Ibrahim Juneidiab,
Maan Hayyan*bc and
Mohd Ali Hashimab
aDepartment of Chemical Engineering, University of Malaya, Kuala Lumpur 50603, Malaysia
bUniversity of Malaya Centre for Ionic Liquids (UMCiL), University of Malaya, Kuala Lumpur 50603, Malaysia. E-mail: maan_hayyan@yahoo.com; maan@um.edu.my
cDepartment of Civil Engineering, University of Malaya, Kuala Lumpur 50603, Malaysia
First published on 25th September 2015
This study presented the toxicological and biodegradable assessment of different cholinium-based deep eutectic solvents (DESs). They were formed from choline chloride (ChCl) and N,N-diethyl ethanol ammonium chloride (EAC) as salts and four hydrogen bond donors, namely ethylene glycol (EG), glycerol (Gly), urea (U), malonic acid (MA), in addition to a metal salt, i.e. zinc chloride (ZnCl2), and a hydrated metal salt, i.e. zinc nitrate hexahydrate (ZnN). The toxicity towards Aspergillus niger of pure and aqueous DESs was evaluated by observing the inhibition zone using an agar well diffusion assay, and the minimum inhibition concentration (MIC) using a broth dilution assay, respectively. The MIC values of the DESs varied from 1 to 650 mg mL−1, whereas the inhibition zones changed according to the DES dose amount. Another test for acute toxicity was performed by evaluating the lethal concentration at 50% (LC50) of the same DESs on Cyprinus carpio fish. The LC50 of DESs ranged from practically harmless (e.g. ChCl:EG-DESaq) to highly toxic (e.g. EAC:ZnCl2-DESaq). The toxicity profile of the DESs depended on their concentration, type of individual components, and interaction with living organisms. Moreover, the DESs recorded higher toxicity compared to their individual components on fungi. However, lower toxicity was found for the DESs tested on Cyprinus carpio. Types I (organic salts and metal salt) and II (organic salt and hydrate metal salt) eutectics exhibited significantly higher toxicity than type III (organic salts and HBD). This was due to the presence of the innate toxicity of the metal salts. The biodegradability was appraised by a closed bottle test in which all the DESs were found to be readily biodegradable. To the best of our knowledge, there are no previous studies reported regarding the toxicity of cholinium-based DESs on freshwater fish or fungi and the biodegradability of EAC-based DESs. Therefore, this investigation can be used as a benchmark for future development of DESs.
DESs and ILs share a few interesting characteristics (low melting point, low volatility, high thermal stability, and high solubility).4 This has led to some authors to consider them as a new generation of ILs.5,6 However, ILs carry some limitations which are higher cost (around 10 times higher than organic solvents), possess similar or higher toxicity than organic solvents, and has generally low biodegradability.7 On the contrary, due to its low-cost, biodegradable, and environmental-friendly starting material, DESs are believed to be more effective and environmental-friendly.8,9 Cholinium based DESs (which is also known as choline) are receiving considerable attention as a solvent for many processes and applications such as catalysis, electrochemistry,10 material chemistry, nanomaterials,11 biomass treatment and enzyme-catalysed reactions.1,4,12–14 N,N-Diethyl ethanol ammonium chloride (EAC) and 2-hydroxy-N,N,N-trimethylethanaminium chloride known as (choline chloride) (ChCl)-based DESs were previously reported in different studies along with their physical properties.15–18 Most of the earlier studies focused on ChCl-based DESs. However, EAC as salt has superior advantages compared to other salts. It was proven that maximum solubility of NaCl was achieved in some of EAC-based DESs.19 The densities, refractive indices, viscosities, electrolyte applicability, electrical conductivity and other physical properties for different cholinium-based DESs (e.g. glycerol and ethylene glycol) were well studied and explained.20–22
