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Acute toxicity of ionic liquids for three freshwater organisms: Pseudokirchneriella subcapitata, Daphnia magna and Danio rerio
ARTICLE IN PRESS Ecotoxicology and Environmental Safety 72 (2009) 1170–1176
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Acute toxicity of ionic liquids for three freshwater organisms: Pseudokirchneriella subcapitata, Daphnia magna and Danio rerio Carlo Pretti a,, Cinzia Chiappe b,1, Ilaria Baldetti b,1, Sara Brunini a,2, Gianfranca Monni a,2, Luigi Intorre c,3 a b c
` di Pisa, Viale delle Piagge 2, Pisa, Italy Dipartimento di Patologia Animale, Profilassi ed Igiene degli Alimenti, Universita ` di Pisa, via Bonanno 33, Pisa, Italy Dipartimento di Chimica Bioorganica e Biofarmacia, Universita ` di Pisa, Viale delle Piagge 2, Pisa, Italy Dipartimento di Clinica Veterinaria, Universita
a r t i c l e in f o
a b s t r a c t
Article history: Received 18 February 2008 Received in revised form 29 August 2008 Accepted 7 September 2008 Available online 29 October 2008
The static acute toxicities of 18 ionic liquids (ILs) were determined for three representative freshwater organisms, the cladoceran Daphnia magna, the green alga Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum), and the fish Danio rerio (formerly known as zebrafish). The test kit compounds contained three widely used ILs (1-butyl-3-methylimidazolium bis(triflimide), [bmim][Tf2N], butylpyridinium bis(triflimide), [bpy][Tf2N], and N,N-methylbutylpyrrolidinium bis(triflimide), [bmpyrr][Tf2N]) and 15 less common salts. These latter comprised a range of five anions, four positively charged head groups (ammonium, morpholinium, thiophenium, and sulfonium), five 1-methyl-3-alkyl imidazolium derivatives bearing a specific functional group on the longer alkyl chain (Cl, OH, or (CH3)3Si) and three imidazolium derivatives characterized by the presence of a hydrogen atom on the imidazolium nitrogen (‘‘Brønsted acidic imidazolium’’-based ILs). Generally, long-chain ammonium salts showed higher toxicity to algae, cladocerans, and fish, whereas very low toxicities characterized sulfonium- and morpholinium-based ILs. In imidazolium-based ILs, the substitution of one or two carbon atoms of the longer alkyl chain with a more electronegative atom (chlorine or oxygen) reduced the acute toxicity for algae and cladocerans. Low toxicity also characterized the ‘‘Brønsted acidic imidazolium’’-based ILs. Structural information for a rational designer of safer ILs can be obtained from these studies. & 2008 Elsevier Inc. All rights reserved.
Keywords: Daphnia magna Pseudokirchneriella subcapitata Danio rerio Ionic liquids Acute toxicity
1. Introduction The use of bioassays on standard test organisms represents a fundamental approach in the definition of ecological risk in the aquatic environment for promising chemicals as ionic liquids (ILs). The environmental hazard assessment of chemicals consists of the identification of the effects that a chemical may have on organisms in the environment and the determination of the concentration of the chemical below which adverse effects in the environmental sphere of concern (e.g., aquatic) are not expected to occur. This concentration is known as the predicted no-effect concentration (PNEC). Starting from the available data, the PNEC may be calculated by applying an appropriate assessment factor to the effect values [e.g., median lethal concentration (LC50) or
Corresponding author. Fax: +39 50 221 6941.
E-mail addresses: [email protected] (C. Pretti), [email protected] (C. Chiappe), [email protected] (L. Intorre). 1 Fax: +39 50 221 9669. 2 Fax: +39 50 221 6941. 3 Fax: +39 50 221 6813. 0147-6513/$ - see front matter & 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2008.09.010
median effective concentration (EC50) or no observed effect concentration (NOEC)] derived from tests on organisms, such as crustaceans, algae, and fish. Environmental hazard assessment has been regulated by constantly updated EU directives and regulations (Dir. 67/548, 88/379, and 76/769; Reg. 793/93), which take into account scientific and technical progress. In order to improve the protection of human health and the environment through better and earlier identification of the properties of chemical substances, EU proposed a new regulatory framework for the Registration, Evaluation, and Authorisation of Chemicals (REACH) on 29 October 2003 (COM(03) 644). After 3 years of negotiation, REACH Regulation was adopted by the Plenary of the European Parliament at the end of 2006. According to the REACH Regulation, the registration process requires (eco)toxicological data for all chemicals produced in or imported into the European Union above one metric tonne per year. Really, REACH has the aim not only of improving the protection of human health and the environment, but also of maintaining the competitiveness and enhancing the innovative capability of the EU chemicals industry. Over the past 10 years or so, research of new chemicals able to substitute for many environmental
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unfriendly compounds has been a field of intense investigation in the area of green chemistry and engineering. In particular, ILs have received considerable attention as potential green solvents for a wide range of applications. This is mainly due to their superior properties compared with conventional organic solvents, such as nonvolatility, nonflammability, and high thermal stability. However, some important claims about ILs have been recently discredited. It has been shown that a large group of ILs are combustible (Smiglak et al., 2006) and that some commercially available ILs may be toxic for different aquatic organisms, from bacteria to fish (Bernot et al., 2005a, b; Couling et al., 2006; Latala et al., 2005; Pretti et al., 2006; Wells and Coombe, 2006). Furthermore, although it has been evidenced that selected families of commonly used aprotic ILs can be distilled at 200–300 1C and low pressure (Earle et al., 2006), this feature practically has no effect on the environmental impact of ILs on the air compartment. The possibility to diffuse ILs into the atmosphere is extremely low. On the other hand, the water solubility of many ILs may allow their entering into the aquatic compartment. This may have important consequences, in particular if their application in large-scale processes increases (e.g., accidental spills, effluent discarges). Recently, even if with a relevant delay with respect to their application as solvents for synthesis, catalysis, and extraction, important data (both experimental and calculated) on the environmental effect of ILs have been published, showing the increasing interest in the subject. To assess the aquatic toxicity of ILs on eukaryotic organisms, tests have been carried out mainly on imidazolium-based ILs related to the crustacean Daphnia magna (Bernot et al., 2005a; Garcia et al., 2005; Couling et al., 2006; Wells and Coombe, 2006; Samorı´ et al., 2007), the marine microalga Oocystis submarina, the diatom Cyclotella meneghiniana (Latala et al., 2005), the freshwater algae Scenedesmus quadricauda (Kulacki and Lamberti, 2008), and Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum) (Wells and Coombe, 2006; Cho et al., 2008). Some information on common ILs is available also on molluscs such as the freshwater snail Physa acuta (Bernot et al., 2005b) and on the marine bacteria Vibrio fischeri (Couling et al., 2006; Matzke et al., 2007; Stolte et al., 2007), whereas the acute toxicity of several ILs on fish has been assessed using the zebrafish (Danio rerio) (Pretti et al., 2006). Although limited in number, these studies have shown that ILs have different degrees of toxicity to aquatic organisms ranging from bacteria to fish, and that toxicity is primarily determined by the cationic moiety. More in particular, it is strongly affected by the side-chain length (Bernot et al., 2005a). Recently, there has also been evidence that anions can contribute to toxicity, but in most cases anion effects are less dramatic compared with the side-chain effect (Stolte et al., 2007; Matzke et al., 2007). In crustaceans and algae, the mechanism of toxicity induced by ILs is still unknown. However, it has been suggested that toxicity in D. magna could be related to enzymatic inhibition and membrane disruption (Bernot et al., 2005a), whereas in fish toxic effects towards the branchial epithelium, with a supposed alteration of membrane stability, have been demonstrated for two ammonium ILs, having structural features similar to common cationic surfactants (Pretti et al., 2006). An extensive review on sustainability of ILs reporting a complete overview on (eco)toxicological data of ILs has been recently published (Ranke et al., 2006). One of the main features of ILs is the possibility to tune the physico-chemical properties of these salts via the selection of specific anions and cations, making it possible, at least in principle, to design the best IL for any specific application. In order to obtain information on the possibility of modifying the structures of ILs to improve chemical properties, and reduce (eco)toxicity, the acute toxic effects of 18 ILs were investigated on
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three organisms from three different trophic levels: primary producers (green algae), primary consumers (cladocerans), and predators (fish). The test kit compounds comprised several aromatic, heterocyclic and non-cyclic quaternary nitrogencontaining compounds (ammonium, imidazolium, pyridinium, pyrrolidium, and morpholinium), and two sulfonium salts. In the case of the imidazolium salts, toxic effects were investigated in relation to the presence of specific functional groups on the longer alkyl chain or the substitution of an alkyl group on nitrogen with a hydrogen atom (Brønsted acidic ILs).
