ACUTE AND CHRONIC TOXICITY OF IMIDAZOLIUM-BASED IONIC LIQUIDS ON DAPHNIA MAGNA

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1 Environmental Toxicology and Chemistry, Vol. 24, No. 1, pp , SETAC Printed in the USA /05 $ ACUTE AND CHRONIC TOXICITY OF IMIDAZOLIUM-BASED IONIC LIQUIDS ON DAPHNIA MAGNA RANDALL J. BERNOT,* MICHAEL A. BRUESEKE, MICHELLE A. EVANS-WHITE, and GARY A. LAMBERTI Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana , USA (Received 19 November 2003; Accepted 1 June 2004) Abstract Room-temperature ionic liquids (ILs) are considered to be green chemicals that may replace volatile organic solvents currently used by industry. However, IL effects on aquatic organisms and ecosystems are currently unknown. We studied the acute effects of imidazolium-based ILs on survival of the crustacean Daphnia magna and their chronic effects on number of first-brood neonates, total number of neonates, and average brood size. Lethal concentrations of imidazolium ILs with various anions (X ) ranged from a median lethal concentration (LC50) of 8.03 to mg L 1, whereas salts with a sodium cation (Na X ) were more than an order of magnitude higher (NaPF 6 LC50, 9, mg L 1 ; NaBF 4 LC50, mg L 1 ). Thus, toxicity appeared to be related to the imidazolium cation and not to the various anions (e.g., Cl,Br, PF6, and BF4). The toxicity of imidazoliumbased ILs is comparable to that of chemicals currently used in manufacturing and disinfection processes (e.g., ammonia and phenol), indicating that these green chemicals may be more harmful to aquatic organisms than current volatile organic solvents. We conducted 21-d chronic bioassays of individual D. magna exposed to nonlethal IL concentrations at constant food-resource levels. Daphnia magna produced significantly fewer total neonates, first-brood neonates, and average neonates when exposed to lower concentrations (0.3 mg L 1 ) of imidazolium-based ILs than in the presence of Na-based salts at higher concentrations (400 mg L 1 ). Such reductions in the reproductive output of Daphnia populations could cascade through natural freshwater ecosystems. The present study provides baseline information needed to assess the potential hazard that some ILs may pose should they be released into freshwater ecosystems. Keywords 1-Butyl-3-methylimidazolium Daphnia magna Bioassay Ionic liquid Life history INTRODUCTION Organic solvents are used by industry and consumers for a variety of manufacturing, processing, and cleaning purposes [1]. However, the volatility of most solvents leads to evaporation into the atmosphere that contributes to smog formation, ozone depletion, and global climate change [2]. Other proximate dangers of solvent volatility include human inhalation exposure and fires or explosions because of their lower flash points. The recent search for less harmful compounds has led to a surge of research into room-temperature ionic liquids (ILs) [1]. Room-temperature ILs are a class of chemicals that are rapidly being adopted by industry as environmentally friendly solvent substitutes [3 5]. The properties of ILs that make them interesting for chemical synthesis and catalysis have been reviewed by Chauvin and Olivier-Bourbigou [6], and these include their unique solvent properties and the fact that they do not evaporate. Ionic liquids also have low melting points because of the bulkiness and asymmetry of the molecules, thereby preventing the molecule packing that promotes crystallization [1]. Some ILs react with water, whereas others, including imidazolium- (Fig. 1) and pyridinium-based ILs, are water stable. Importantly, varying the cation and anion constituents also significantly alters the chemical and physical properties of an IL [1]. Useful applications of IL reactions include metal refining [7], fuel-cell electrolytes [8], removal of metal contaminants from aqueous waste [9], lubrication, and sequestration of CO 2 [10]. Because they are nonvolatile, ILs are relatively benign to the atmosphere, but their impacts on aquatic organisms and communities are largely unknown [5]. Recent * To whom correspondence may be addressed (rbernot@nd.edu). studies have investigated IL toxicity to mammalian cell lines [11] and the marine bacteria Vibrio fischeri [11] as well