoromethylsulfonyl)imide 350 (30 min)[30c]

14 O-methyl sulfate >395 (30 min)[30q]

cations (bromide as anion)15 1-butyl-3-methylimidazolium 3359[30s]

16 4-butyl-4-methylmorpholinium >20 000(30 min)[30c]

17 1-butyl-1-methylpyrrolidinium bro-mide

25 119[93]

18 1-butyl-1-methylpiperidinium bro-mide

18 600 (30 min)[30c]

19 1-butylpyridinium 5248[93]

20 tetrabutylammonium 1862[49]

21 tetrabutylphosphonium 509[30s]

[a] 24 MIC. [b] 15 min EC50, luminescence inhibition if not specified otherwise. [c] 24 MIC; E. faecium, E. hirae.



www.chemsuschem.org

by Fourier transform infrared (FTIR) spectroscopy.[94] The toxicILs produced significantly more pronounced changes in theFTIR patterns of the cellular chemicals than the biocompatibleILs, and subcellular fractionation demonstrated that trihexylte-tradecylphosphonium bis(trifluoromethylsulfonyl)imide accu-mulated specifically in the membrane fraction of the cells.[94]

2.7. Impact on enzymatic activity and protein stability

The above-mentioned four structural factors influencing IL tox-icity also have an effect on the IL impact on enzymatic activity,as demonstrated for acetylcholinesterase (from the electric eelElectrophorus electricus)[30a,q, 34a, 95] and luciferase (from the fireflyPhotinus pyralis)[96] (Tables 15–17). As shown for imidazolium-based ILs, the bis(trifluoromethyl)imide anion displayed thehighest inhibition effect towards acetylcholinesterase (IC50

40 mmol L�1),[30a] whereas tetrafluoroborate was the weakest in-hibitor (IC50 540 mmol L�1)[34a] (see Table 17, entries 3 and 6). Incase of cations, morpholinium-based ILs were the least toxic,whereas quinolinium-based were the most toxic (Table 17, en-tries 16 and 17).

Ammonium- and imidazolium-based ILs with long alkylchains inhibited the activity of the HIV-1 integrase (both the 3’-processing and strand transfer steps of the integration reac-tion), and a correlation of their effect with chain length of analkyl substituent was observed. In contrast, the phosphonium-,pyrrolidinium-, pyridinium-, and guanidinium-based ILs had noeffect on HIV-1 integrase activity.[97] 1-Allyl-3-methylimidazoli-um and 1-octyl-3-methylimidazolium chlorides inhibited ade-nosine deaminase (from calf intestinal mucosa), the effect ofthe latter IL being more potent owing to its higher hydropho-bicity. According to molecular dynamics (MD) simulations andfluorescence spectrophotometry, ILs reduced intermolecularhydrogen bonds and caused unfolding of the protein.[98]

On the other hand, ILs were shown to increase the stabilityand activity of several enzymes, including the bovine a-chymo-trypsin. Hydrophobic imidazolium and phosphonium cationscarrying long alkyl chains proved to be weaker stabilizers fora-chymotrypsin, whereas small alkyl chain molecules of triethy-lammonium salts produced a stronger effect.[99] Thus, triethy-lammonium acetate attenuated the denaturing action of ureaon a-chymotrypsin.[100] The a-chymotrypsin activity also de-pended on the type of the IL anion.[101] Overall, the initial rateof peptide production was improved 16-fold by changing anorganic solvent to 1-ethyl-3-methylimidazolium bis(fluorosulfo-nyl)imide,[101] whereas 1-ethyl-3-methylimidazolium bromideimproved the activity of trypsin in cationic reverse micelles ofcetyltrimethylammonium bromide.[102] The authors suggestedthat the imidazolium moiety of the ILs resembled the histidineamino acid component of the hydrolase active site, whereas itsbromide counter ion formed a hydrogen bond that promotedthe enzyme-catalyzed hydrolysis.[102]

Investigation of free and immobilized lipases from Candidaantarctica, Thermomyces lanuginosus, and Rhizomucor mieheicatalyzing the synthesis of butyl propionate by transesterifica-tion in nine ionic liquids demonstrated that enzyme activitiesdepended on the nature of the ions and improved with in-

Table 15. Influence of alkyl side-chain length on IL toxicity (IC50) on en-zymes.