2. Materials and methods 2.1. Ionic liquids AMMOENG 100 and AMMOENG 130 were purchased from Solvent Innovation (GMBH). 1-Butyl-3-methylimidazolium bis(triflimide) ([bmim][Tf2N]), butylpyridinium bis(triflimide) ([bpy][Tf2N]), and N,N-methylbutylpyrrolidinium bis(triflimide) ([bmpyr][Tf2N]) were prepared following reported procedures (Cammarata et al., 2001). 1-(2-Cloroethyl)imidazolium chloride ([HC2Clim]Cl), methylimidazolium chloride ([Hmim]Cl), 1,10 -(1,2-ethanediyl)bis-imidazolium chloride [(C2(Him)2]2Cl), 1-methyl-3-[(trimethylsilyl)methyl]-imidazolium bromide ([TMSiMmim]Br), 3-(2-hydroxyethyl)-1-methylimidazolium bis-triflimide ([C2OHmim][Tf2N]), 3-(2-cloroethyl)-1-methylimidazolium chloride ([C2Clmim]Cl), 3-(2-cloroethyl)-1-methylimidazolium bis-triflimide ([C2Clmim][Tf2N]), choline hexafluorophosphate ([Chol][PF6]), 3-(2-cloropropyl)-1-methylimidazolium chloride ([C3OHmim]Cl), ethyl-methylmorpholinium bromide ([emmor]Br), butylethylmorholinium bromide ([ebmor]Br), triethylsulfonium bromide ([C2C2C2S]Br), and ethyltetrahydrothiophenium bromide ([ETHT]Br) were prepared following reported procedures (Bini et al., 2007). For all synthesized ILs, purity (499%) was checked by NMR (Bruker, Avance 250) and ESI–MS (LCQ Advantage spectrometer equipped with an ion-trap analyzer, ThermoElectron Company). Halide impurities were checked by a silver-nitrate test. All tested ILs are represented in Fig. 1.
2.2. Test methods Acute toxicity of ILs for D. rerio was assessed measuring their lethal effect after 96 h exposure in a static test. The main test was preceded by a limit test performed at the concentration of 100 mg/L in order to demonstrate that the LC50 was greater than this concentration. If mortality occurred in the limit test, the full LC50 study was then carried out. All tests were performed according to the OECD Guideline no. 203 (OECD, 1992). Procedures for the care and management of animals were performed in accordance with the provisions of the EC Council Directive 86/609 EEC, recognised, and adopted by the Italian Government (DL 27.01.1992, no. 116). The water temperature was 2371 1C, and fish were kept under normal laboratory illumination with a daily photoperiod of 12 h. No food was provided during the test. On the day of experiment, 10 fish were placed in 5-L-glass aquaria containing test or control solution (rearing water) and aerated to restore the concentration of dissolved oxygen to at least 90% of its air saturation value. ILs were directly dissolved in rearing water without co-solvents or vehicles. The D. magna test was performed following OECD Guideline no. 202 (OECD, 1984a), using the Daphtoxkit FTM (Microbiotests, BE). Tests were performed at 20 1C, in darkness, for 24 and 48 h, after which immobility was recorded. The main test was preceded by a limit test performed at the concentration of 100 mg/L in order to demonstrate that the EC50 was greater than this concentration. If immobility occurred in the limit test, the full EC50 study was then carried out. Twenty animals (divided into batches of 5) for each tested concentration were used, and one control without the test substance was run. Neonates (24 h old) animals were transferred in a multiwell plate system (10 mL/well; 5 animals/well). ILs were directly dissolved in test water without co-solvents or vehicles. As indicated by the guidelines, potassium dichromate was also tested as reference substance as a means of demonstrating that under the laboratory test conditions the sensitivity of the test was not changed significantly. The P. subcapitata test was performed according to OECD Guideline no. 201 (OECD, 1984b), using the Algaltoxkit FTM (Microbiotests, BE). Tests were performed at 24 1C in uniform illumination of 8000 L , for 24, 48, and 72 h, in 25 mL cells, with daily determination of algal growth using absorbance values related to a standard curve measured with a spectrophotometer (model Ultrospec 2000, Pharmacia Biotech) at a wavelength of 670 nm. A preliminary limit test at 100 mg/L was performed for all bioassays in order to demonstrate that the EC50 was greater than this concentration; if lower, a full test was performed on some selected ILs to determine the EC50. Prior to performing both toxicity tests with samples, tests with the reference chemical potassium dichromate were carried out to check the reliability of the test procedures.
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Fig. 1. Structures of ILs tested.
2.3. Data treatment The EC50 (96) and (48 h) values for the D. rerio and D. magna, respectively, reference tests and their 95% confidence limits were determined by probit analysis (OECD 1984a) using computer software (USEPA Probit analysis program, version 1.5). The EC50 (72 h) values for the algal reference test were calculated according to the procedure outlined in OEDC Guideline 201 for the calculation of growth rates (OECD, 1984b); the test endpoint was the inhibition of growth, expressed as logarithmic algal biomass increase (average growth rate) during the exposure period. Calculation was performed by the use of the software Prism, GraphPad Software (San Diego, CA, USA).
The ILs tested had been categorized from practically harmless to highly toxic using the different EC50 values according to an acute toxicity rating developed by Passino and Smith (1987). Practically harmless chemicals produce an acute toxicity measure (expressed by EC50 values) ranging from 100 to 1000 mg/L, moderately toxic chemicals from 10 to 100 mg/L, slightly toxic chemicals from 1 to 10 mg/L, and highly toxic from 0.1 to 1 mg/L.
3. Results The data for all tested ILs are summarized in Tables 1–3. All the investigated ILs can be considered as practically harmless for
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D. rerio (EC50 4100 mg/L, limit test) and, therefore, according to OECD guidelines for these latter ILs, the full test was not performed. In contrast, toxicity picture of the investigated ILs to D. magna was more complex (Table 2). Most ILs (IL6–IL8, IL11–IL18) had an EC50 4100 mg/L (full test was not performed) and could be categorized as practically harmless. On the other hand, IL1–IL5, IL9, and IL10 showed 48 h EC50s ranging from 0.55 to 59.87 mg/L. The more toxic compounds were the quaternary ammonium-based ILs IL1 and IL2 that were characterized by EC50 of 1.57 mg/L (slightly toxic) and 0.55 mg/L (highly toxic), respectively. Imidazolium-based ILs like IL5, IL9, and IL10 showed, respectively, an EC50 of 18.91, 59.87, and 17.53 mg/L and could be classified as moderately toxic, whereas the pyridinum (IL3)- and pyrrolidium (IL4)-based ILs showed EC50s of 1.73 mg/L (slightly toxic) and 37.15 mg/L (moderately toxic), respectively. Related to the toxicity to P. subcapitata, only the pyrrolidiumbased IL, IL4, and the morpholinium-based ILs, IL15 and IL16, showed an EC50 4100 mg/L (limit test). Again, according to OECD guidelines, the full test was not performed on these ILs since they can be categorized as practically harmless. Full tests were performed on all the other ILs, for which 72-h EC50s ranging from 0.119 to 82.13 mg/L (Table 3) were found. It is noteworthy that the algal test also confirmed that the more toxic ILs were the quaternary ammonium-based salts IL1 and IL2 (EC50 0.119 and 0.831 mg/L, respectively). These ILs can be classified as highly toxic to algal cells, whereas the pyridinium-based IL, IL3, showing an EC50 of 7.055 mg/L (Table 3), may be considered as slightly toxic. All the other ILs had EC50s ranging from 10.71 (IL10) to 82.13(IL11) mg/L and could be classified as moderately toxic.
4. Discussion The intention of the present study was to provide further information about the (eco)toxicological impacts of structural variations in ILs (considering the positively charged head groups, the substitution with one or more different side chains, and the corresponding anionic species) using relatively simple, quick, and inexpensive bioassays. This information may be particularly useful in the case of ILs, considering that it is a goal of many researchers to tune the physico-chemical properties of ILs via the choice of certain anionic and cationic components. Moreover, dose–response bioassays on test aquatic organisms such as
Table 1 EC50s of ILs (D. rerio) ILs
[C2Clmim]Cl [C2Clmim [Tf2N] [C2OHmim] [TF2N] [C3OHmim]Cl [TMSiMmim]Br [Hmim]Cl [HC2Clim]Cl [C2(Him)2]2Cl [Chol][PF6] [emmor]Br [ebmor]Br [ETHT]Br [C2C2C2S]Br
IL6 IL7 IL8 IL9 IL10 IL11 IL12 IL13 IL14 IL15 IL16 IL18 IL17
Limit test/full test EC50 48 h (mg/L)
(mM)
4100 4100 4100 4100 4100 4100 4100 4100 4100 4100 4100 4100 4100
4552 4234 4246 4566 4402 4844 4599 4425 4401 4476 4397 4508 4505
Hazard rankinga
+ + + + + + + + + + + + +
a Hazard ranking and EC50s range: + practically harmless: 100–1000 mg/L; ++ moderately toxic: 10–100 mg/L,; +++ slightly toxic: 1–10 mg/L; ++++ highly toxic 0.1–1 mg/L (Passino and Smith, 1987).
