Accepted Manuscript Title: The role of the anion in the toxicity of imidazolium ionic liquids Author: Robert Biczak Barbara Pawłowska Piotr Bałczewski Piotr Rychter PII: DOI: Reference:
S0304-3894(14)00197-6 http://dx.doi.org/doi:10.1016/j.jhazmat.2014.03.021 HAZMAT 15794
To appear in:
Journal of Hazardous Materials
Received date: Revised date: Accepted date:
3-12-2013 8-3-2014 10-3-2014
Please cite this article as: R. Biczak, B. Pawlowska, P. Balczewski, P. Rychter, The role of the anion in the toxicity of imidazolium ionic liquids, Journal of Hazardous Materials (2014), http://dx.doi.org/10.1016/j.jhazmat.2014.03.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The role of the anion in the toxicity of imidazolium ionic liquids Robert Biczaka, Barbara Pawłowskaa, Piotr Bałczewskia,b, Piotr Rychtera,* a
Jan Dlugosz University in Czestochowa, Institute of Chemistry, Environmental Protection and Biotechnology, 13/15 Armii Krajowej Av., 42-200 Czestochowa, Poland
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Corresponding author. Tel.: +48 343614918 int.195; fax: +48 343665322 E-mail address: [email protected] (P. Rychter).
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Center of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363 Lodz, Poland
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ABSTRACT
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From the environmental protection point of view, the growing interest of ionic liquids in various industrial branches has raised concerns for the toxicity assessment of these compounds. The paper discusses the effect of salts containing the shared 1-ethyl-3methylimidazolium [EMIM] cation as coupled with five different anions: bromide [Br], nitrate [NO3], p-toluenesulfonate (tosylate) [Ts], dimethylphosphate [dMP] and methanesulfonate [MS] on the growth and development of higher land plants – spring barley and common radish. The experiment was done according to the ISO Standard 11269-2:1995 and the OECD/OCDE Guide 208/2006. As the indications of phytotoxicity, the percentage of sprouts and the level of dry and fresh plant mass were used; in addition, the visual assessment of any signs of damage to the examined plant species, such as growth inhibition and chlorotic changes, was also made. Results of our study has proved the negative impact of ILs on the tested plants and the toxic effect of imidazolium salts was dependent primarily on the applied ionic liquids concentration. The common radish revealed the higher tolerance to the imidazolium as compared to spring barley. The anion type of ionic liquid was crucial for the toxicity against common radish. Keywords: ionic liquids, phytotoxicity, terrestrial plants, yield, dry weight 1. Introduction
The principle of “Green Chemistry” is the design of chemical products and processes that reduce or eliminate the use or generation of hazardous substances. Ionic liquids have been generating increasing interest over the last decade as an alternative for classic but often toxic reagents or solvents. Ionic liquids (ILs) make a very interesting class of chemicals, which principally have their melting point below 100 °C, which can be achieved by combining an asymmetric large-size cation with an anion of weak coordination properties. Thanks to their physicochemical properties, such as low vapour pressure, incombustibility, high thermal stability, high ionic conductivity and good catalytic properties, as well as almost perfect ability to dissolve organic and inorganic materials, ionic liquids have been finally used on a large scale in industry in recent years [1-4]. These unique properties of ionic liquids compared to classic solvents have enabled these compounds to be used in many branches of chemical industry, such as catalysis and 1 Page 1 of 25
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biocatalysis [5,6], extraction and separation [7,8] and electrochemistry [9], as well in the pharmaceutical and food industries and biotechnology [10,11]. Currently, research on ILs are also mostly focused on the possibility of using ionic liquids for controlling the growth of bacteria and fungi as well as for eliminating pathogenic microbes resistant to antibiotics and other medicines [12, 13]. These substances have been already used as additives for delayed-action drugs, medical products, including dressing materials, as well as disinfectant detergents [14]. ILs have found a great interest in medicine for controlling neoplastic cells in tumor therapy and the preliminary results of such research are promising [15-17]. Nevertheless, the most important reason for that wide use of ionic liquids in laboratories and industry is the possibility of modifying their individual physicochemical properties to meet the requirements of specific chemical reactions or entire technological processes. This can be achieved thanks to their virtually unlimited capabilities to exchange cations or anions – hence, these salts has been named designer solvents [18-20]. It seems highly probable that this feature will allow also the toxicity of ionic liquids to be reduced, which will result in such technologies that, on the one hand, will assure the optimization of industrial processes and, on the other hand, will