Entry Alkyl chain IC50 [mmol L�1]acetylcholinesterase luciferase

1-alkyl-3-methylimidazolium chlorides1 ethyl 115–120[33b, 95b] 150 000[96]

2 butyl 81[95b] 123 000[96]

3 hexyl 83[95b] 90 800[96]

4 octyl 40[24b] 19 200[96]

5 decyl 12[95b]


7 butyl 90[30a]

8 hexyl 140[24b]

9 octyl 107[24b]

Table 16. Influence of functionalized side chain on the toxicity of 1-alkyl-3-methylimidazolium chlorides (IC50) on acetylcholinesterase.

Entry Alkyl chain IC50 [mmol L�1]

1 ethyl 115–120[33b, 95b]

2 butyl 81[95b]

3 3-hydroxypropyl 977[95b]

4 ethoxymethyl 407[95b]

5 2-methoxyethyl 380[95b]

6 3-methoxypropyl 407[95b]

7 cyanomethyl 776[95b]

Table 17. Influence of ionic moiety on IL toxicity (IC50) on acetylcholines-terase.

Entry Ionic moiety IC50 [mmol L�1]

anions1-butyl-3-methylimidazolium ILs1 bromide 80[95a]

2 chloride 81[95b]

3 tetrafluoroborate 540[34a]

4 hexafluorophosphate 140[95a]

5 bis(trifluoromethylsulfonyl)imide 90[30a]

6 bis(trifluoromethyl)imide 40[30a]

7 dicyanamide 89[95a]

8 O-methyl sulfate 89[24b]

9 O-octyl sulfate 96[24b]

10 trifluoromethanesulfonate 85[24b]

1-butyl-1-methylpyrrolidinium Ils11 bromide 85[24b]

12 chloride 83[95b]

13 bis(trifluoromethylsulfonyl)imide 135[24b]

14 O-methyl sulfate 158[30q]

cations (bromide as anion)15 1-butyl-3-methylimidazolium 80[95a]

16 1-butylquinolinium 6[95b]

17 4-butyl-4-methylmorpholinium 513[95b]

18 1-butyl-1-methylpyrrolidinium 85[24b]

19 1-butyl-1-methylpiperidinium 68[95b]

20 1-butylpyridinium 59[24b]

21 tetrabutylammonium 200[95b]

22 tetrabutylphosphonium 407[95b]



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creasing length of the imidazolium alkyl chain of the cation.[103]

It was also observed that catalytic activity of lipases dependedon the type of the IL anion.[104] Interestingly, ILs modified theclassical regioselectivity of the transglycosylation reaction cata-lyzed by b-galactosidase from Thermus thermophilus, possiblyby inducing conformational changes in the enzyme struc-ture.[105]

It is worth mentioning that the protic ILs ethylammoniummesylate and diethylammonium mesylate stabilized the nativeparticle of the Tobacco mosaic virus, whereas triethylammoni-um mesylate imposed changes in its secondary structure.[106]

As for the mechanism of the IL effects imposed on activityand stability of proteins, Huang and colleagues studied interac-tions between imidazolium-based chlorides and bovine serumalbumin (BSA) and showed that ILs interacted with tryptophanand tyrosine residues, thus changing the structure and hydro-phobic conformation of BSA; strength of this interaction in-creased with lengthening of the cation side chain.[107]

2.8. Common tendencies of biological activity of ionic liq-uids

It has been observed that the IL toxicity increases with increas-ing lipophilicity.[24a] This general trend has been repeatedlyconfirmed for ILs of different classes. Thus, Ranke and co-work-ers demonstrated a correlation between lipophilicity (ex-pressed as cation retention time tR in RP-HPLC experiments)and toxicity and concluded that the cation lipophilicity wasthe dominating factor for the IL cytotoxicity.[33a] Still, a moredetailed correlation is somewhat difficult to extract. The impor-tant fact is that lipophilicity should not be confused with hy-drophobicity.[108] Lipophilicity may play a major role in definingtoxicity towards cell cultures,[33a] where it is thought that IL at-taches to the cell lipid membrane and disrupts it,[40] whereashydrophobicity seems to be important with regard to ecotoxic-ity towards water organisms.[108] Thus, Ventura and co-workersproposed the designing of new hydrophobic ILs with low tox-icity.[108] Figure 7 shows the relationship between lipophilicityor hydrophobicity of ILs and their inhibitory activity.