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freshwater algae, crustaceans, and fish are always required for defining (eco)toxicological endpoints of chemicals. They were demanded by EU regulations (Dir. 67/548, 88/379, and 76/769; Reg. 793/93), and they are required by the new EU regulatory framework REACH. As evidenced by the data reported in Tables 1–3, ILs having structures more similar to those of surfactants (IL1 and IL2) are as toxic, or more so, as common organic solvents (Tables 4–6) to freshwater algae, crustaceans, and fish; their hazard ranking range from highly toxic to slightly toxic. Considering that these are the sole ILs today found to be toxic to fish (Pretti et al., 2006), these data confirm that the use of these salts as solvents should be avoided, if possible. All the other ILs generally show a lower toxicity. For D. magna, most of the investigated compounds may be considered practically harmless (EC50 4100 mg/L) and four ILs show a moderate toxic effect. For the algae P. subcapitata, although the number of harmless ILs is lower (only three), most of the compounds are characterized by EC50 values ranging in the interval of moderately toxic compounds (10–100 mg/L). As expected, the anion and cation structure affects the toxicity of the investigated ILs. Related to the anion effect on (eco)toxicity, it is interesting to note that although this feature has been recently investigated (Stolte et al., 2006; Matzke et al., 2007; Cho et al., 2008), in most of the studied systems it appears not to be as distinct as the demonstrated cation effect. Nevertheless, whereas it is generally considered that halides (Br and Cl) do not exhibit an intrinsic anion effect, for the[Tf2N] anion a clear contribution to toxicity has been demonstrated at least for algae (Scenedesmus vacuolatus) and some other systems (Stolte et al., 2007; Matzke et al., 2007). When expressed as mM concentration, the EC50 values for P. subcapitata found for ILs IL6 and IL7 confirm this behaviour, as well as the comparison of the values characterizing IL3 and IL5 with the data reported in the literature for the corresponding chloride salts ([bmim]Cl 221 mM, bpy]Cl 368 mM, from values in Table 5 expressed as mg/L). Cationic head groups and functionalized side chains significantly contribute to the aquatic toxicity. The ILs IL3, IL4, IL5, IL7, and IL8, all having the polyatomic bis(trifluoromethanesulfonyl)imide [Tf2N] species as anion, show EC50 values for D. magna and P. subcapitata that allow these compounds to be classified in a range going from practically harmless to slightly toxic. For both systems, the following toxicity order can be evidenced: [bpy]+4[bmim]+4[bmpyr]+. However, it is noteworthy that the introduction on the longer alkyl chain of the imidazolium ring of a more electronegative atom (chlorine or oxygen, IL7 and IL8) drastically reduces the acute toxicity to D. magna and P. subcapitata. IL7 and IL8 are less toxic than the widely used [bmim][Tf2N], which is characterized by the same anion and a very similar molecular weight. The effect of the functionalisation of imidazolium ring can also be evidenced comparing with each other the EC50 values found for the ILs IL6, IL9, IL10, IL11, IL12, and IL13. In the case of the methylimidazolium-based ILs (IL6, IL9, and IL10), toxicity to P. subcapitata and D. magna increases in the order IL6oIL9oIL10; IL10 has a toxicity slightly lower than [bmim]Br. Related to ILs IL11, IL12, and IL13, which are practically harmless to D. magna and show a toxicity P. subcapitata, increasing in the order IL11oIL12oIL13, it is noteworthy that the substitution of the butyl chain with a hydrogen atom reduces toxicity both to D. magna and P. subcapitata (compare IL11 with [bmim]Cl). A similar effect can be evidenced also in the substitution of a methyl group (compare IL6 and IL 12 for toxicity to D. magna). Finally, it is noteworthy that even the morpholium salts IL15 and IL16, the pyrrolidinium salt IL4, the choline derivative IL14, and the sulfonium salts IL17 and IL18 are practically harmless to daphnids, and harmless or moderately toxic to P. subcapitata.
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Table 2 EC50s of ILs (D. magna) ILs
AMMOENG 100 AMMOENG 130 [bpy] [Tf2N] [bmpyr] [Tf2N] [bmim] [Tf2N] [C2Clmim]Cl [C2Clmim] [Tf2N] [C2OHmim] [Tf2N] [C3OHmim]Cl [TMSiMmim]Br [Hmim]Cl [HC2Clim]Cl [C2(Him)2]2Cl [Chol][PF6] [emmor]Br [ebmor]Br [ETHT]Br [C2C2C2S]Br
IL1 IL2 IL3 IL4 IL5 IL6 IL7 IL8 IL9 IL10 IL11 IL12 IL13 IL14 IL15 IL16 IL17 IL18
Hazard rankinga
Limit test/full test EC50 48 h mg/L (lower limit; upper limit)
mM (lower limit; upper limit)
1.57 (1.13; 1.91) 0.55 (0.43; 0.64) 1.73 (1.15; 2.69) 37.15 (29.21;48.01) 18.91 (15.77;22.68) 4100 4100 4100 59.87 (54.39; 65.66) 17.53 (4.75; 62.97) 4100 4100 4100 4100 4100 4100 4100 4100
0.9 (0.7; 1.1) 4.2 (2.8; 6.5) 88.0 (69.2;113.8) 45.1 (37.6; 54.1) 4552 4233 4245 340.2 (309.0; 373.1) 70.4 (19.1; 252.8) 4844 4599 4425 4401 4476 4397 4507 4502
b
+++ ++++ +++ ++ ++ + + + ++ ++ + + + + + + + +
a Hazard ranking and EC50s range: + practically harmless: 100–1000 mg/L; ++ moderately toxic: 10–100 mg/L,; +++ slightly toxic: 1–10 mg/L; ++++ highly toxic 0.1–1 mg/L (Passino and Smith, 1987). b Molecular weight cannot be calculated being an unknown isomer mixture.
Table 3 EC50s of ILs (P. subcapitata) ILs
AMMOENG 100 AMMOENG 130 [bpy] [Tf2N] [bmpyr] [Tf2N] [bmim] [Tf2N] [C2Clmim]Cl [C2Clmim [Tf2N] [C2OHmim] [Tf2N] [C3OHmim]Cl [TMSiMmim]Br [Hmim]Cl [HC2Clim]Cl [C2(Him)2]2Cl [Chol][PF6] [emmor]Br [ebmor]Br [ETHT]Br [C2C2C2S]Br
IL1 IL2 IL3 IL4 IL5 IL6 IL7 IL8 IL9 IL10 IL11 IL12 IL13 IL14 IL15 IL16 IL17 IL18
Hazard rankinga
Limit test/full test EC50 72 h mg/L (lower limit; upper limit)
mM (lower limit; upper limit)
0.12 (0.08; 0.17) 0.83 (0.30; 2.29) 7.05 (6.06; 8.21) 4100 26.49 (22.29; 31.49) 76.22 (69.45; 86.32) 71.15 (64.27; 75.11) 56.65 (49.32; 61.68) 29.82 (21.12; 33.52) 10.71 (8.07; 12.22) 82.13 (75.02; 89.36) 79.18 (61.19; 87.91) 67.92 (59.03; 78.58) 37.74 (34.65; 39.57) 4100 4100 59.61 (52.25; 65.14) 43.74 (39.22; 47.96)
1.4 (0.5; 3.9) 16.9 (14.6; 19.7) 4237 63.2 (53.2; 75.1) 421.1 (383.7; 476.9) 167.2 (151.1; 176.5) 139.2 (121.2; 151.5) 169.4 (120.0; 190.4) 43.0 (32.4; 49.1) 693.3 (633.3; 754.4) 474.0 (366.4; 526.40) 289.0 (251.1; 334.4) 151.6 (139.1; 158.9) 4476 4397 302.7 (265.2; 330.6) 219.8 (197.0; 241.0)
b
++++ ++++ +++ + ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + + ++ ++
a Hazard ranking and EC50s range:+ practically harmless: 100–1000 mg/L; ++ moderately toxic: 10–100 mg/L,; +++ slightly toxic: 1–10 mg/L; ++++ highly toxic 0.1–1 mg/L (Passino and Smith, 1987). b Molecular weight cannot be calculated being an unknown isomer mixture.