Several researchers reported the green characteristics of DESs. Their assumption was based on the pharmaceutically acceptable profile that was reported in the material safety data sheets individual components.23,24 However, the first studies on the toxicity of DESs conducted by our group have proved DESs exhibit some toxic profile.25,26 It was found that phosphonium-based DESs possessed some antibacterial activity and cytotoxicity towards Brine shrimp and different strains of bacteria. In addition, it was found that the toxicity of the DESs tested was much higher than their individual components on Brine shrimp.25 However, no inhibition was observed for ChCl-based DESs on all of the bacteria strains tested. Later, Wen et al.27 studied the toxicity of ChCl and choline acetate (ChAc) as the salt, and urea (U), acetamide (A), glycerol (Gly), ethylene glycol (EG) as the HBDs on E. coli, garlic, and Hydra. The tested DESs showed inhibition of the bacteria due to their interactions with bacterial cellular membranes. Moreover, the effect of DESs on fish and human cell line were evaluated using three types of ChCl-based DESs.28 The cytotoxicity of the DESs tested ranged from low to moderate, while the results on wheat phytotoxicity implied that the DESs tested are non-toxic with seed germination. Recently, another study of our group29 on the toxicity and cytotoxicity in vitro cell lines and in vivo animal model of ChCl-based DESs with glycerine (Gl), EG, TEG, and U as the HBDs was conducted. It was found that the cytotoxicity effect of DESs was varied depending on the types of cell lines. However, both cytotoxicity and selectivity can be influenced by different combinations of salt/HBD and their molar ratio. Cvjetko Bubalo et al.30 reported that the percentage of yeast cells that survived for 24 h within aqueous ChCl:EG-DES are almost identical to those within a conventional buffer. So far, only two studies have tested the biodegradability of DESs where both focused on ChCl-based DESs. However, different results were obtained. Radošević et al.28 reported that all DESs tested (ChC:Gly, ChCl:Glc, ChCl:OA) were classified as (68–96%) readily biodegradable. Meanwhile, Wen et al.27 found that only two DESs (ChCl:U and ChCl:A) were biodegradable among the group tested.
The common freshwater fish, carp (Cyprinus carpio) is a typical cyprinid which can tolerate low oxygen levels for extended periods of time. It was used before in numerous ecotoxicological studies on different materials and was suggested by OECD 203 fish: acute toxicity test.31,32 Cyprinus carpio fish are important consumers in the aquatic food chain and play a vital role in keeping the balance of the aquatic ecosystem.32 The toxicity assessment DESs on Cyprinus carpio in particular is very useful for future involvement of DESs in the industry. Cyprinus carpio is the largest freshwater teleost family33 and most extensively cultured fish species in Asia with 70% total freshwater aquaculture production.34 The fungi strain Aspergillus niger (A. niger) was selected based on their common usage in fermentation processes, enzyme production, and biochemical applications.35,36 Lately, choline based DESs were considered to be enzyme activators and stabilizers. Cvjetko Bubalo et al.30 found that cholinium-based DESs are promising candidates for green whole-cell biocatalysis. Therefore, providing the toxicity assessments of DESs on these particular fungi strain may open the door for future applications using DESs as solvents in fungi biofermentation processes and enzyme production.
Based on the aforementioned, the toxicity and biodegradability of DESs should be studied in order for DESs to be safely implemented in industrial applications.26,37 This study was conducted to evaluate the toxicity and biodegradability of cholinium-based DESs. The tested DESs are presented in Tables 1 and 2 with their individual names, structures, and abbreviations. The DESs ratios selected are the most common ratios reported in literatures and used in diverse applications.11,15–19,38,39
DES name | Molar ratio |
---|---|
ChCl:EG | 1:2 |
ChCl:Gly | 1:2 |
ChCl:U | 1:2 |
EAC:EG | 1:2 |
EAC:Gly | 1:2 |
EAC:MA | 1:1 |
EAC:ZnN | 1:1 |
EAC:ZnCl2 | 1:2 |
Smith et al.37 presented a new classification for all existed DESs and divided them into four main categories which are:
• Organic salts and metal (type I).
• Organic salt and hydrate metal salt (type II).
• Organic salt and HBD (type III).
• Metal salt with HBD (type VI).