Numerous investigations conducted in the past years sug-gest that there are four major internal factors influencing theIL toxicity. It also should be noted that the toxic effect of ILscan change significantly depending on time.[37b, 67a]

The first factor affecting the IL toxicity is the length of thecation alkyl side chain. Tables 2, 5, 7, 11, 12, and 15 summarizethe recent data on this effect for imidazolium-, quinolinium-,and cholinium-based ILs. The general trend appears to be thefollowing: the longer the alkyl chain, the higher the IL toxicity.Still, some other correlations seem to exist. Thus, in case of thequinolinium- and cholinium-based ILs, increasing the side-chain length resulted in leveling off of the toxicity upon reach-ing a limit, after which the toxicity sometimes started to de-crease.[36, 87a, 91] Another observation was that the rates of thetoxicity increase upon side-chain lengthening could vary signif-icantly as, for example, was demonstrated in D. polymorpha :upon elongation of the side chain from four to eight carbonatoms, 96 h LC50 of 1-alkyl-3-methylimidazolium and 1-alkyl-3-

methylpyridinium bromides decreased 74-fold and 52-fold, re-spectively.[51] Results obtained with imidazolium- and pyridini-um-based ILs suggested that the position of substitution couldalso affect toxicity. Thus, 1-octyl-3-methylimidazolium and 1-methyl-3-octylimidazolium chlorides showed different toxicitiestowards E. coli (24 h MIC>722 and 4000 mmol L�1, respectively;however, it should be noted that the values were obtained fordifferent E. coli strains).[30i, 81] Similarly, 1-butyl-2-methylpyridini-um, 1-butyl-3-methylpyridinium, and 1-butyl-4-methylpyridini-um tetrafluoroborates produced different effects in IPC-81 cells(48 h EC50 1778, 1995, and 933 mmol L�1, respectively).[33a] Still,this effect may also depend on the test system. When Venturaand coworkers investigated the effect of isomerism usingD. magna, V. fischeri, and S. capricornutum as test models, theyfound that the location of a cation alkyl chain had no signifi-cant influence on the IL toxicity, although a branched alkylchain decreased the toxic effect.[108]

Figure 7. Relationship between a) lipophilicity and b) water solubility of dif-ferent ILs and their inhibitory activity towards acetylcholinesterase (a) andV. fischeri (b) [a: relationship between the decadic logarithms of the lipophi-licity parameter k0 and of the IC50 values (mmol L�1) of a series of imidazoliumheadgroups; b: relationship between water solubility and toxicity towardsV. fischeri, expressed as EC50 (mg L

�1) obtained by Microtox assays; AChE:acetylcholinesterase, R2 : correlation coefficient, RSE: residual standard error ;[C3C1pyrr]: 1-methyl-1-propylpyrrolidinium, [C3C1pip]: 1-methyl-1-propylpiper-idinium, [C3C1pyr]: 3-methyl-1-propylpyridinium, [C3C1im]: 1-methyl-3-propyli-midazolium, [PF6]: hexafluorophosphate, [NTf2]: bis(trifluoromethylsulfonyl)i-mide] (a from [95b] ; b from [108]).