5. Conclusions In conclusion, the results based on the data of the present work and of literature demonstrated that (i) ILs show a different degree of acute toxicity to aquatic organisms, and the cation plays an important role. Long-chain quaternary ammonium salts having structures more similar to those of surfactants are toxic to freshwater algae, crustaceans, and fish. Among those tested, ILs not having a surfactant-like structure were generally not toxic for D. rerio. However, many show a toxicity to D. magna and P. subcapitata, which is strongly affected by the cationic head group. It decreases on going from aromatic heterocyclic
nitrogen-containing compounds (pyridinium and imidazolium) to non-aromatic cyclic and acyclic compounds (pyrrolidinium, ammonium, and morpholinium). A low toxicity characterizes also cyclic and acyclic sulfonium salts; the investigated sulfonium- and thiophenium-based ILs are practically harmless in all three tested systems. In the case of the imidazolium-based ILs the data here reported seem to suggest that (i) maintaining practically constant the molecular weight, the substitution of one or two carbon atoms of the longer alkyl chain with a more electronegative atom (chlorine or oxygen) reduces the acute toxicity to D. magna and P. subcapitata; (ii) the substitution of an alkyl chain with a hydrogen atom reduces toxicity, too; and (iii) the presence of
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Table 4 Data from literature on acute toxicity (EC50) to D. magna ILs
Head group
EC50 48 h (mg/L) (lower limit; upper limit)
Information source
Hazard rankinga
[bmim] [Br] [bmim][Br] [bmim] [Cl] [bmim] Cl [bmim] Cl [C12mim] Cl [C16mim] Cl [C18mim] Cl [bmim][PF6] [bmim][PF6] [bmim][PF6] [bmim][BF4] [bmim][BF4] [Na][PF6] [Na][BF4] [3-picolinium] [Br] [TBA][Br] [TBP][Br] [bpy]Cl [C4C4C4C2P] [(EtO)2PO2] [C6C6C6C14P] Cl [C8C8C8CN] Cl EcoEng500 Methanol Dichloromethane Acetonitrile Aniline Triethylamine Chlorine Ammonia (NH3) Phenol
Imidazolium Imidazolium Imidazolium Imidazolium Imidazolium Imidazolium Imidazolium Imidazolium Imidazolium Imidazolium Imidazolium Imidazolium Imidazolium
8.03 (6.62; 9.40) 13.2 (0.25)b 14.80 (12.75; 17.03) 6.5 (5.4; 7.8) 12.3 (9.2)b 0.0043 (0.0034; 0.0056) 0.0034 (0.0027; 0.004) 0.0017 (0.0013; 0.002) 19.91 (16.30; 26.33) 24 (21; 27) 25.3 (0.3)b 10.68 (8.78; 14.60) 13.9 (0.2)b 9,344 (8,707; 100,051) 4,765 (4,228; 5,323) 13.24 9.51 3.02 20 (17; 24) 11 (8.7; 14) 0.072 (0.054; 0.093) 0.2 (0.14; 0.27) 1 (0.60; 1.6) 24,500 1,682 3,600 80–380 200 0.12–0.15 2.90–6.93 10–17
Bernot et al. (2005a, b) Garcia et al (2005) Bernot et al. (2005a, b) Wells and Coombe (2006) Garcia et al (2005) Wells and Coombe (2006) Wells and Coombe (2006) Wells and Coombe (2006) Bernot et al. (2005a, b) Wells and Coombe (2006) Garcia et al (2005) Bernot et al. (2005a, b) Garcia et al (2005) Bernot et al. (2005a, b) Bernot et al. (2005a, b) Couling et al. (2006) Couling et al. (2006) Couling et al. (2006) Wells and Coombe (2006) Wells and Coombe (2006) Wells and Coombe (2006) Wells and Coombe (2006) Wells and Coombe (2006) Sigma-Aldrich MSDS Sigma-Aldrich MSDS Sigma-Aldrich MSDS Sigma-Aldrich MSDS Sigma-Aldrich MSDS Kaniewska-Prus (1982) Kaniewska-Prus (1982) Cowgill and Milazzo (1991)
+++ ++ ++ +++ ++ ++++ ++++ ++++ ++ ++ ++ ++ ++ + + ++ +++ +++ ++ ++ ++++ ++++ ++++ + + + ++/+ + ++++ +++ ++
Picolinium
Piridinium Phosphonium Phosphonium Ammonium Ammonium
a Hazard ranking and EC50s range: + practically harmless: 100–1000 mg/L; ++ moderately toxic: 10–100 mg/L,; +++ slightly toxic: 1–10 mg/L; ++++ highly toxic 0.1–1 mg/L (Passino and Smith, 1987). b D. magna 24 h immobilization tests.
Table 5 Data from literature on acute toxicity (EC50) to P. subcapitata ILs
Head group
EC50 72 h (mg/L) (lower limit; upper limit)
Information source
Hazard rankingb
[bmim] [PF6] [bmim] Cl [C12mim] Cl [C16mim] Cl [C18mim] Cl [bpy]Cl [C4C4C4C2P] [(EtO)2PO2] [C6C6C6C14P] Cl [C8C8C8CN] Cl EcoEng500 Dichloromethane Aniline Phenola
Imidazolium Imidazolium Imidazolium Imidazolium Imidazolium Pyridimum Phosphonium Phosphonium Ammonium Ammonium
45 (42; 49) 38.5 (38.1; 38.8) 0.0011 (0.0011; 0.0013) 0.0041 (0.0034; 0.0052) 0.0129 (0.0129) 63 (55; 73) 6.2 (5.6; 6.8) 0.042 (0.037; 0.048) 0.058 (0.052; 0.066) 0.088 (0.079; 0.097) 662 19 370
Wells and Coombe (2006) Wells and Coombe (2006) Wells and Coombe (2006) Wells and Coombe (2006) Wells and Coombe (2006) Wells and Coombe (2006) Wells and Coombe (2006) Wells and Coombe (2006) Wells and Coombe (2006) Wells and Coombe (2006) EPA (EPA/600/8-82/004F) (1985) Sigma-Aldrich MSDS Sigma-Aldrich MSDS
++ ++ ++++ ++++ ++++ ++ ++ ++++ ++++ ++++ + ++ +
a
Algal species: C. vulgaris. Hazard ranking and EC50s range: + practically harmless: 100–1000 mg/L; ++ moderately toxic: 10–100 mg/L; +++ slightly toxic: 1–10 mg/L; ++++ highly toxic 0.1–1 mg/L (Passino and Smith, 1987). b
[Tf2N] anion increases the toxicity, compared to the halides, for P. subcapitata. Therefore, the toxicity data here reported can be considered as evidence that only some ILs are not ‘‘environmentally benign’’ solvents. An appropriate choice of cation and anion
structure is important not only to design the IL with improved physico-chemical properties but also to obtain inherently safer ILs. Research on the ILs having the best solvent properties, including lower toxicity, should be the challenge of future research.
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Table 6 Data from literature on acute toxicity (EC50) to (D. rerio)
AMMOENG 100 AMMOENG 130 [bpy] [Tf2N] [bmpyr] [Tf2N] [bmim] [Tf2N] Methanol Dichloromethane Acetonitrile Aniline Triethylamine
ILs
Limit test/full test EC50 48-h (mg/L) (lower limit; upper limit)
Hazard rankinga
Information source
IL1 IL2 IL3 IL4 IL5
5.9 (4.3; 8.0) 5.2 (3.8; 7.0) 4100 4100 4100 12,700–29,400 4100 4100 4100 44
+++ +++ + + + + + + + ++
Pretti et al. (2006) Pretti et al. (2006) Pretti et al. (2006) Pretti et al. (2006) Pretti et al. (2006) Sigma-Aldrich MSDS Sigma-Aldrich MSDS Sigma-Aldrich MSDS Sigma-Aldrich MSDS Sigma-Aldrich MSDS
a Hazard ranking and EC50s range: + practically harmless: 100–1000 mg/L; ++ moderately toxic: 10–100 mg/L; +++ slightly toxic: 1–10 mg/L; ++++ highly toxic 0.1–1 mg/L (Passino and Smith, 1987).
Acknowledgments This study was supported by the grants from the University of Pisa and from the Italian Ministry of Higher Education and Research (MIUR). References Bernot, R.J., Brueseke, M.A., Evans-White, M.A., Lamberti, G.A., 2005a. Acute and chronic toxicity of imidazolium-based ionic liquids on Daphnia magna. Environ. Toxicol. Chem. 24, 87–92. Bernot, R.J., Kennedy, E.E., Lamberti, G.A., 2005b. Effects of ionic liquids on the survival, movement, and feeding behavior of the freshwater snail Physa acuta. Environ. Toxicol. Chem. 24, 1759–1765. Bini, R., Bortolini, O., Chiappe, C., Pieraccini, D., Siciliano, T., 2007. Development of cation/anion interaction scales for ionic liquids through ESI–MS measurements. J. Phys. Chem. B 111, 598–604. Cammarata, L., Kazarian, S.G., Salter, P.A., Welton, T., 2001. Molecular states of water in room temperature ionic liquids. Phys. Chem. Chem. Phys. 3, 5192–5200. Cho, C-W., Pham, T.P.T., Jeon, Y.-C., Yun, Y.-S., 2008. Influence of anions on the toxic effects of ionic liquids to a phytoplankton Selenastrum capricornutum. Green Chem. 10, 67–72. Couling, D.J., Bernot, R.J., Docherty, K.M., Dixon, J.K., Maginn, E.J., 2006. Assessing the factors responsible for ionic liquid toxicity to aquatic organisms via quantitative structure—property relationship modeling. Green Chem. 8, 82–90. Cowgill, U.M., Milazzo, D.P., 1991. The sensitivity of Ceriodaphnia dubia and Daphnia magna to seven chemicals utilizing the three-brood test. Arch. Environ. Contam. Toxicol. 20, 211–217. Earle, M.J., Esperanc-a, J.M.S.S., Gilea, M.A., Canonjı´a Lopes, J.N., Rebelo, L.N.R., Magee, J.W., Seddon, K.R., Widegren, J.A., 2006. The distillation and volatility of ionic liquids. Nature 439, 831–834. Garcia, M.T., Gathergood, N., Scammells, P.J., 2005. Biodegradable ionic liquids. Part II. Effect of the anion and toxicology. Green Chem. 7, 9–14. Kaniewska-Prus, M., 1982. The effect of ammonia, chlorine, and chloramines toxicity on the mortality of Daphnia magna Straus. Pol. Arch. Hydrobiol. 29, 607–624.