According to this classification, the tested DESs represent three out of four types. EAC:ZnCl2 is a combination of metal and organic salt, EAC:ZnN is a combination of hydrate metal salt and organic salt, while the rest are a combination of organic salts and HBDs. It is important to clarify that the toxicity of DESs were assessed by aqueous and non-aqueous (pure DES) systems. Considering water dilution may lead to a partial destruction of the DESs structure, we differentiated the designations given for each system. Pure DES, denoted as e.g. (EAC:Gly-DES) and aqueous DES denoted as e.g. (EAC:Gly-DES)aq. The antifungal activity for pure DESs was determined by measuring the inhibition zone diameters using agar well diffusion method. Meanwhile, aqueous DESs toxicity was determined by evaluating the minimum inhibitory concentration (MIC) using broth dilution method. The toxicity on freshwater organisms was assessed by calculating the 50% of the lethal concentration (LC50) on Cyprinus carpio fish and the biodegradability using wastewater microorganisms in a closed bottle test. The data obtained would expand knowledge about the environmental fate of DESs and could be beneficial to design a more environmentally friendly and biodegradable solvent.
Reconstituted water was distributed equally (10 L) to each container (static system). The testing was conducted in three phases.
Phase 1: single addition of each individual component to the water aquarium, denoted by their abbreviations, Table 1.
Phase 2: simultaneous addition of the two components in the same DESs ratio, denoted as e.g. (EAC:EG-Mix)aq.
Phase 3: direct addition of the pure formed DESs, denoted as e.g. (EAC:EG-DES)aq.
Different range of doses was added for each substance in order to cover the maximum and minimum lethal doses. The tested DESs and their mixture components were equally measured according to the DES ratio. The solution was then mixed properly to ensure the solubility of the DESs. Saturated oxygen level was provided through a central air pump, which distributed oxygen equally through pipes to each container. The pH of the solution was measured accordingly at 1 h, 24 h, 48 h, 72 h and 96 h, and before the fish is added to each container (ten fishes). In addition, a temperature control system was provided to keep the temperature within the preferable range (20 °C–24 °C). Oxygen concentration was maintained above 90% of the saturation. The containers were kept under daylight for 16 h and were not fed during the study. Finally, controlled blank containers were added to the test. Mortalities were recorded at 1 h, 24 h, 48 h, 72 h and 96 h and subsequently removed from the container. The total number of mortalities after 96 h at different DESs concentrations was used to calculate the LC50 using US-EPS's Probit analysis program. The toxicity of the DESs toward Cyprinus carpio was represented by LC50 values which indicate the concentrations that kill 50% of the population. The results were referred to the aqueous solutions of the DESs.
Solvent | MIC mg mL−1 |
---|---|
a The minimum inhibitory concentration (MIC) expressed as the mean value of three replicates ± SD (mg DES mL−1 broth). | |
ChCl:EG-DESaq | 325.3 ± 34 |
ChCl:Gly-DESaq | 550.4 ± 51 |
ChCl:U-DESaq | 138.5 ± 23 |
EAC:EG-DESaq | 314.8 ± 44 |
EAC:Gly-DESaq | 495.4 ± 63 |
EAC:MA-DESaq | 64.4 ± 14 |
EAC:ZnN-DESaq | <2.2 |
EAC:ZnCl2-DESaq | <1.3 |
ChCl | 628.2 ± 54 |
EAC | 614.6 ± 41 |
EG | 322.7 ± 32 |
Gly | 534.1 ± 71 |
U | 126.1 ± 24 |
MA | 31.2 ± 8 |
ZnN | <2.9 |
ZnCl2 | <1.36 |
It is worth to mention that at low concentrations (8–45 mg mL−1) of ChC:U-DESaq and ChCl:Gly-DESaq, higher A. niger spore growth compared to the blank was observed virtually. This indicates that these DESs might be used as a supplementary material for fungal growth. Studies52,53 reported that U can be used as a nitrogen source and fertilizer for fungi, incorporating it into the cell through specific transporters. In addition, the same studies mentioned that some mould fungi such as Chrysosporium keratinophilum favour growth in the presence of U. Nevertheless, an inhibition was observed as the concentration increased beyond 400 mM which was also detected in this study for all of the DESs tested and its components.