as enzyme activity [12], but studies of IL toxicity to eukaryotic organisms are essential to determine the impacts that ILs may have on aquatic ecosystems. Ionic liquids have the potential to affect aquatic ecosystems through a variety of mechanisms, including mortality of individual organisms, altered population demographic rates, modified species interactions propagated through communities, altered biogeochemical processes, and bioaccumulation in higher trophic levels of food webs. Because water-stable ILs have been widely known only since 1992 [13], their potential direct and indirect impacts on aquatic organisms, communities, and ecosystems are unknown [5]. Therefore, fundamental information about the effects of ILs on single species is necessary to lay the groundwork for more comprehensive community and ecosystem investigations. In the present study, we investigated the acute and chronic toxicities of imidazolium-based ILs to the water flea Daphnia magna. Acute toxicity of ILs (48-h median lethal concentration [LC50] value [14]) was determined using standard U.S. Environmental Protection Agency (U.S. EPA) protocols. We also assessed the chronic toxicity of ILs to Daphnia first-brood neonates, total number of neonates, and average brood size using 21-d static exposures with unlimited food resources. Test chemicals MATERIALS AND METHODS Ionic liquids are salts comprised of an organic cation and a variety of anions and that have melting points below 100 C. Imidazolium-based ILs consist of an imidazolium cation and a variety of anion species (Fig. 1) that have a relatively low 87

2 88 Environ. Toxicol. Chem. 24, 2005 R.J. Bernot et al. Table 1. Lethal concentrations of different ionic liquids to Daphnia magna in 48-h acute toxicity bioassays a Ionic liquid LC5 (mg L 1 ) LC50 (mg L 1 ) Fig. 1. Chemical structure of imidazolium salt. Both R 1 and R 3 represent substituents that may be attached from the nitrogen atoms in the aromatic ring. The anions (X ) used in Daphnia magna acute and chronic toxicity bioassays were Cl, PF, PF, and Br. 6 4 [bmim]br [bmim]cl [bmim]pf 6 [bmim]bf 4 NaPF 6 NaBF ( ) 8.82 ( ) 8.00 ( ) 2.28 ( ) 7, (6, ,200.40) 2, (1, ,004.23) 8.03 ( ) ( ) ( ) ( ) 9, (8, ,051.68) 4, (4, ,323.46) octanol water partition coefficient (K ow ), ranging from to [15]. We tested the toxicity of a family of ILs containing 1-butyl-3-methylimidazolium (bmim) as a cation with Cl, PF6, BF4, and Br as anions. We also tested the toxicity of salts with Na as the cation and PF6 and BF4 as anions to determine if the imidazolium cation or the various anions influenced toxicity. Ionic liquids used in the bioassays were synthesized at the University of Notre Dame (Notre Dame, IN, USA); the Na -based salts were obtained commercially (Sigma-Aldrich, St. Louis, MO, USA). Test organisms and test water Daphnia magna neonates (age, 24 h) were obtained from batch cultures maintained in groundwater-filled laboratory aquaria at 20 2 C and a 16:8-h light:dark photoperiod with a 15-min transition period. Stock animals were originally obtained from Carolina Biological Supply (Burlington, NC, USA). Cultures were fed a mixture of pond water and Spirulina Dry Powder Microalga (Petaluma, CA, USA). Dilutions and water quality Test concentrations of ILs were prepared by pipetting the required amount of stock solution (ILs dissolved in water) into known volumes of test water. Water-quality parameters (temperature, ph, and dissolved oxygen) were recorded daily. Dissolved oxygen and temperature were measured with an oxygen meter (Orion Research, Beverly, MA, USA), and ph was measured with a ph tester (Oakton Instruments, Beverly, MA, USA). Acute toxicity The IL-exposure bioassays were conducted as 48-h static acute tests according to standard procedures [14]. Each IL exposure used D. magna neonates (age, 24 h) born from parthenogenic females grown in batch cultures. Eight neonates were placed in each of 30 glass beakers (250 ml), with five replicates for each of six treatment concentrations (control plus five IL concentrations). The number of living and dead neonates was noted at 24 and 48 h after the initiation of each trial. Neonates observed as motionless and without a discernable heartbeat were considered to be dead. Each