eliminate or reduce to a minimum the undesirable effects of these chemical compounds on the natural environment [1]. However, at present, the increasing interest of the industry in ionic liquids leads to a rapid increase in their production and practical use, which in turn results in these substances getting into the environment. The low pressure of ionic liquid vapours was initially equated (wrongly, as it turned out later) with the lack of toxicity of these salts. Now it is known above all doubt that ionic liquids do exhibit toxicity and thus they may pose hazards to human health and the quality of the natural environment [2, 18-20]. Initial research on the ecotoxicity of ionic liquids was focused on the water environment due to the fact that the majority of ionic liquids exhibits almost perfect solubility in water. The obtained results have proved negative impact of the examined substances on many groups of aquatic organisms, including algae, water microorganisms, water crustaceans, mollusks and some vertebrates. In this reason the term of “ecological or green solvents” assigned to ionic liquids started to become doubtful [19-26]. The common use of ionic liquids in many branches of industry may create a huge amount of ILs wastes leading finally to their accumulation in soil, therefore behaviour of these substances especially in agriculture field seems to be crucial from the environmental protection and public health point of view. At present, thanks to their antistatic, antibacterial and fungicidal properties, ionic liquids are gradually becoming substitutes for traditional pesticides (bactericides, fungicides) or compounds used for wood preservation [14, 20, 27, 28]. Comprehensive studies are also conducted on the possibility of using ionic liquids as herbicidal substances and plant growth regulators, which would make it possible to eliminate many high-toxicity herbicidal compounds from use, which long linger in the soil [29-31]. Therefore, ionic liquids that will pass to the soil environment may become absorbed by soil colloids, which will not only have an impact on the development of the edaphon, but will also influence on the crops and the quality of plants grown on these soils, because the phytotoxicity may serve a good indicator of the hazardous nature of the ionic liquids [32]. With this respect, if for some reason ionic liquids contaminate the soil, they may be adsorbed by soil colloids affecting the development and growth and development upon both the plants and all living organisms of the soil surface [15, 18, 33-41]. The toxic impact of ionic liquids on environmental organisms is dependent on many factors such as their structure, alkyl chain length, concentration in environment or plant resistance. According to literature the structure 2 Page 2 of 25
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of cation of ionic liquids is the most important agent affecting their toxicity [34, 38, 39, 41]. The toxicity assessment of ILs with dimethylpiridinium, pyridinium, imidazolium, piperidinum, pyrrolidinium, morpholinium and ammonium cations containing the same side chain and halogens as a counterions against marine bacteria (Vibrio fischeri), algae (Scenedesmus vacuolatus) and duckweed (Lemna minor) has been reported by Stolte et al. [39]. They have noticed that the most toxic were ionic liquids containing aromatic cations as compared to quaternary ammonium salts and morpholinium cations. Toxicity results of ionic liquids containing aromatic cations were ambiguous, however, the highest toxic effect has been observed for the ILs with dimethylpyridinium cations. Toxicity of these compounds is also strongly correlated with the length of side chain and the more the carbon atoms in chain, the higher toxicity impact is observed [34, 37-41]. Matzke et al. [38] has assessed the acute toxicity of 1-alkyl-3-methylimidazolium bonded with six organic and inorganic anions against marine and land organisms living on various level of food chain including marine bacteria (Vibrio fischeri), algae (Scenedesmus vacuolatus), duckweed (Lemna minor), wheat (Triticum aestivum) and cress (Lepidium sativum). They have proved that regardless the bonded anion, the lengthening of alkyl chain negatively affect the viability of organisms. Results of ecotoxicity evaluation of 1-ethyl-, 1-buthyl- and 1-hexyl-3-methylimidazolium against cress conducted by Studzińska et al. [37] confirm fact, that the extent of toxicity of used ILs was dependent on the length of alkyl side chain and hydrophobic behavior of cations. However the phenomena of growing toxic impact with lengthening the side chain is still unclear. It is generally considered that the toxicity mechanism of ionic liquids due to their structure is similar to detergents, antibiotics and pesticides resulting in dysfunction of double lipid bilayer. Long, hydrophobic alkyl chains of ionic liquids easily interact with double phospholipid bilayer containing hydrophobic protein domains which may cause the damage and final lysis of cells [39, 41]. Described till now toxicity mechanisms of ionic liquids take a place mostly in water environment. However in soil, the degree of ecotoxicity of any chemical substances including ionic liquids is strongly