In order to have a better analysis for this study, the MIC values were investigated for some of the individual components of DESs, namely ChCl, EAC, EG, G, MA and U, and compared to their formed DESs. ChCl and EAC possess very high MIC values, which means a very low toxicity. These results corresponds to a previous study for ammonium salt on A. niger fungi which reported that the MIC value was above the range tested (800 μmol L−1).54 DESs possess anti-fungal property higher than their individual components. For instance, the MIC was 322.7 mg mL−1 for EG and 628.2 mg mL−1 for ChCl, whereas for the formed DES ChCl:EG-DESaq was 325.3 mg L−1. This is in line with DESs toxicity on E. coli reported earlier.27 It was found that DESs exhibited higher inhibition index at different concentrations compared to their individual components. This phenomenon could be due to the charge delocalization through the hydrogen bonding between the DESs components, and it was found that chemicals with delocalized charges are usually more toxic than those with localized charges.25,27,55 Moreover, the HBD might denature the proteins in a living cell.56 The relatively high toxicity of EAC:MA-DESaq is due to their acidity (pH 4.14), as the concentration of DESs increase the solution becomes more acidic which is an unfavourable environment for A. niger growth. It was observed that EAC:ZnCl2 and EAC:ZnN possess very high toxic effect by presenting the lowest MIC (i.e. <2.0 mg mL−1). This result could be ascribed to the innate toxicity of the metal salt which make types I, II, and IV DESs more toxic than type III which contains amides and polyols such as U, Gly, EG.57 EAC:ZnCl2 and EAC:ZnN made up of organic salt (EAC) and metal (ZnCl2)/hydrate metal (ZnN). Their combination resulted in ionic interaction, which exhibits a high toxic effect on the cell surface, inhibition of respiration growth and germination of spores.58 The same study has reported that the MIC for zinc on A. niger and E. coli less than to 0.5 mM.59
Considering partial destruction of the DESs structure, agar well diffusion test for pure DESs was also conducted. The results presented in Table 4. Agar well diffusion assays also confirmed the results obtained in broth dilution method. The activity of DESs was found to follow the same toxicity pattern. EAC:ZnCl2-DES exhibited the highest toxicity with strong 18 ± 4 mm inhibition zone diameter at the lowest DES dose tested (10 mg). This is followed by EAC:ZnN-DES and EAC:MA-DES with 11 ± 2 and no inhibition at the same concentration, respectively. The ChCl:U-DES showed no inhibition for the doses between 10 to 150 mg. However, inhibition of 14 ± 2 mm was observed at 200 mg. On the other hand, the least antifungal activity was exhibited by ChCl:Gly-DES and EAC. The inhibition zone was only observed after addition of 300 mg dose of their formed DESs.
Amount (mg) | Zone of inhibition in millimeter (mm)/agar disc | ||||||
---|---|---|---|---|---|---|---|
10 | 50 | 100 | 150 | 200 | 300 | 400 | |
a Antifungal activity detected as the mean zones of inhibition ± SD of three replicates in millimeter (mm), No IN = no inhibition, Full IN = full inhibition, N.D = not determined. | |||||||
ChCl:EG-DES | No IN | No IN | No IN | No IN | No IN | 10 ± 3 | 28 ± 4 |
ChCl:Gly-DES | No IN | No IN | No IN | No IN | No IN | 4 ± 1 | 20 ± 4 |
ChCl:U-DES | No IN | No IN | No IN | No IN | 14 ± 2 | 25 ± 3 | N.D |
EAC:EG-DES | No IN | No IN | No IN | No IN | No IN | 14 ± 4 | 33 ± 5 |
EAC:Gly-DES | No IN | No IN | No IN | No IN | No IN | 7 ± 3 | 24 ± 2 |
EAC:MA-DES | No IN | 8 ± 1 | 14 ± 3 | 22 ± 3 | 29 ± 4 | 37 ± 4 | N.D |
EAC:ZnN-DES | 11 ± 2 | 25 ± 4 | 36 ± 5 | Full IN | N.D | N.D | N.D |
EAC:ZnCl2-DES | 18 ± 4 | 31 ± 5 | Full IN | Full IN | N.D | N.D | N.D |
In our previous studies E. coli as bacterium was used together with some other bacteria strains for testing the toxicity of some DESs, ChCl-based DESs showed no inhibition to all bacterial strains tested (E. coli, S. aureus, P. aeuriginosa, and B. subtilis).26 However, some inhibition was observed for phosphonium-based DESs.25 Wen et al.27 reported the toxicity of cholonium-based DESs on E. coli. similar to the finding of this study on both fungi strains. They observed that DESs toxicity increased on E. coli as the concentration increased which indicated that the toxicity of DESs towards fungi was due to higher concentrations.60 There were two common DESs used in the study of Wen et al.,27 i.e. ChCh:U and ChCl:EG, with determined LC50 on E. coli 295.9 mM and 532 mM, respectively. However, these results were not comparable with our findings due to the different organisms used. Nevertheless, both studies shared a similarity in which the ChCl:EG was reported as one of the least toxic DESs toward E. coli and A. niger.