trial was conducted at 20 1 C in the laboratory with a 16:8-h light:dark photoperiod. Ionic liquid test concentrations were based on preliminary range-finding tests of acute toxicity and ranged from 1 to 100 mg L 1. The Na -based salt concentrations ranged from 1 to 10,000 mg L 1. The 5% lethal concentration and LC50 values as well as the associated 95% confidence intervals (CIs) were obtained by fitting the dose response curves to a a Values in parentheses are the 95% confidence intervals. bmim 1- butyl-3-methylimidazolium. normal model using the maximum-likelihood method [16]. All analyses were performed using SAS Version 8.02 statistical software (SAS, Cary, NC, USA). Chronic toxicity Chronic tests of IL toxicity to D. magna generally followed the procedures recommended by the U.S. EPA [17]. Animals were raised in 25 ml of test solution in 30-ml glass vials. Test Daphnia were neonates (age, 24 h) from the third generation of a single parthenogenetic female. Chronic tests with [bmim]cl, [bmim]pf 6, NaBF 4, and NaPF 6 used a single Daphnia clone (clone A), whereas tests with [bmim]br and [bmim]bf 4 used a different Daphnia clone (clone B). Both clones exhibited similar acute sensitivities to KCl in qualityassurance bioassays (clone A: 48-h LC50, 405 mg L 1 ; 95% CI, mg L 1 ; clone B: LC50, 415 mg L 1 ; 95% CI, mg L 1 ). Ten replicates with one neonate per vial were used for each of six treatment concentrations as described for the acute bioassays. Ionic liquid test concentrations were 0, 0.2, 0.4, 0.8, 1.6, and 3.2 mg L 1. The Na -based salt test concentrations were 0, 187.5, 375, 750, 1,500, and 3,000 mg L 1. Animals were transferred to fresh media (test solution cells ml 1 of Spirulina Dry Powder) daily, and the number of neonates was recorded. Each experiment was terminated either when all control females completed their third brood or at 21 d. The IL treatment effects on number of first-brood neonates, total number of neonates, and average brood size were analyzed with multivariate analysis of variance, because responses are not independent of each other. When significant multivariate effects were found, each response variable was analyzed separately using one-way analysis of variance. Tukey s multiple-comparison test was used to detect differences between pairs of treatments. Each trial (i.e., different IL) was analyzed separately. Acute toxicity RESULTS The most toxic material in the present study was [bmim]br (LC50, 8.03 mg L 1 ), and the least toxic was NaPF 6 (LC50, 9, mg L 1 ) (Table 1). Daphnia magna lethal concentrations were much lower for ILs with imidazolium as the cation than they were for salts with Na as the cation (Table 1), suggesting that toxicity was related to the imidazolium cation and not to the various anions tested.

3 Ionic liquid toxicity Environ. Toxicol. Chem. 24, Table 2. Multivariate analysis of variance results for chronic effects of ionic liquids on Daphnia magna life-history traits (total neonates, first-brood neonates, and average neonates) a Ionic liquid df F p b [bmim]br [bmim]bf 4 [bmim]cl [bmim]pf 6 NaBF 4 NaPF 6 15, 30 15, , , , 90 10, a bmim 1-butyl-3-methylimidazolium; df degrees of freedom. b Wilks s lambda. Chronic toxicity Ionic liquids had significant effects on D. magna life-history traits (Table 2). In general, higher concentrations of salts having Na as the cation were needed to observe significant effects on D. magna compared to imidazolium-based ILs. However, all ILs, regardless of cation, negatively affected reproductive output, with the lowest number of neonates being produced in the highest concentrations of IL. Total number of Daphnia neonates produced ranged from 18 to 62, depending on the IL and its concentration. The total number of neonates and the average number of neonates differed among concentrations of [bmim]br (total neonates: F 5, , p 0.01; brood size: F 5, , p 0.01), but the number of first-brood neonates did not differ among treatments (F 5, , p 0.34) (Figs. 2 4). All concentrations of [bmim]br reduced the total number of neonates relative to controls (Tukey s, p 0.01) (Fig. 2). Average brood size was also reduced in the presence of [bmim]br at concentrations of 0.2, 1.6, and 3.2 mg L 1 (Tukey s, p 0.01) but was not significantly