Kulacki, K.J., Lamberti, G.A., 2008. Toxicity of imidazolium ionic liquids to freshwater algae. Green Chem. 10, 104–110. Latala, A., Stepnowski, P., Nedzi, M., Mrozik, W., 2005. Marine toxicity assessment of imidazolium ionic liquids: acute effects on the Baltic algae Oocystis submarina and Cyclotella meneghiniana. Aquat. Toxicol. 73, 91–98. Matzke, M., Stolte, S., Thiele, K., Juffernholz, T., Arning, J., Ranke, J., Welz-Biermann, U., Jarstoff, B., 2007. The influence of an anion species on the toxicity of 1-alkyl3-methylimidazolium ionic liquids observed in an (eco)toxicological test battery. Green Chem. 9, 1198–1207. OECD, 1984a. Daphnia sp., acute immobilization test and reproduction test. OECD Guideline for testing chemicals, no. 202. Organization for Economic Cooperation and Development. OECD, 1984b. Alga growth inhibition test. OECD Guideline for testing chemicals, no. 201. Organization for Economic Co-operation and Development. OECD, 1992. Fish acute toxicity test. OECD Guideline for testing chemicals, no. 203. Organization for Economic Co-operation and Development. Passino, D.R.M., Smith, S.B., 1987. Acute bioassays and hazard evaluation of representative contaminants detected in Great Lakes fish. Environ. Toxicol. Chem. 6, 901–907. Pretti, C., Chiappe, C., Pieraccini, D., Gregori, M., Abramo, F., Monni, G., Intorre, L., 2006. Acute toxicity of ionic liquids to the zebrafish (Danio rerio). Green Chem. 8, 238–240. Ranke, J., Stolte, S., Stormann, R., Arning, J., Jastorff, B., 2006. Design of sustainable chemical products—the example of ionic liquids. Chem. Rev. 107, 2183–2206. Samorı´, C., Pasteris, A., Galletti, P., Tagliavini, E., 2007. Acute toxicity of oxygenated and nonoxygenated imidazolium-based ionic liquids to Daphnia magna and Vibrio fischeri. Environ. Toxicol. Chem. 26, 2379–2382. Smiglak, M., Reichert, W.M., Holbrey, J.D., Wilkes, J.S., Sun, L., Thrasher, J.T., Kirichenko, K., Singh, S., Katrinsky, A.R., Rogers, R.D., 2006. Combustible ionic liquids by design: is laboratory safety another ionic liquid myth? Chem. Commun. 24, 2554–2556. Stolte, S., Arning, J., Bottin-Weber, U., Matzke, M., Stock, F., Thiele, K., Uerdingen, M., Welz-Biermann, U., Jarstoff, B., Ranke, J., 2006. Anion effects on the cytotoxicity of ionic liquids. Green Chem. 8, 621–629. Stolte, S., Matzke, M., Arning, J., Boschen, A., Pittner, W.R., Welz-Biermann, U., Jarstoff, B., Ranke, J., 2007. Effect of different head groups and functionalised side chains on the aquatic toxicity of ionic liquids. Green Chem. 9, 1170–1179. Wells, A.S., Coombe, V.T., 2006. On the freshwater ecotoxicity and biodegradation properties of some common ionic liquids. Org. Proc. Res. Dev. 10, 794–798.
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Acute toxicity of ionic liquids for three freshwater organisms: Pseudokirchneriella subcapitata, Daphnia magna and Danio rerio Carlo Pretti a,, Cinzia Chiappe b,1, Ilaria Baldetti b,1, Sara Brunini a,2, Gianfranca Monni a,2, Luigi Intorre c,3 a b c
` di Pisa, Viale delle Piagge 2, Pisa, Italy Dipartimento di Patologia Animale, Profilassi ed Igiene degli Alimenti, Universita ` di Pisa, via Bonanno 33, Pisa, Italy Dipartimento di Chimica Bioorganica e Biofarmacia, Universita ` di Pisa, Viale delle Piagge 2, Pisa, Italy Dipartimento di Clinica Veterinaria, Universita
a r t i c l e in f o
a b s t r a c t
Article history: Received 18 February 2008 Received in revised form 29 August 2008 Accepted 7 September 2008 Available online 29 October 2008
The static acute toxicities of 18 ionic liquids (ILs) were determined for three representative freshwater organisms, the cladoceran Daphnia magna, the green alga Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum), and the fish Danio rerio (formerly known as zebrafish). The test kit compounds contained three widely used ILs (1-butyl-3-methylimidazolium bis(triflimide), [bmim][Tf2N], butylpyridinium bis(triflimide), [bpy][Tf2N], and N,N-methylbutylpyrrolidinium bis(triflimide), [bmpyrr][Tf2N]) and 15 less common salts. These latter comprised a range of five anions, four positively charged head groups (ammonium, morpholinium, thiophenium, and sulfonium), five 1-methyl-3-alkyl imidazolium derivatives bearing a specific functional group on the longer alkyl chain (Cl, OH, or (CH3)3Si) and three imidazolium derivatives characterized by the presence of a hydrogen atom on the imidazolium nitrogen (‘‘Brønsted acidic imidazolium’’-based ILs). Generally, long-chain ammonium salts showed higher toxicity to algae, cladocerans, and fish, whereas very low toxicities characterized sulfonium- and morpholinium-based ILs. In imidazolium-based ILs, the substitution of one or two carbon atoms of the longer alkyl chain with a more electronegative atom (chlorine or oxygen) reduced the acute toxicity for algae and cladocerans. Low toxicity also characterized the ‘‘Brønsted acidic imidazolium’’-based ILs. Structural information for a rational designer of safer ILs can be obtained from these studies. & 2008 Elsevier Inc. All rights reserved.
Keywords: Daphnia magna Pseudokirchneriella subcapitata Danio rerio Ionic liquids Acute toxicity
1. Introduction The use of bioassays on standard test organisms represents a fundamental approach in the definition of ecological risk in the aquatic environment for promising chemicals as ionic liquids (ILs). The environmental hazard assessment of chemicals consists of the identification of the effects that a chemical may have on organisms in the environment and the determination of the concentration of the chemical below which adverse effects in the environmental sphere of concern (e.g., aquatic) are not expected to occur. This concentration is known as the predicted no-effect concentration (PNEC). Starting from the available data, the PNEC may be calculated by applying an appropriate assessment factor to the effect values [e.g., median lethal concentration (LC50) or
Corresponding author. Fax: +39 50 221 6941.
E-mail addresses: [email protected] (C. Pretti), [email protected] (C. Chiappe), [email protected] (L. Intorre). 1 Fax: +39 50 221 9669. 2 Fax: +39 50 221 6941. 3 Fax: +39 50 221 6813. 0147-6513/$ - see front matter & 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2008.09.010
median effective concentration (EC50) or no observed effect concentration (NOEC)] derived from tests on organisms, such as crustaceans, algae, and fish. Environmental hazard assessment has been regulated by constantly updated EU directives and regulations (Dir. 67/548, 88/379, and 76/769; Reg. 793/93), which take into account scientific and technical progress. In order to improve the protection of human health and the environment through better and earlier identification of the properties of chemical substances, EU proposed a new regulatory framework for the Registration, Evaluation, and Authorisation of Chemicals (REACH) on 29 October 2003 (COM(03) 644). After 3 years of negotiation, REACH Regulation was adopted by the Plenary of the European Parliament at the end of 2006. According to the REACH Regulation, the registration process requires (eco)toxicological data for all chemicals produced in or imported into the European Union above one metric tonne per year. Really, REACH has the aim not only of improving the protection of human health and the environment, but also of maintaining the competitiveness and enhancing the innovative capability of the EU chemicals industry. Over the past 10 years or so, research of new chemicals able to substitute for many environmental
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unfriendly compounds has been a field of intense investigation in the area of green chemistry and engineering. In particular, ILs have received considerable attention as potential green solvents for a wide range of applications. This is mainly due to their superior properties compared with conventional organic solvents, such as nonvolatility, nonflammability, and high thermal stability. However, some important claims about ILs have been recently discredited. It has been shown that a large group of ILs are combustible (Smiglak et al., 2006) and that some commercially available ILs may be toxic for different aquatic organisms, from bacteria to fish (Bernot et al., 2005a, b; Couling et al., 2006; Latala et al., 2005; Pretti et al., 2006; Wells and Coombe, 2006). Furthermore, although it has been evidenced that selected families of commonly used aprotic ILs can be distilled at 200–300 1C and low pressure (Earle et al., 2006), this feature practically has no effect on the environmental impact of ILs on the air compartment. The possibility to diffuse ILs into the atmosphere is extremely low. On the other hand, the water solubility of many ILs may allow their entering into the aquatic compartment. This may have important consequences, in particular if their application in large-scale processes increases (e.g., accidental spills, effluent discarges). Recently, even if with a relevant delay with respect to their application as solvents for synthesis, catalysis, and extraction, important data (both experimental and calculated) on the environmental effect of ILs have been published, showing the increasing interest in the subject. To assess the aquatic toxicity of ILs on eukaryotic organisms, tests have been carried out mainly on imidazolium-based ILs related to the crustacean Daphnia magna (Bernot et al., 2005a; Garcia et al., 2005; Couling et al., 2006; Wells and Coombe, 2006; Samorı´ et al., 2007), the marine microalga Oocystis submarina, the diatom Cyclotella meneghiniana (Latala et al., 2005), the freshwater algae Scenedesmus quadricauda (Kulacki and Lamberti, 2008), and Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum) (Wells and Coombe, 2006; Cho et al., 2008). Some information on common ILs is available also on molluscs such as the freshwater snail Physa acuta (Bernot et al., 2005b) and on the marine bacteria Vibrio fischeri (Couling et al., 2006; Matzke et al., 2007; Stolte et al., 2007), whereas the acute toxicity of several ILs on fish has been assessed using the zebrafish (Danio rerio) (Pretti et al., 2006). Although limited in number, these studies have shown that ILs have different degrees of toxicity to aquatic organisms ranging from bacteria to fish, and that toxicity is primarily determined by the cationic moiety. More in particular, it is strongly affected by the side-chain length (Bernot et al., 2005a). Recently, there has also been evidence that anions can contribute to toxicity, but in most cases anion effects are less dramatic compared with the side-chain effect (Stolte et al., 2007; Matzke et al., 2007). In crustaceans and algae, the mechanism of toxicity induced by ILs is still unknown. However, it has been suggested that toxicity in D. magna could be related to enzymatic inhibition and membrane disruption (Bernot et al., 2005a), whereas in fish toxic effects towards the branchial epithelium, with a supposed alteration of membrane stability, have been demonstrated for two ammonium ILs, having structural features similar to common cationic surfactants (Pretti et al., 2006). An extensive review on sustainability of ILs reporting a complete overview on (eco)toxicological data of ILs has been recently published (Ranke et al., 2006). One of the main features of ILs is the possibility to tune the physico-chemical properties of these salts via the selection of specific anions and cations, making it possible, at least in principle, to design the best IL for any specific application. In order to obtain information on the possibility of modifying the structures of ILs to improve chemical properties, and reduce (eco)toxicity, the acute toxic effects of 18 ILs were investigated on
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three organisms from three different trophic levels: primary producers (green algae), primary consumers (cladocerans), and predators (fish). The test kit compounds comprised several aromatic, heterocyclic and non-cyclic quaternary nitrogencontaining compounds (ammonium, imidazolium, pyridinium, pyrrolidium, and morpholinium), and two sulfonium salts. In the case of the imidazolium salts, toxic effects were investigated in relation to the presence of specific functional groups on the longer alkyl chain or the substitution of an alkyl group on nitrogen with a hydrogen atom (Brønsted acidic ILs).