It can be observed from the obtained results that most DESs showed no inhibition at low concentrations. As a result, it was necessary to add relatively large doses of some DESs to expose their antifungal activity. In consistence with this finding, Cardellini et al.61,62 reported the first toxicity studies of pure DESs toward yeast fungi strain. They evaluated the toxicity of some novel DESs on Saccharomyces cerevisiae cells via an FTIR-bioassay, the DESs studied composed of zwitter ionic trimethylglycine and carboxylic acids as well as mixtures of (1S)-(+)-10-camphorsulfonic acid (CSA) and differently structured sulfobetaines (SB). The results indicated the green profile of the tested DESs. They proved that the toxicity resulted was due to the high concentration of these compounds which led to dehydrating effect on the cells. The normalized spectra from cells treated with DESs and CaCl2 (dehydrating agent known not to be toxic) were found to be almost identical.
The results obtained in this study are comparable with other studies on IL toxicity using different strains of fungi as a model test. Petkovic et al.43 used filamentous fungi to evaluate the toxicity of different ILs. Similar to the MIC values for most of the DESs tested, ILs showed active growth in media containing relatively high IL concentrations, up to 2 M for cholinium chloride alkanoates.63 The antifungal activities of eight yeasts and filamentous fungi were evaluated for imidazolium-based IL. Some of them showed no inhibition at the highest concentration screened (i.e. 2.0 mM).64 IL proved to have toxic effects on the fungi and different types of bacteria. The antimicrobial activity is governed by the molecular structure of each IL.42 In general, the antimicrobial activity of IL increases with the extension of substituent alkyl chain.63 Consequently, lipophilicity was increased. In general, primarily, imidazolium, pyridinium, and quaternary ammonium-based head groups are the most antimicrobial IL reported.65
Aspergillus nidulans canidia was used as a model test for the toxicity of alkyl tributyl phosphonium chloride.43 It was found that the toxicity occurred as a result of the interaction with the plasma membrane and the cell wall. In addition, the availability for a chemical uptake by the tested species depends on both the species tested and the chemical form and mobility.59 It might be difficult to explain the toxicity mechanisms of DESs on fungi. However, in general, the major toxicity mechanism often results in strong coordinating abilities, which will cause several effects, such as blocking biologically functional groups, interaction with the membrane structure, disruption of cell wall, and denaturation of enzymes. Consequently, every aspect of fungi growth and metabolism will be affected.66 More specifically, the mechanism of DESs toxicity towards yeast fungi was studied by Cardellini et al.61,62 They found that DESs challenged the cells by inducing dehydrating effects similar to that produced by CaCl2. This indicated that biological action for their tested DESs is rather independent of the compound structure. On the other hand, consistent correlation between the toxicity of the pure and aqueous DESs and their individual components were observed in our study, indicating that different types of DESs can affect the cell growth in different ways.
Fig. 1 shows microscopic investigation for MIC sample (3700 mM, 325.3 mg L−1) and the lower concentration sample (3600 mM) was conducted using the light microscope (Leica DM1000 LED) to ensure the inhibition effect. Fig. 1B represents the microscopic image of the sample taken from the (3600 mM) tube where the fungi show growth and colony formation. Fig. 1C represents the (3600 mM, 316.5 mg L−1) tube where no growth or colonies can be observed.