affected at concentrations of 0.4 mg L 1 (Tukey s, p 0.14) and 0.8 mg L 1 (Tukey s, p 0.10) (Fig. 4). Daphnia raised in any concentration of [bmim]bf 4 had significantly lower total number of neonates (F 5, , p 0.01), number of first-brood neonates (F 5, , p 0.01), and average number of neonates (F 5, , p 0.01) than did controls (Figs. 2 4). However, no differences were found among [bmim]bf 4 concentrations, exclusive of controls, for these traits (Tukey s, p 0.05). The IL [bmim]cl significantly affected Daphnia total neonates (F 5, , p 0.01), first-brood neonates (F 5, , p 0.03), and average number of neonates (F 5, , p 0.01). Total neonates differed between controls and all other concentrations (Tukey s, p 0.01) (Fig. 2) and were significantly lower in 1.6 and 3.2 mg L 1 than in 0.2 and 0.4 mg L 1 treatments (Tukey s, p 0.04 for all comparisons). The highest concentration of [bmim]cl (3.2 mg L 1 ) produced Fig. 2. Total number of neonates produced by single Daphnia magna over a 21-d period exposed to different concentrations of ionic liquid. Different letters represent significant differences between treatment concentrations (Tukey s multiple-comparison test, 0.05). bmim 1- butyl-3-methylimidazolium.

4 90 Environ. Toxicol. Chem. 24, 2005 R.J. Bernot et al. Fig. 3. Number of neonates in the first brood of single Daphnia magna exposed to different concentrations of ionic liquid. Different letters represent significant differences between treatment concentrations (Tukey s multiple-comparison test, 0.05). bmim 1-butyl-3-methylimidazolium. significantly fewer first-brood neonates than did the 0.2 mg L 1 [bmim]cl treatment (Tukey s, p 0.01) (Fig. 3) but did not differ from controls (Tukey s, p 0.21) or other treatments (Tukey s, p 0.10 for all comparisons). Average brood size was significantly reduced in the presence of any concentration of [bmim]cl relative to controls (Tukey s, p 0.01) (Fig. 4), and the highest concentration resulted in the lowest average brood sizes (Tukey s, p 0.01). The IL [bmim]pf 6 did not significantly affect total neonates (F 5, , p 0.10) and first-brood neonates (F 5, , p 0.06), but average brood size was largest for controls (F 5, , p 0.01). Salts with a sodium cation (NaPF 6 and NaBF 4 ) generally affected Daphnia reproduction at higher concentrations. Total number of neonates (F 5, , p 0.01), first-brood neonates (F 5, , p 0.01), and average brood size (F 5, , p 0.01) differed between controls and some of the higher concentrations of NaBF 4 and NaBF 4, with each trait generally declining with increasing IL concentration. DISCUSSION The present study represents, to our knowledge, the first toxicological tests of ILs on aquatic eukaryotes. Imidazoliumbased ILs were more toxic to D. magna than were Na -based salts. Acute toxicity (i.e., LC50) occurred at concentrations comparable to those of phenol, tetrachloromethane, and trichloromethane but at lower concentrations than those of benzene, methanol, and acetonitrile (Table 3). However, imidazolium-based ILs were less toxic than other common chemicals, such as chlorine and ammonia (Table 3). Lethal concentrations for the four ILs tested were similar (Table 1) despite their having different anions, indicating that the anion does not influence toxicity relative to bmim. Sublethal effects on Daphnia life-history traits occurred at IL concentrations an order of magnitude lower than concentrations eliciting acute effects, indicating the potential for reductions in population vital rates at low IL concentrations. The mechanisms of IL toxicity to Daphnia are unknown, but potential modes of action include enzyme inhibition [18,19], disruption of membrane permeability [5], and structural DNA damage [18]. For other chemicals, enzymatic inhibition is most commonly cited as the toxic mode of action on Daphnia [19,20]. In natural systems, several algal taxa produce toxins that can affect zooplankton, the metabolites of which are feeding deterrents [20], protease inhibitors [19], and direct toxins [21]. Additionally, imidazolium compounds or metabolites may be lipid permeable, potentially entering cells and disrupting normal cellular function or oxidizing and causing DNA damage [5]. However, low K ow values [15] indicate low lipid permeability of these ILs, which also makes bioaccumulation unlikely. Bacteria potentially able to break down imidazolium into different metabolites may be inhibited by the IL itself [22]. However, IL antimicrobial activity is dependent on alkyl chain lengths, with longer alkyl chains showing greater antimicrobial activity [22]. The bmim cation used in the present study has relatively short alkyl chains. Compared to Na salts, imidazolium ILs have a greater likelihood of harm-