2. Materials and methods 2.1. Ionic liquids AMMOENG 100 and AMMOENG 130 were purchased from Solvent Innovation (GMBH). 1-Butyl-3-methylimidazolium bis(triflimide) ([bmim][Tf2N]), butylpyridinium bis(triflimide) ([bpy][Tf2N]), and N,N-methylbutylpyrrolidinium bis(triflimide) ([bmpyr][Tf2N]) were prepared following reported procedures (Cammarata et al., 2001). 1-(2-Cloroethyl)imidazolium chloride ([HC2Clim]Cl), methylimidazolium chloride ([Hmim]Cl), 1,10 -(1,2-ethanediyl)bis-imidazolium chloride [(C2(Him)2]2Cl), 1-methyl-3-[(trimethylsilyl)methyl]-imidazolium bromide ([TMSiMmim]Br), 3-(2-hydroxyethyl)-1-methylimidazolium bis-triflimide ([C2OHmim][Tf2N]), 3-(2-cloroethyl)-1-methylimidazolium chloride ([C2Clmim]Cl), 3-(2-cloroethyl)-1-methylimidazolium bis-triflimide ([C2Clmim][Tf2N]), choline hexafluorophosphate ([Chol][PF6]), 3-(2-cloropropyl)-1-methylimidazolium chloride ([C3OHmim]Cl), ethyl-methylmorpholinium bromide ([emmor]Br), butylethylmorholinium bromide ([ebmor]Br), triethylsulfonium bromide ([C2C2C2S]Br), and ethyltetrahydrothiophenium bromide ([ETHT]Br) were prepared following reported procedures (Bini et al., 2007). For all synthesized ILs, purity (499%) was checked by NMR (Bruker, Avance 250) and ESI–MS (LCQ Advantage spectrometer equipped with an ion-trap analyzer, ThermoElectron Company). Halide impurities were checked by a silver-nitrate test. All tested ILs are represented in Fig. 1.
2.2. Test methods Acute toxicity of ILs for D. rerio was assessed measuring their lethal effect after 96 h exposure in a static test. The main test was preceded by a limit test performed at the concentration of 100 mg/L in order to demonstrate that the LC50 was greater than this concentration. If mortality occurred in the limit test, the full LC50 study was then carried out. All tests were performed according to the OECD Guideline no. 203 (OECD, 1992). Procedures for the care and management of animals were performed in accordance with the provisions of the EC Council Directive 86/609 EEC, recognised, and adopted by the Italian Government (DL 27.01.1992, no. 116). The water temperature was 2371 1C, and fish were kept under normal laboratory illumination with a daily photoperiod of 12 h. No food was provided during the test. On the day of experiment, 10 fish were placed in 5-L-glass aquaria containing test or control solution (rearing water) and aerated to restore the concentration of dissolved oxygen to at least 90% of its air saturation value. ILs were directly dissolved in rearing water without co-solvents or vehicles. The D. magna test was performed following OECD Guideline no. 202 (OECD, 1984a), using the Daphtoxkit FTM (Microbiotests, BE). Tests were performed at 20 1C, in darkness, for 24 and 48 h, after which immobility was recorded. The main test was preceded by a limit test performed at the concentration of 100 mg/L in order to demonstrate that the EC50 was greater than this concentration. If immobility occurred in the limit test, the full EC50 study was then carried out. Twenty animals (divided into batches of 5) for each tested concentration were used, and one control without the test substance was run. Neonates (24 h old) animals were transferred in a multiwell plate system (10 mL/well; 5 animals/well). ILs were directly dissolved in test water without co-solvents or vehicles. As indicated by the guidelines, potassium dichromate was also tested as reference substance as a means of demonstrating that under the laboratory test conditions the sensitivity of the test was not changed significantly. The P. subcapitata test was performed according to OECD Guideline no. 201 (OECD, 1984b), using the Algaltoxkit FTM (Microbiotests, BE). Tests were performed at 24 1C in uniform illumination of 8000 L , for 24, 48, and 72 h, in 25 mL cells, with daily determination of algal growth using absorbance values related to a standard curve measured with a spectrophotometer (model Ultrospec 2000, Pharmacia Biotech) at a wavelength of 670 nm. A preliminary limit test at 100 mg/L was performed for all bioassays in order to demonstrate that the EC50 was greater than this concentration; if lower, a full test was performed on some selected ILs to determine the EC50. Prior to performing both toxicity tests with samples, tests with the reference chemical potassium dichromate were carried out to check the reliability of the test procedures.
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Fig. 1. Structures of ILs tested.
2.3. Data treatment The EC50 (96) and (48 h) values for the D. rerio and D. magna, respectively, reference tests and their 95% confidence limits were determined by probit analysis (OECD 1984a) using computer software (USEPA Probit analysis program, version 1.5). The EC50 (72 h) values for the algal reference test were calculated according to the procedure outlined in OEDC Guideline 201 for the calculation of growth rates (OECD, 1984b); the test endpoint was the inhibition of growth, expressed as logarithmic algal biomass increase (average growth rate) during the exposure period. Calculation was performed by the use of the software Prism, GraphPad Software (San Diego, CA, USA).
The ILs tested had been categorized from practically harmless to highly toxic using the different EC50 values according to an acute toxicity rating developed by Passino and Smith (1987). Practically harmless chemicals produce an acute toxicity measure (expressed by EC50 values) ranging from 100 to 1000 mg/L, moderately toxic chemicals from 10 to 100 mg/L, slightly toxic chemicals from 1 to 10 mg/L, and highly toxic from 0.1 to 1 mg/L.
3. Results The data for all tested ILs are summarized in Tables 1–3. All the investigated ILs can be considered as practically harmless for
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D. rerio (EC50 4100 mg/L, limit test) and, therefore, according to OECD guidelines for these latter ILs, the full test was not performed. In contrast, toxicity picture of the investigated ILs to D. magna was more complex (Table 2). Most ILs (IL6–IL8, IL11–IL18) had an EC50 4100 mg/L (full test was not performed) and could be categorized as practically harmless. On the other hand, IL1–IL5, IL9, and IL10 showed 48 h EC50s ranging from 0.55 to 59.87 mg/L. The more toxic compounds were the quaternary ammonium-based ILs IL1 and IL2 that were characterized by EC50 of 1.57 mg/L (slightly toxic) and 0.55 mg/L (highly toxic), respectively. Imidazolium-based ILs like IL5, IL9, and IL10 showed, respectively, an EC50 of 18.91, 59.87, and 17.53 mg/L and could be classified as moderately toxic, whereas the pyridinum (IL3)- and pyrrolidium (IL4)-based ILs showed EC50s of 1.73 mg/L (slightly toxic) and 37.15 mg/L (moderately toxic), respectively. Related to the toxicity to P. subcapitata, only the pyrrolidiumbased IL, IL4, and the morpholinium-based ILs, IL15 and IL16, showed an EC50 4100 mg/L (limit test). Again, according to OECD guidelines, the full test was not performed on these ILs since they can be categorized as practically harmless. Full tests were performed on all the other ILs, for which 72-h EC50s ranging from 0.119 to 82.13 mg/L (Table 3) were found. It is noteworthy that the algal test also confirmed that the more toxic ILs were the quaternary ammonium-based salts IL1 and IL2 (EC50 0.119 and 0.831 mg/L, respectively). These ILs can be classified as highly toxic to algal cells, whereas the pyridinium-based IL, IL3, showing an EC50 of 7.055 mg/L (Table 3), may be considered as slightly toxic. All the other ILs had EC50s ranging from 10.71 (IL10) to 82.13(IL11) mg/L and could be classified as moderately toxic.