Fig. 2 LC50 of aqueous DESs, their mixtures and individual components determined on Cyprinus carpio fish in water with 95% CI error bar. |
The investigated aqueous DESs ranged from slightly toxic LC50, 10–100 mg L−1 to relatively harmless LC50, >1000 mg L−1 according to the acute toxicity rating scale by Fish and Wildlife service (FSW) (see Table 5). EAC:ZnCl2-DESaq was the most toxic (LC50, 33.9 mg L−1) followed by EAC:ZnN-DESaq (111 mg L−1), while EAC:MA-DESaq showed slightly lower toxicity (LC50, 190 mg L−1). However, DESs formed by EG and Gly as HBDs (ChCl:EG-DESaq, ChCl:Gly-DESaq, EAC:EG-DESaq and EAC:Gly-DESaq) were relatively harmless, obtaining an LC50 range from 5400 to 12500 mg L−1. A moderate toxicity was exhibited by ChCl:U-DESaq (LC50, 5310.47 mg L−1). The effective concentration was higher (less toxic) for ChCl-based DESs than EAC-based DESs.
Relative toxicity | Aquatic LC50 (mg L−1) |
---|---|
Super toxic | 0.01–0.1 |
Highly toxic | 0.1–1 |
Moderately toxic | 1–10 |
Slightly toxic | 10–100 |
Practically nontoxic | 100–1000 |
Relatively harmless | >1000 |
Interestingly, all of the aqueous DESs (phase 3) were clearly less toxic on Cyprinus carpio than their individual components (see Fig. 2). The individual components mixtures (phase 2) of the DES solutions were prepared by adding them simultaneously to the distilled water. They showed higher toxicity in all of the DESs tested, for example, the LC50 for ChCl:EG-DESaq is 7342.4 mg L−1 whereas ChCl:EG-Mixaq is 4974.1 mg L−1. Other DESs followed the same pattern as well. The difference in toxicity between DESs and their individual components could be attributed to the hydrogen bonding or the complexation created after the formation of DESs. This is in agreement with previous studies for ChCl:Uaq toxicity on the growth of Hydra and garlic roots.27,67 It was found that Hydra lived longer inside the DES solutions compared to its mixture solution or individual components. This is similarly observed for the growth of garlic roots. The higher toxicity of the components mixture solution was ascribed to the salt but this effect was weakened by the extensive hydrogen bonding network after DESs formation, in which the hydrogen bonding network throughout the DES was sufficiently strong to prevent the DESs from dissociation in aqueous solution.27,68 It was also reported that the hydrogen-bond network in DESs lowers the chemical potential of the components of DESs and thereby affected the toxicological profile.25
In this study, the salts and HBD or metal salts were tested separately. Both ammonium salts showed very benign behaviour whereas metal salts or HBD showed higher toxicity in all cases. This indicated that the toxicity shown by the tested DESs was ascribed to the metal salts or HBDs. However, the strong association during the formation of DESs decreased their reactivity, making them inert in most cases, and thus, decreased their toxicity.69 The LC50 for ZnCl2 and ZnN as individual materials, their DESs and aqueous solutions were very low, ranging from 27–111 mg L−1. Zinc is known as a heavy metal and most of its derivatives are very toxic. Many studies reported that the toxicity of zinc compounds is harmful to fish such as the rainbow fish and other organisms.70–72 However, the pH of the solvent plays a vital role as well. It was observed that EAC:MA-DESaq possesses a high acidity value (pH, 4.14). Consequently, it possesses a relatively low LC50. According to literature, a pH of 6–8 is the preferable range for C. carpio growth.