5 Ionic liquid toxicity Environ. Toxicol. Chem. 24, Fig. 4. Average number of neonates per brood produced by single Daphnia magna over a 21-d period exposed to different concentrations of ionic liquid. Different letters represent significant differences between treatment concentrations (Tukey s multiple-comparison test, 0.05). bmim 1-butyl-3-methylimidazolium. ing Daphnia and related organisms through multiple modes of action because of their aromatic ring and range of potentially harmful metabolites [5]. Dose response approaches to estimating the lethal effects of toxicants on organisms have been criticized for lacking real ecological meaning [16,23]. Nonetheless, regulatory norms have been built around LC50 values that can be compared across toxicants and organisms [17,23]. Thus, LC50 values from dose response bioassays necessarily become starting points for more ecologically relevant studies of toxicant effects Table 3. Acute toxicity (48-h LC50) to Daphnia magna for ionic liquids and common chemicals used in manufacturing and disinfection a Chemical name Chlorine Ammonia (NH 3 ) Imidazolium ionic liquids Phenol Trichloromethane Tetrachloromethane Benzene Methanol Acetonitrile Acute toxicity (mg L 1 ) ,289 3,600 a Arranged in order of declining toxicity. Reference [30] [31] Present study [32] [33] [33] [34] [35] [36] on populations [24,25], communities [26], and ecosystems [27]. The acute and chronic data of the present study provide baseline information needed to develop models of IL effects on ecological systems. Total neonates produced were generally greater in tests with Na -based salts than in tests with imidazolium (Fig. 2), but controls also produced more neonates in Na tests at consistent resource levels. Average brood size was similar in controls with those in [bmim]cl, [bmim]pf 6, NaBF 4, and NaPF 6 treatments, indicating that we may have used a more productive clone of D. magna in these tests. Clonal variation in Daphnia toxicity bioassays has been cited as a reason for low reproducibility of Daphnia toxicity tests across laboratories and investigators [28]. Thus, generalizations about IL effects on absolute values of Daphnia life-history traits should be made with caution. Nonetheless, the chronic effects of imidazolium ILs on Daphnia occurred at concentrations an order of magnitude lower than the effects of Na -based salts. Ionic liquids have been known since 1914 [1] and were conceived as a useful addition to the solvents that are available for synthetic chemistry [1]. Only recently, however, have ILs been studied intensively for their practical applications [29]. Whereas ILs are being touted as green chemicals because of their low volatility and subsequent improvements in air quality compared to industrial solvents [3], more information is needed to assess their potential impacts on aquatic environments.

6 92 Environ. Toxicol. Chem. 24, 2005 R.J. Bernot et al. Acknowledgement We thank J. Brennecke and A. Sudhir for providing ILs and E. Kennedy, M. Henrick, and J. Brenay for laboratory assistance. Valuable input to our studies was provided by J. Brennecke, D. Lodge, C. Kulpa, R. Gillespie, and P. Landrum. Funding was provided by the National Oceanic and Atmospheric Administration and a U.S. Department of Education Graduate Assistance in Areas of National Need Fellowship to M.A. Evans-White (P200A010448). REFERENCES 1. Welton T Room-temperature ionic liquids. Solvents for synthesis and catalysis. Chem Rev 99: Allen D, Sinclair-Rosselot K Pollution Prevention for Chemical Processes. John Wiley, New York, NY, USA. 3. Freemantle M Eyes on ionic liquids. Chemical Engineering News 78: Chang K With a splash of salt, industry may reap environmental advantages. 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