www.chemsuschem.org

The second factor affecting the IL toxicity is the presence ofa functionalized side chain in the IL cation. It was observedthat an introduction of a functionalized side chain couldreduce the IL toxicity (Tables 3, 8, 10, 13, and 16). This effectseems to depend on the side-chain structure. Thus, the ethoxy-methyl side chain clearly increased the cytotoxicity for all headgroups except for the piperidinium cation compared to theisomeric methoxyethyl side chain; nevertheless, the toxicitieswere lower than that for ILs with alkyl side chains [in IPC-81,48 h EC50 of 1-(ethoxymethyl)-3-methylimidazolium, 1-(2-me-thoxyethyl)-3-methylimidazolium and 1-butyl-3-methylimidazo-lium chlorides were 4000, >20 000 and 3600 mmol L�1, respecti-vely] .[30b] Similar results were obtained for imidazolium-basedbis(trifluoromethylsulfonyl)imides.[30b,c] The effect was also seenfor pyridinium-based ILs when studying their inhibitory activitytowards acetylcholinesterase [IC50 125.9, 50.1, 446.7, 117.5, and114.8 mmol L�1 for 1-ethylpyridinium, 1-butylpyridinium, 1-(3-hydroxypropyl)pyridinium, 1-(2-methoxyethyl)pyridinium, and1-(ethoxymethyl)pyridinium chlorides, respectively] .[95b]

1-Cyanomethyl-3-methylimidazolium chloride also demon-strated low toxicity (in IPC-81, 48 h EC50 was>20 000 mmol L�1),[30b] and the same was observed for 1-(cya-nomethyl)pyridinium chloride (IC50 295.1 mmol L�1 compared to125.9 mmol L�1 for 1-ethylpyridinium chloride when studyinginhibition of acetylcholinesterase),[95b] whereas an inclusion ofthe nitrile or dimethyldisulfide functional group into the cationdecreased the toxicity by nearly an order of magnitude in com-parison to ILs without these functional groups.[30g] Toxicity of 1-benzyl-3-methylimidazolium chloride, as well as 1-p-fluoroben-zyl-3-methylimidazolium chloride, towards CaCo-2 cells wasalso low (24 h EC50 30 760 and 20 370 mmol L�1, respectively) ;however, it should be noted that 1-p-chlorobenzyl-3-methyl-imidazolium chloride was significantly more toxic than 1-butyl-3-methylimidazolium chloride (24 h EC50 4580 and28 690 mmol L�1, respectively).[37b] 1,3-Dibenzylimidazoiliumchloride demonstrated no toxicity towards CaCo-2 cells atshort exposition times (4 h).[30f] Pretti and colleagues studiedthe toxicity of 3-(2-chloroethyl)-1-methylimidazolium chloridetowards D. magna and D. rerio and ranked it as practicallyharmless, whereas towards S. capricornutum it showed moder-ate toxicity.[26] 1-Methyl-3-(phenylmethyl)-imidazolium chlorideand 1-methyl-3-(2-phenylethyl)-imidazolium chloride demon-strated relatively low toxicity towards IPC-81 cells (48 h EC50>

1000 mmol L�1) ; the authors suggested that an increase in tox-icity compared to 1-butyl-3-methylimidazolium chloride de-pended on a higher lipophilicity of the phenyl-containingILs.[33a]

Therefore, according to the accumulated data, the presenceof oxygen in the cation side chain seems to decrease the toxic-ity, whereas the influence of other functional groups dependstrongly on their nature and can be evaluated only experimen-tally. It also should be noted that in some cases the nature ofthe biological system seems to govern the observed effect;thus, 1-(ethoxymethyl)-3-methylimidazolium chloride demon-strated relatively low toxicity compared to 1-butyl-3-methylimi-dazolium chloride, but the effect depended on the systemused: toxicities of these two ILs were similar in IPC-81, but dif-

fered by as much as eight times in L. minor (Table 3, entries 2and 4, and Table 10, entries 4 and 6).[30b,c] The opposite was ob-served for 1-(2-methoxyethyl)-3-methylimidazolium chloride,which had a very low toxicity in IPC-81 cells, but proved to bemore toxic in L. minor (Table 3, entries 2 and 5, and Table 10,entries 4 and 7).[30b, c]