Up to date, there is no specific investigation on the toxicity of cholinium-based DESs on fish. Therefore, the comparison of our results with the literature data is limited. However, some investigations were carried out for DESs using different marine organisms such as Vibrio fischeri,73 Brine shrimp25 and Hydra.27 Other toxicity studies were carried on E. coli bacteria, garlic, and cell line toxicity.29,73 However, several attempts to justify the ecotoxicity were provided on conventional IL and cholinium salts by several research groups using Daphnia magna as the model test.74,75 A similar conclusion was reported. Firstly, the ILs ecotoxicity varied widely with different organisms across the trophic levels in which the fish are less sensitive to ILs toxicity.76,77 Secondly, the chemical structures of ILs anion and cation core may cause completely different effects. For example, the cholinium cation has higher toxicity compared to other quaternary ammonium,76,78 and a clear contribution from anions was noticed on algae toxicity (Scenedesmus vacuolatus).77 Thirdly, many researchers reported that the extension of the cation alkyl side chain is responsible for the increment on the ecotoxicity until a certain number of carbons.76,79,80 However, it is noteworthy that the functionalization of cation side chain decreases the toxicity of ILs. Another group reported that the longer alkyl chain of the imidazolium ring of a more electronegative atom drastically reduces the acute toxicity of Daphnia magna.73 As a result, it can be concluded that, similar to ILs, the toxicity of DESs highly depended on the salt and HBD in which it will most likely result in a toxic DES. However, the strong hydrogen bonding in DESs has a positive effect of lowering their toxicity by incorporating the components into eutectic mixtures and making them inert rather than by physical mixing (aqueous) which showed almost no change in their toxicity. This is in agreement with a study conducted on the Hydra where it lived longer inside DESs compared to their individual components.27,68,81
Similar conclusion was reported by Gork et al.14 and Lindberg et al.82 to explain the positive enzyme activity of ChCl-based DESs despite the presence of chloride and hydrogen bonding. They hypothesized that strong hydrogen bonds between DES components lowered their reactivity which helped in dampening their toxic effects. The DESs were found to possess higher toxicity than their individual components toward fungi. Similar results were observed earlier by Wen et al.27 for E. coli bacteria. In contrast, our results showed that DESs possess lower toxicity than their individual components on the Cyprinus carpio fish, which was the same behaviour observed earlier on Hydra.68
The higher toxicity of the aqueous solution can be ascribed to the existence of cholinium salts, but incorporating it into DESs can weaken its toxicity by the establishment of an extensive hydrogen bonding network throughout the DES. This network is sufficiently strong to prevent the DESs from a serious dissociation in aqueous solution. On the other hand, the cytotoxicity for ChCl and phosphonium-based DESs were found to be higher than their individual components on Brine shrimp and bacteria.25,26 Later, Radošević et al. confirmed this hypothesis by comparing the EC50 values determined for ChCl-based DESs with their individual compounds (e.g. ChCl and oxalic acid) on the CCO fish cell line and MCF-7 human tumour cell line. Both authors attributed this behaviour to the charge delocalization occurring through hydrogen bonding which makes the DESs more toxic compared to their components. Therefore, DESs and their individual components exhibit different toxicological profile based on the method followed and tested organisms, in which each system exhibits different response, and the results, cannot be generalized over other systems or organisms.
The use of DESs in the biological or biochemical applications is common with aqueous solutions. Therefore, information on lethal concentration and minimum inhibitory concentration of DESs in aqueous medium is essential. For instance, in enzyme catalysed reactions, DESs can be applied as additive and co-solvent for lipase hydrolysis in aqueous medium. This can potentially be a stepping stone for further application of DESs as solvent for enzyme productions, in which non-toxic DESs can be used as solvents for fungi biotransformation process in the future.
As shown in Fig. 3, the biodegradability was more than 60% for all of the tested DESs in water, therefore all of them can be referred to as ‘readily biodegradable’. The highest biodegradability is shown by ChCl:Gly-DESaq (91%) followed by ChCl:U 85%. The lowest result was observed with EAC:MA-DESaq (61%). ChCl-based aqueous DESs were more biodegradable (77–91%) in comparison to EAC-based except for EAC:ZnN-DESaq (80%). Comparing among the HBDs, Gly, U, and EG were more liable for biodegradation than ZnCl2 and MA. The biodegradability of the conventional IL [BMPyr][NTf2] was tested. Most of the DESs showed either similar or higher biodegradability except for EAC:ZnCl2-DESaq and EAC:M-DESaq.