4. Discussion The intention of the present study was to provide further information about the (eco)toxicological impacts of structural variations in ILs (considering the positively charged head groups, the substitution with one or more different side chains, and the corresponding anionic species) using relatively simple, quick, and inexpensive bioassays. This information may be particularly useful in the case of ILs, considering that it is a goal of many researchers to tune the physico-chemical properties of ILs via the choice of certain anionic and cationic components. Moreover, dose–response bioassays on test aquatic organisms such as
Table 1 EC50s of ILs (D. rerio) ILs
[C2Clmim]Cl [C2Clmim [Tf2N] [C2OHmim] [TF2N] [C3OHmim]Cl [TMSiMmim]Br [Hmim]Cl [HC2Clim]Cl [C2(Him)2]2Cl [Chol][PF6] [emmor]Br [ebmor]Br [ETHT]Br [C2C2C2S]Br
IL6 IL7 IL8 IL9 IL10 IL11 IL12 IL13 IL14 IL15 IL16 IL18 IL17
Limit test/full test EC50 48 h (mg/L)
(mM)
4100 4100 4100 4100 4100 4100 4100 4100 4100 4100 4100 4100 4100
4552 4234 4246 4566 4402 4844 4599 4425 4401 4476 4397 4508 4505
Hazard rankinga
+ + + + + + + + + + + + +
a Hazard ranking and EC50s range: + practically harmless: 100–1000 mg/L; ++ moderately toxic: 10–100 mg/L,; +++ slightly toxic: 1–10 mg/L; ++++ highly toxic 0.1–1 mg/L (Passino and Smith, 1987).
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freshwater algae, crustaceans, and fish are always required for defining (eco)toxicological endpoints of chemicals. They were demanded by EU regulations (Dir. 67/548, 88/379, and 76/769; Reg. 793/93), and they are required by the new EU regulatory framework REACH. As evidenced by the data reported in Tables 1–3, ILs having structures more similar to those of surfactants (IL1 and IL2) are as toxic, or more so, as common organic solvents (Tables 4–6) to freshwater algae, crustaceans, and fish; their hazard ranking range from highly toxic to slightly toxic. Considering that these are the sole ILs today found to be toxic to fish (Pretti et al., 2006), these data confirm that the use of these salts as solvents should be avoided, if possible. All the other ILs generally show a lower toxicity. For D. magna, most of the investigated compounds may be considered practically harmless (EC50 4100 mg/L) and four ILs show a moderate toxic effect. For the algae P. subcapitata, although the number of harmless ILs is lower (only three), most of the compounds are characterized by EC50 values ranging in the interval of moderately toxic compounds (10–100 mg/L). As expected, the anion and cation structure affects the toxicity of the investigated ILs. Related to the anion effect on (eco)toxicity, it is interesting to note that although this feature has been recently investigated (Stolte et al., 2006; Matzke et al., 2007; Cho et al., 2008), in most of the studied systems it appears not to be as distinct as the demonstrated cation effect. Nevertheless, whereas it is generally considered that halides (Br and Cl) do not exhibit an intrinsic anion effect, for the[Tf2N] anion a clear contribution to toxicity has been demonstrated at least for algae (Scenedesmus vacuolatus) and some other systems (Stolte et al., 2007; Matzke et al., 2007). When expressed as mM concentration, the EC50 values for P. subcapitata found for ILs IL6 and IL7 confirm this behaviour, as well as the comparison of the values characterizing IL3 and IL5 with the data reported in the literature for the corresponding chloride salts ([bmim]Cl 221 mM, bpy]Cl 368 mM, from values in Table 5 expressed as mg/L). Cationic head groups and functionalized side chains significantly contribute to the aquatic toxicity. The ILs IL3, IL4, IL5, IL7, and IL8, all having the polyatomic bis(trifluoromethanesulfonyl)imide [Tf2N] species as anion, show EC50 values for D. magna and P. subcapitata that allow these compounds to be classified in a range going from practically harmless to slightly toxic. For both systems, the following toxicity order can be evidenced: [bpy]+4[bmim]+4[bmpyr]+. However, it is noteworthy that the introduction on the longer alkyl chain of the imidazolium ring of a more electronegative atom (chlorine or oxygen, IL7 and IL8) drastically reduces the acute toxicity to D. magna and P. subcapitata. IL7 and IL8 are less toxic than the widely used [bmim][Tf2N], which is characterized by the same anion and a very similar molecular weight. The effect of the functionalisation of imidazolium ring can also be evidenced comparing with each other the EC50 values found for the ILs IL6, IL9, IL10, IL11, IL12, and IL13. In the case of the methylimidazolium-based ILs (IL6, IL9, and IL10), toxicity to P. subcapitata and D. magna increases in the order IL6oIL9oIL10; IL10 has a toxicity slightly lower than [bmim]Br. Related to ILs IL11, IL12, and IL13, which are practically harmless to D. magna and show a toxicity P. subcapitata, increasing in the order IL11oIL12oIL13, it is noteworthy that the substitution of the butyl chain with a hydrogen atom reduces toxicity both to D. magna and P. subcapitata (compare IL11 with [bmim]Cl). A similar effect can be evidenced also in the substitution of a methyl group (compare IL6 and IL 12 for toxicity to D. magna). Finally, it is noteworthy that even the morpholium salts IL15 and IL16, the pyrrolidinium salt IL4, the choline derivative IL14, and the sulfonium salts IL17 and IL18 are practically harmless to daphnids, and harmless or moderately toxic to P. subcapitata.
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Table 2 EC50s of ILs (D. magna) ILs
AMMOENG 100 AMMOENG 130 [bpy] [Tf2N] [bmpyr] [Tf2N] [bmim] [Tf2N] [C2Clmim]Cl [C2Clmim] [Tf2N] [C2OHmim] [Tf2N] [C3OHmim]Cl [TMSiMmim]Br [Hmim]Cl [HC2Clim]Cl [C2(Him)2]2Cl [Chol][PF6] [emmor]Br [ebmor]Br [ETHT]Br [C2C2C2S]Br
IL1 IL2 IL3 IL4 IL5 IL6 IL7 IL8 IL9 IL10 IL11 IL12 IL13 IL14 IL15 IL16 IL17 IL18
Hazard rankinga
Limit test/full test EC50 48 h mg/L (lower limit; upper limit)
mM (lower limit; upper limit)
1.57 (1.13; 1.91) 0.55 (0.43; 0.64) 1.73 (1.15; 2.69) 37.15 (29.21;48.01) 18.91 (15.77;22.68) 4100 4100 4100 59.87 (54.39; 65.66) 17.53 (4.75; 62.97) 4100 4100 4100 4100 4100 4100 4100 4100
0.9 (0.7; 1.1) 4.2 (2.8; 6.5) 88.0 (69.2;113.8) 45.1 (37.6; 54.1) 4552 4233 4245 340.2 (309.0; 373.1) 70.4 (19.1; 252.8) 4844 4599 4425 4401 4476 4397 4507 4502
b
+++ ++++ +++ ++ ++ + + + ++ ++ + + + + + + + +
a Hazard ranking and EC50s range: + practically harmless: 100–1000 mg/L; ++ moderately toxic: 10–100 mg/L,; +++ slightly toxic: 1–10 mg/L; ++++ highly toxic 0.1–1 mg/L (Passino and Smith, 1987). b Molecular weight cannot be calculated being an unknown isomer mixture.
Table 3 EC50s of ILs (P. subcapitata) ILs
AMMOENG 100 AMMOENG 130 [bpy] [Tf2N] [bmpyr] [Tf2N] [bmim] [Tf2N] [C2Clmim]Cl [C2Clmim [Tf2N] [C2OHmim] [Tf2N] [C3OHmim]Cl [TMSiMmim]Br [Hmim]Cl [HC2Clim]Cl [C2(Him)2]2Cl [Chol][PF6] [emmor]Br [ebmor]Br [ETHT]Br [C2C2C2S]Br
IL1 IL2 IL3 IL4 IL5 IL6 IL7 IL8 IL9 IL10 IL11 IL12 IL13 IL14 IL15 IL16 IL17 IL18
Hazard rankinga
Limit test/full test EC50 72 h mg/L (lower limit; upper limit)
mM (lower limit; upper limit)
0.12 (0.08; 0.17) 0.83 (0.30; 2.29) 7.05 (6.06; 8.21) 4100 26.49 (22.29; 31.49) 76.22 (69.45; 86.32) 71.15 (64.27; 75.11) 56.65 (49.32; 61.68) 29.82 (21.12; 33.52) 10.71 (8.07; 12.22) 82.13 (75.02; 89.36) 79.18 (61.19; 87.91) 67.92 (59.03; 78.58) 37.74 (34.65; 39.57) 4100 4100 59.61 (52.25; 65.14) 43.74 (39.22; 47.96)
1.4 (0.5; 3.9) 16.9 (14.6; 19.7) 4237 63.2 (53.2; 75.1) 421.1 (383.7; 476.9) 167.2 (151.1; 176.5) 139.2 (121.2; 151.5) 169.4 (120.0; 190.4) 43.0 (32.4; 49.1) 693.3 (633.3; 754.4) 474.0 (366.4; 526.40) 289.0 (251.1; 334.4) 151.6 (139.1; 158.9) 4476 4397 302.7 (265.2; 330.6) 219.8 (197.0; 241.0)
b
++++ ++++ +++ + ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + + ++ ++
a Hazard ranking and EC50s range:+ practically harmless: 100–1000 mg/L; ++ moderately toxic: 10–100 mg/L,; +++ slightly toxic: 1–10 mg/L; ++++ highly toxic 0.1–1 mg/L (Passino and Smith, 1987). b Molecular weight cannot be calculated being an unknown isomer mixture.