The third factor is the anion moiety (Tables 4, 6, 9, 10,14, and 17). In general, halides demonstrated lower tox-icities than bis(trifluoromethylsulfonyl)imides, as shownfor imidazolium-[30a,d,i,s, 32c, 33a, 67, 85a] and pyrrolidinium-based[26, 30a,b,i, 32c, 109] ILs in different biological systems. The hightoxicity of ILs with fluoride-containing anions towards some or-ganisms can be explained by the hydrolytic cleavage resultingin the formation of free fluoride ions, which are potent inhibi-tors of Na+-K+-ATPase.[30g] No fluoride formation was foundwith hexafluorophosphate, whereas small amounts of fluorideions produced by 1-butyl-3-methylimidazolium tetrafluorobo-rate might not affect algal growth; however, the fluoride ionformation increased with incubation time.[30d] The effect of flu-orinated anions had no significant influence in case of pyridini-um-based ionic liquids, which implies that the impact of thearomatic ring of a pyridinium cation on the toxic mechanism isconsiderably stronger than that of the anion.[30g]

Therefore, the third and fourth factors influencing the IL tox-icity—the anion and cation—appear to be interdependent,suggesting that the resulting toxicity is a sum of both cationand anion impacts, not forgetting the external factor of theobject’s nature. Thus, in contrast to imidazolium- and pyrrolidi-nium-based ILs, trihexyl(tetradecyl)phosphonium chloride hadhigher cytotoxic effects than trihexyl(tetradecyl)phosphoniumbis(trifluoromethylsulfonyl)imide in S2 cells.[30n] The presence ofthe bis(trifluoromethylsulfonyl)imide anion decreased the tox-icity, independently of the cation, in HT-29 and CaCo-2 cells.[30-

f, 37a] The impact of a hydrophobic anion was particularly signifi-cant when the length of an alkyl substituent in the pyrrolidini-um or piperidinium cations was small. For pyrrolidinium-basedILs containing butyl chains, replacing bromide with the bis(tri-fluoromethylsulfonyl)imide anion resulted in an increase in tox-icity. On the other hand, for pyrrolidinium-based ILs containingoctyl chains, no increase in toxicity was observed when replac-ing bromide with bis(trifluoromethylsulfonyl)imide.[30g]

Comparison of the cytotoxic data on ILs with 1-alkoxymeth-yl-3-hydroxypyridinium cations showed the EC50 for the chlor-ides to be 2–3-fold lower than the corresponding values foracesulfamates and saccharinates, suggesting that acesulfa-mates and saccharinates with short alkoxylmethyl chains couldeffectively moderate the toxic effect of the cation.[30e] Imidazoli-um and ammonium saccharinates inhibited growth of E. coli ;however, the cations were also toxic when combined with thehalide anions, implying that the IL toxicity could be dominatedby the most toxic component, in this case, the cation. More-over, the saccharinate ILs caused a drop in the pH value from7.5 to 5.0, which also might influence the viability of microor-ganisms.[30p]

Apparently, the length of the linear chain of an anion canalso affect the toxicity, as demonstrated for cholinium alka-noates in Penicillium sp.[30j] Investigation of the cytotoxicity of



www.chemsuschem.org

choline-based ILs in the murine macrophage cell line J774showed a strong correlation between the EC50 value and anionmass fraction, indicating that the anion size and presence ofmoderately long and/or branched alkyl chains may affect viabi-lity.[30k] An increase of cytotoxicity with alkyl chain length wasalso demonstrated for N,N,N-trialkylammoniododecaborateanions in V79 cells.[35]

Tables 4, 6, 9, 14, and 17 summarize data concerning the in-fluence of cation nature on IL toxicity. It was shown thata cyclic cation could influence the toxicity of ILs depending onthe length of alkyl chain substituents.[30g] The effect seems todepend strongly on the nature of the test object and theanion: in MCF-7 cells 1-butyl-1-methylpyrrolidinium bromidewas less toxic than 1-butyl-1-methylpiperidinium bromide andchanging bromide to bis(trifluoromethylsulfonyl)imide en-hanced the difference,[30g] whereas the opposite was observedin IPC-81 cells.[30b, 33a] In general, phosphonium-based ILs dem-onstrated the highest toxicity towards cell cultures, bacteria,and algae, whereas morpholinium-based ILs were the leasttoxic. The effects also apparently depended on the test object.Studying the toxicity of ethyl-, butyl-, octyl-, benzyl-, and allyl-substituted 1-alkyl-3-methylimidazolium, alkylpyridinium, alkyl-dimethyl(2-hydroxyethyl)ammonium (choline derivatives), andalkyltriethylammonium ILs in HeLa cells, Wang and co-workersobserved that although all ILs were more toxic than commonwater-miscible solvents, choline derivatives and alkyltriethylam-monium ILs were less toxic than their pyridinium and imidazo-lium analogues.[32c] Ventura and co-workers observed that ILswith aromatic (imidazolium- and pyridinium-based) cationswere more toxic than nonaromatic (pyrrolidinium- and piperi-dinium-based) ones and that hexamerous cations were moretoxic than pentamerous (Figure 7 b).[108] This observation is tosome degree confirmed by tests with other objects, such asIPC-81 cells and acetylcholinesterase (Tables 4, 9, 14, and 17).Interestingly, bicyclic quinolinium-based ILs showed significant-ly higher toxicities than other ILs with cyclic cations, whereasmorpholinium-based ILs were the least toxic, suggesting thatadding a heteroatom into the cycle might decrease the toxici-ty.