The high biodegradation level for the DESs tested can be credited to the ammonium salts and HBDs that form these DESs. ChCl is readily biodegradable. According to OECD-criteria (MITI-I test; BOD measurements), it reached 93% within 14 days.28 Similar results were shown for different HBDs where 92% biodegradation was reported after 30 days for glycerol.84 In addition, the BOD5/COD ratio was 0.86 and the fact that it was greater than 0.5 further supports the ready biodegradability of glycerol. EG is readily biodegradable under both aerobic and anaerobic conditions in standard tests using sewage sludge.85 Urea is biodegradable on an average of 93–98% according to EPA.46 Moreover, MA is 86–87% biodegradable according to the Material Safety Data Sheet. The potential of cholinium-based solvents to be biodegradable was recently confirmed by Hou et al.78 They evaluated the biodegradability of 18 cholinium amino acid ILs via closed bottle tests and CO2 headspace tests using wastewater microorganisms.
Differences in aerobic biodegradability between ChCl and EAC-based DESs can be noticed. This could be accounted on the structures of ChCl and EAC (see Table 1). The presence of less carbon atoms of methyl groups (CH3) in ChCl might be more favourable for biodegradation than ethyl groups in EAC (C2H5).86,87 The structures of HBDs play an important role in the biodegradation level. A slight improvement in biodegradation can be noticed with the existence of the hydroxyl group, which was also reported previously in different ILs.87 For instance, due to the presence of three hydroxyl groups in glycerol, the level of biodegradation was marginally higher (ChCl:Gly-DESaq 91% and EAC:Gly-DESaq 74%) than that in EG with two hydroxyl groups (ChCl:EG-DESaq 77% EAC:EG-DESaq 70% respectively).28
A relatively high biodegradability was showed by ChCl:U-DESaq (85%) and IL, this is due to the carboxylic acid and amide groups which showed excellent degradability. These results are in agreement with Boethling's rules of thumb where the presence of carboxylate groups or hydrolysable bonds such as ester and amide generally increase the aerobic biodegradability.78 ChCl:MA-DESaq was just about the passing level for ready biodegradability (61%). This relatively poor biodegradation stemmed from its acidity after its addition to the mineral solution with pH of 4.14, which is not a favoured growth environment for all types of microorganisms.
Recently, only two studies have covered the biodegradability of DESs. However, they obtained contradicting results. According to Radošević et al.,28 all the tested aqueous DESs (ChCl:Gly, ChCl:glucose, and ChCl:oxalic acid) were readily biodegradable (68–96%). Moreover, ChCl:Gly-DESaq already reached 84% biodegradation level after 7 days. The author attributed the high level of DESs biodegradation to the components that form them which has been reported in many papers as readily biodegradable. In contrast, Wen et al.27 tested the biodegradability of 8 DESs combined from ChCl and choline acetate (ChAc) as the salt and U, Gly and EG as HBD with 1:1 molar ratio. The result showed that all the tested DESs were not readily biodegradable (less than 60%) except for ChCl:U and ChCl:A. This difference might be caused due to the different reaction of conditions used, different sources of the wastewater microorganisms obtained and different molar ratio of the DESs used.
Several studies reported on the biodegradability of ILs. Recently, Hou et al.74 studied the potential of 18 cholinium-based ILs as biodegradable solvents using the closed bottle test and CO2 headspace tests. It was found that all ILs tested were readily biodegradable (62–87%). In addition, choline as a cation was completely decomposed within 5 days. These results are in agreement with our DESs tested as ChCl and EAC are cholinium-based. Another study reviewed by Coleman and Gathergood showed that different ranges of biodegradation were based on the anion and cation structure and the existing functional groups.50
It is worth mentioning that the DESs tested are considered ‘readily biodegradable’. Previously, in classical ILs, a conflict between the toxicity and biodegradability always occurs; toxic ILs were found to be the most biodegradable whereas greener ILs pose lack of biodegradability.78 This conflict between the toxicity and biodegradability did not occur in this study. All the tested DESs were biodegradable regardless of their toxicological profile. The results of this study strongly indicate that DESs are sufficiently compatible as solvents to be implemented in industrial applications.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra12425e |
This journal is © The Royal Society of Chemistry 2015 |