5. Conclusions In conclusion, the results based on the data of the present work and of literature demonstrated that (i) ILs show a different degree of acute toxicity to aquatic organisms, and the cation plays an important role. Long-chain quaternary ammonium salts having structures more similar to those of surfactants are toxic to freshwater algae, crustaceans, and fish. Among those tested, ILs not having a surfactant-like structure were generally not toxic for D. rerio. However, many show a toxicity to D. magna and P. subcapitata, which is strongly affected by the cationic head group. It decreases on going from aromatic heterocyclic
nitrogen-containing compounds (pyridinium and imidazolium) to non-aromatic cyclic and acyclic compounds (pyrrolidinium, ammonium, and morpholinium). A low toxicity characterizes also cyclic and acyclic sulfonium salts; the investigated sulfonium- and thiophenium-based ILs are practically harmless in all three tested systems. In the case of the imidazolium-based ILs the data here reported seem to suggest that (i) maintaining practically constant the molecular weight, the substitution of one or two carbon atoms of the longer alkyl chain with a more electronegative atom (chlorine or oxygen) reduces the acute toxicity to D. magna and P. subcapitata; (ii) the substitution of an alkyl chain with a hydrogen atom reduces toxicity, too; and (iii) the presence of
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Table 4 Data from literature on acute toxicity (EC50) to D. magna ILs
Head group
EC50 48 h (mg/L) (lower limit; upper limit)
Information source
Hazard rankinga
[bmim] [Br] [bmim][Br] [bmim] [Cl] [bmim] Cl [bmim] Cl [C12mim] Cl [C16mim] Cl [C18mim] Cl [bmim][PF6] [bmim][PF6] [bmim][PF6] [bmim][BF4] [bmim][BF4] [Na][PF6] [Na][BF4] [3-picolinium] [Br] [TBA][Br] [TBP][Br] [bpy]Cl [C4C4C4C2P] [(EtO)2PO2] [C6C6C6C14P] Cl [C8C8C8CN] Cl EcoEng500 Methanol Dichloromethane Acetonitrile Aniline Triethylamine Chlorine Ammonia (NH3) Phenol
Imidazolium Imidazolium Imidazolium Imidazolium Imidazolium Imidazolium Imidazolium Imidazolium Imidazolium Imidazolium Imidazolium Imidazolium Imidazolium
8.03 (6.62; 9.40) 13.2 (0.25)b 14.80 (12.75; 17.03) 6.5 (5.4; 7.8) 12.3 (9.2)b 0.0043 (0.0034; 0.0056) 0.0034 (0.0027; 0.004) 0.0017 (0.0013; 0.002) 19.91 (16.30; 26.33) 24 (21; 27) 25.3 (0.3)b 10.68 (8.78; 14.60) 13.9 (0.2)b 9,344 (8,707; 100,051) 4,765 (4,228; 5,323) 13.24 9.51 3.02 20 (17; 24) 11 (8.7; 14) 0.072 (0.054; 0.093) 0.2 (0.14; 0.27) 1 (0.60; 1.6) 24,500 1,682 3,600 80–380 200 0.12–0.15 2.90–6.93 10–17
Bernot et al. (2005a, b) Garcia et al (2005) Bernot et al. (2005a, b) Wells and Coombe (2006) Garcia et al (2005) Wells and Coombe (2006) Wells and Coombe (2006) Wells and Coombe (2006) Bernot et al. (2005a, b) Wells and Coombe (2006) Garcia et al (2005) Bernot et al. (2005a, b) Garcia et al (2005) Bernot et al. (2005a, b) Bernot et al. (2005a, b) Couling et al. (2006) Couling et al. (2006) Couling et al. (2006) Wells and Coombe (2006) Wells and Coombe (2006) Wells and Coombe (2006) Wells and Coombe (2006) Wells and Coombe (2006) Sigma-Aldrich MSDS Sigma-Aldrich MSDS Sigma-Aldrich MSDS Sigma-Aldrich MSDS Sigma-Aldrich MSDS Kaniewska-Prus (1982) Kaniewska-Prus (1982) Cowgill and Milazzo (1991)
+++ ++ ++ +++ ++ ++++ ++++ ++++ ++ ++ ++ ++ ++ + + ++ +++ +++ ++ ++ ++++ ++++ ++++ + + + ++/+ + ++++ +++ ++
Picolinium
Piridinium Phosphonium Phosphonium Ammonium Ammonium
a Hazard ranking and EC50s range: + practically harmless: 100–1000 mg/L; ++ moderately toxic: 10–100 mg/L,; +++ slightly toxic: 1–10 mg/L; ++++ highly toxic 0.1–1 mg/L (Passino and Smith, 1987). b D. magna 24 h immobilization tests.
Table 5 Data from literature on acute toxicity (EC50) to P. subcapitata ILs
Head group
EC50 72 h (mg/L) (lower limit; upper limit)
Information source
Hazard rankingb
[bmim] [PF6] [bmim] Cl [C12mim] Cl [C16mim] Cl [C18mim] Cl [bpy]Cl [C4C4C4C2P] [(EtO)2PO2] [C6C6C6C14P] Cl [C8C8C8CN] Cl EcoEng500 Dichloromethane Aniline Phenola
Imidazolium Imidazolium Imidazolium Imidazolium Imidazolium Pyridimum Phosphonium Phosphonium Ammonium Ammonium
45 (42; 49) 38.5 (38.1; 38.8) 0.0011 (0.0011; 0.0013) 0.0041 (0.0034; 0.0052) 0.0129 (0.0129) 63 (55; 73) 6.2 (5.6; 6.8) 0.042 (0.037; 0.048) 0.058 (0.052; 0.066) 0.088 (0.079; 0.097) 662 19 370
Wells and Coombe (2006) Wells and Coombe (2006) Wells and Coombe (2006) Wells and Coombe (2006) Wells and Coombe (2006) Wells and Coombe (2006) Wells and Coombe (2006) Wells and Coombe (2006) Wells and Coombe (2006) Wells and Coombe (2006) EPA (EPA/600/8-82/004F) (1985) Sigma-Aldrich MSDS Sigma-Aldrich MSDS
++ ++ ++++ ++++ ++++ ++ ++ ++++ ++++ ++++ + ++ +
a
Algal species: C. vulgaris. Hazard ranking and EC50s range: + practically harmless: 100–1000 mg/L; ++ moderately toxic: 10–100 mg/L; +++ slightly toxic: 1–10 mg/L; ++++ highly toxic 0.1–1 mg/L (Passino and Smith, 1987). b
[Tf2N] anion increases the toxicity, compared to the halides, for P. subcapitata. Therefore, the toxicity data here reported can be considered as evidence that only some ILs are not ‘‘environmentally benign’’ solvents. An appropriate choice of cation and anion
structure is important not only to design the IL with improved physico-chemical properties but also to obtain inherently safer ILs. Research on the ILs having the best solvent properties, including lower toxicity, should be the challenge of future research.
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Table 6 Data from literature on acute toxicity (EC50) to (D. rerio)
AMMOENG 100 AMMOENG 130 [bpy] [Tf2N] [bmpyr] [Tf2N] [bmim] [Tf2N] Methanol Dichloromethane Acetonitrile Aniline Triethylamine
ILs
Limit test/full test EC50 48-h (mg/L) (lower limit; upper limit)
Hazard rankinga
Information source
IL1 IL2 IL3 IL4 IL5
5.9 (4.3; 8.0) 5.2 (3.8; 7.0) 4100 4100 4100 12,700–29,400 4100 4100 4100 44
+++ +++ + + + + + + + ++
Pretti et al. (2006) Pretti et al. (2006) Pretti et al. (2006) Pretti et al. (2006) Pretti et al. (2006) Sigma-Aldrich MSDS Sigma-Aldrich MSDS Sigma-Aldrich MSDS Sigma-Aldrich MSDS Sigma-Aldrich MSDS
a Hazard ranking and EC50s range: + practically harmless: 100–1000 mg/L; ++ moderately toxic: 10–100 mg/L; +++ slightly toxic: 1–10 mg/L; ++++ highly toxic 0.1–1 mg/L (Passino and Smith, 1987).
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