Fatemi and Izadiyan used a set of 227 ILs (94 imidazolium,53 pyridinium, 23 pyrrolidinium, 22 ammonium, 15 piperidini-um, 10 morpholinium, 5 phosphonium, and 5 quinolinium cat-ions in combination with 25 anion types) with data on IL toxic-ity in the rat leukemia cell line IPC-81 for developing linearand nonlinear models using genetic algorithms (GA), multiplelinear regressions (MLR), and multilayer perceptron neural net-work (MLP NN) approaches.[45] According to these models, themain molecular structural factors governing cytotoxicity of ILsare symmetry, substituent, and charge distribution in the cat-ions and heavy atom count in the anions. The models suggestthat an increase of substituents increases IL cytotoxicity,whereas anion properties have a secondary effect on cytotoxic-ity in comparison with the influence of the cation. For the ratleukemia cell line, the IL cytotoxicity was predicted to increaseas the cation symmetry decreased. According to Fatemi andIzadiyan, in most cases the imidazolium-based ILs were moretoxic than ILs containing the pyridinium cations.[45]

Apparently, external factors can also influence the toxicity ofILs, and the object’s nature is only one of them. Thus, studyingcytotoxicity of various ILs towards HeLa cells, Wang and co-workers observed that when cells were cultured in the pres-ence of foetal bovine serum, the toxicities were higher than inthe absence of the serum, with the exception of ILs containingan ethyl group, suggesting that they might have a differentmechanism of action.[32c] In case of algae, increasing the salinitydecreased the IL toxicity, probably because of the reducedpenetration of the IL cations through the algal cell walls,[71]

whereas low concentrations of natural dissolved organicmatter also reduced the toxicity of the imidazolium-basedILs,[68a, 75] and an increase in concentration of organic matter insoil decreased the toxicity of 1-alkyl-3-methylimidazolium tetra-fluoroborates towards wheat (T. aestivum) and cress (Lepidiumsativum).[78]

3. Conclusions

The summarized data on biological activity has clearly shownthat an analysis of possible environment penetration pathwaysshould be performed for reliable risk assessment. Significantlydifferent aftereffects can be expected depending on what typeof organisms the ionic liquids will be in contact with. An esti-mation of the level of irreversible ecological response to con-tamination with widely used ionic liquids (Table 18) identifies

D. magna and diatom algae, such as B. paxillifer, as the mostsensitive organisms, whereas bacteria, such as E. coli, and fungishow considerably higher tolerance. It should be also empha-sized that the exposition time–toxicity correlation exists, atleast for some ionic liquids.

In spite of massive data accumulated on toxicity of ionic liq-uids, we still lack complete understanding of their interactionswith the environment. Therefore, apart from studying biologi-cal activities of widely known and new ionic liquids in as manybiological systems as possible, we should concentrate on re-vealing molecular mechanisms underlying the toxic effect ofthese chemical compounds. Once known, this information willfacilitate the design of new eco-friendly ionic liquids, as well as

Table 18. Sensitivity of different biological systems to the influence ofanion of 1-butyl-3-methylimidazolium ILs.[a]

Biologicalobject

IL

chloride trifluoromethanesulfonate tetrafluoroborate

IPC-81 cells 620[30b, 33a] 300[24b] 300[33a]

D. magna 15[49] 6–12[34a, 47b]

S. capricornu-tum

500[30d] 630[30d] 570[30d]

B. paxillifer 1[73] 2[73]

V. fischeri 430[30a, 85a] 1150[92] 790[30a, o]

E. coli 36 240[30i] 18 055[30i] 3550[30i]

S. cerevisiae 36 240[30i] 18 020[30i] 7060[30i]

[a] Expressed as the amount of IL required to kill/inhibit 50 % of a popula-tion or, in case of bacteria and fungi, as MIC, mg L

�1.



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ionic liquids with specified bio-logical activities (as was shownfor ampicillin-containing ionicliquids that displayed highly im-proved antibacterial activity),[82]

which may find medical applica-tion. Understanding molecularmechanisms of the action ofionic liquids is a challengingtopic for interdisciplinary re-search in the nearest future.

An important problem con-cerns incomplete description ofthe measurements and absenceof required information concern-ing systems and assays used forstudies of toxicity. Regretfully,many publications lack the nec-essary description, thus makingreliable comparison and analysisof the measured data hardlypossible. Taking into account thecomplexity of ionic liquid sys-tems, it is crucial to providea uniform description with suffi-cient details of the experimentalprocedure.

Clearly, ILs represent ratherdifficult chemical systems to ad-dress the question of their bio-logical activity. A given IL can beboth nontoxic in one biologicaltest (particular cells or organ-isms) and highly toxic in another.Thus, a careful selection of bio-logical activity data should beperformed for an appropriate as-sessment of chemical technolo-gies involving ILs. For reliableanalysis, we suggest to measurethe biological activity involvingvarious tests rather than relyingon a single toxicity parameter.

A summary of recently report-ed data on the IL toxicity to-wards different levels of life iscompiled in Table 19. Cations arelisted in the rows and anions inthe columns, with the intersec-tion mapping the biological ac-tivity of a particular IL. The fol-lowing principal componentscan be identified from the bio-logical data as major factorsmodulating the toxicity of ILs :i) length of an alkyl chain in thecation; ii) degree and nature of Ta

ble

19.

ILto

xici

tym

app

ing

for

sele

cted

cati

on

/an

ion

com

bin

atio

ns.

[a]

Cat

ion

An

ion

bro

mid

ech

lori

de

bis

(tri

fluo

rom

eth

ylsu

lfon

yl)im

ide

tetr

aflu

oro

bo

rate

hex

aflu

oro

ph

osp

hat

ed

icya

nam

ide

O-m

eth

ylsu

lfate

O-o

ctyl

sul-

fate

1-et

hyl

-3-

met

hyl

imid

azo

lium

8350

[32c

] /–/–

/–/–

7244

[45]

/770

[33b

] /63

30.5

1[30h

] /21

000[3

3b] /

115–

120[3

3b,9

5b]

12[9

5b] /2

30[3

3b] /1

70[3

3b] /8

44[1

08] /

110[3

3b]

9940

[32c

] /–/2

094[3

0o] /

2404

4[30o

] /–83

18[4

5]/–

/–/–

/–66

00[3

3b] /8

60[3

3b] /

430[3

3b] /

1000

0[33b

] /350

[33b

]

1585

0[45]

/–/–

/26

750

0[30i

] /-16

60[4

5]/–

/–/

3010

0[30i

] /–

1-b

uty

l-3-

met

hyl

imid

azo

lium

>91

23[3

4b] /

70[4

7d] /1

02[6

9]/

3359

[30s

] /80[9

5a]

3548

-360

0[30b

,33a

] /–/

1026

.2[3

0h] /2

454–

2500

[30a

,85a

] /81[9

5b]

1170

[32c

] /45.

1[26]

/50[3

0a] /3

39[1

08] /

90[3

0a]

1318

[33a

] /23–

53[3

4a,4

7b] /2

512[3

0d] /

3500

[30a

,30o

] /540

[34a

]

1259

[33a

] /70.

8[49]

/13

18[3

0d] /1

175[4

7g] /

140[9

5a]

1413

[24b

] /78[3

4a] /

5.16

[73]

/467

7[49]

/89

[95a

]

1622

[24b

] /–/1

6.05

[73]

/49

00[8

6]/8

9[24b

]

1698

[24b

] /–/

2239

[30d

] /70

[30a

] /96[2

4b]

1-h

exyl

-3-m

eth

ylim

idaz

oliu

m68

74[3

4b] /

11.5

[47d

] /24

[69]

/–/–

708[3

3a] /-

/64.

5[30h

] /15

1[85a

] /83[9

5b]

–/–/

–/51

[108

] /140

[24b

]95

5[45]

/–/–

/18

45[3

0o] /–

813[4

5]/–

/–/1

29[8

5a] /–

1-o

ctyl

-3-m

eth

ylim

idaz

oliu

m52

0[34b

] /10.

3[47d

] /1.

2[69]

/930

[87b

] /–10

2[24b

] /–/1

5.1[3

0h] /

9[85a

] /40[2

4b]

190[3

2c] /–

/–/7

00[8

7c] /1

07[2

4b]

300[3

2c] /–

/0.0

05[3

0a] /

125.

5[30o

] /46[9

5a]

1460

[37b

] /–/–

/5[8

5a] /–

6076

[30

f]/–

/–/–

/––/

–/–/

72.4

[86]

/-

1-d

ecyl

-3-m

eth

ylim

idaz

oliu

m50

[34b

] /0.5

[47d

] /0.

3[69]

/844

[87b

] /–22

[33a

] /–/3

.68[3

0h] /3

[85b

] /12

[95b

]

6[45]

/–/–

/210

0[30i

] /13

[95a

]

31.6

[45]

/–/–

/–/–

1-(2

-met

ho

xyet

hyl

)-3-

met

hyl

i-m

idaz

oliu

m>

2000

0[30b

] /–/

1820

[30c

] /15

000[3

0c] /

380[9

5b]

1778

[45]

/–/9

5[30c

] /670

[30c

] /––/

80[3

4a] /–

/800

0[34a

] /––/

770[3

4a] /–

/98

00[3

4a] /–

1-b

uty

l-1-

met

hyl

pyr

rolid

iniu

m58

88[3

3a] /–

/93

33[6

7a] /

2511

9[93]

/85[2

4b]

>20

000[3

0b,3

3a] /–

/23

44[3

0c] /2

000

0[30c

] /83

[95b

]

1000

[30b

] /88[2

6]/>

237[2

6]/3

50[3

0c] /

135[2

4b]

794[4

5]/–

/–/–

/–16

982[4

5]/–

/–/–

/–>

3947

[30q

] /395

[30q

] />

395[3

0q] />

395[3

0q] /

158[3

0q]

1-m

eth

yl-1

-oct

ylp

yrro

lidin

ium

80[3

0g] /–

/–/–

/–38

9[45]

/–/–

/–/2

29[9

5b]

85[3

0g] /–

/–/–

/–66

[45]

/–/–

/–/–

1-m

eth

yl-1

-oct

ylp

iper

idin

ium

540[3

0g] /–

/–/–

/–<

25[3

0g] /–

/–/–

/–11

53[3

0g] /–

/–/–

/–53

86[3

0g] /–

/–/–

/–1-

bu

tylp

yrid

iniu

m79

43[4

5]/–

/–/

1730

0[111

] /––/

–/39

0[30c

] /150

0[30c

] /50

[95b

]

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functionalization in the side chain of the cation; iii) nature ofthe anion; iv) nature of the cation; and v) mutual influence ofanion and cation.

These principal components can be used to construct mo-lecular descriptors for task-specific optimization of ionic liquids.In the nearest future, fine tuning for maximizing task-specificadvantageous properties of ILs and minimizing ecological sideeffects can be anticipated to become a necessary step for ap-plication development.

Acknowledgements

The work was supported in part by the Russian Foundation forBasic Research (Projects No. 12-03-33127), grant MD-4969.2012.3,the Ministry of education and science of Russian Federation (Proj-ects 8453,8572) and Programs of Division of Chemistry and Mate-rial Sciences of RAS.

Keywords: biological activity · cytotoxicity · ecologicalactivity · ionic liquids · sustainable technologies

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