dependent on soil sorption capacity and the longer alkyl chain of ILs the higher value of soil sorption is observed [40, 42]. Stepnowski et al. [42] proposed the model of double layer sorption with two partition coefficients where the first one is assigned to layer of ionic liquid directly adhered to surface of soil mineral via electrostatic forces and the second is assigned to outer layer already adhered via hydrophobic interactions. These results do not coincide with those obtained by Matzke et al. [18]. The phytotoxicity evaluation of 1-ethyl-, 1-buthyl-, 1-oktyl-3-imidazolium ionic liquids on the growth and development of cress and wheat has revealed that the degree of ILs toxicity increased with lengthening of side chain but only up to 4 carbon atoms. They state, that this is due to high mobility of ionic liquids with short side chain when compare to ILs with alkyl chain consisted of more than few carbon atoms. The mobility of solutes in soil facilitate the contamination of different layer of soil and ground water. This process is controlled by the flow velocity of the water and by interaction processes in the soil, as cation exchange reactions, solutionprecipitation reactions, content of organic matter, complex formation, biochemical reactions as well as amount of mineral colloids including kaolinite, illite or montmorillonite. 3 Page 3 of 25
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Organic matter is the most important fraction of the soil colloids composed of high molecular weight substances with intricate structure and specific properties. Since the humus is negatively charged colloid may easily bind imidazolium or piridynium cations of ionic liquids inhibiting their toxicity impact [18, 40, 43]. However till now there is a pure information in literature related to sorption of ammonium, phosphonium or pyrrolidynium quaternary salt and ILs anions are not absorbed by soil colloids in such conditions. As could be expected, increasing level or organic carbon in soil results in lowering toxicity of ionic liquids, but the outcomes related to relationship between the soil sorption capacity and the ILs colloids adsorption is still ambiguous [18, 36, 39]. Additional factors influencing on ionic liquids toxicity against terrestrial plants are specimen resistant, presence of other xenobiotics and as could be expected concentration of ILs in soil [15, 18, 33, 34, 37]. In phytotoxicity assessment of imidazolium ionic liquids against algae (Scenedesmus vacuolatus) and wheat seedlings (Triticum aestivum) Matzke et al. [44] have proved synergistic interaction of these ILs with cadmium cations. These results are in accordance with those obtained by Zhang et al. [45] where the synergistic influence of mixture consisted of tetrafluoroborate 1-benzyl-3-methylimidazolium ionic liquids with carbamate pesticides have been revealed. Since the soil is a living, dynamic ecosystem, the fate of chemical substances including the more and more popular newly synthesized ionic liquids must be constantly monitored. The literature concerned with comprehensive assessment of ionic liquids ecotoxicity is soil is scarced, therefore the near future study on relationship between toxicity of ILs and such soil parameters like acidity, moisture, colloids amount, granulometric composition or soil sorption capacity is required. The purpose of the presented study was to assess the toxic action of 5 imidazolium ionic liquids on higher land plants. The effect of the anion type on the phytotoxicity of the examined substances was also determined in the experiment.
2. Experimental 2.1.
Ionic liquids
The ionic liquids was purchased from Sigma-Aldrich Chemical Co. and was used as received without any additional purification: -1-ethyl-3-methylimidazolium bromide [EMIM][Br] ≥ 97.0% (T) ≥ 98.5% (HPLC/T), (impurities: ≤ 200 ppm water); 1-ethyl-3-methylimidazolium nitrate [EMIM][NO3] ≥ 99.0% (NT), (impurities: ≤ 1.0% water); -1-ethyl-3methylimidazolium tosylate [EMIM][Ts] ≥ 98.0% (HPLC), (impurities: ≤ 1.0% water); -1ethyl-3-methylimidazolium dimethyl phosphate [EMIM][dMP] ≥ 98.0% (HPLC), (impurities: ≤ 0,5% water); -1-ethyl-3-methylimidazolium methanesulfonate [EMIM][MS] ≥ 95.0%, (impurities: ≤ 0,5% water). All tested ionic liquids are easily soluble in a water. The presented study has determined the effect of five mentioned above commercial imidazolium ionic liquids on the sprouting and growth of higher plants. The structures of analyzed substances are shown in the Fig. 1. 4 Page 4 of 25
Fig. 1. Structures and abbreviations of the analyzed imidazolium ionic liquids.
2.2. Plant growth inhibition test
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A pot experiment for the determination of the potential phytotoxicity of ionic liquids and the roles of the anion in the magnitude of this adverse effect was carried out in the vegetation hall of the Department of Biochemistry and Bioproduct Technology of Jan Dlugosz University in Czestochowa, based on the OECD/OCDE Guide 208/2006 [46] and the ISO Standard 112692:1995 [47]. A monocotyledonous plant – the spring barley (Hordeum vulgare) and a dicotyledonous plant – the common radish (Raphanus sativus L. subvar. radicula Pers.) were used in the experiment. 90 mm-diameter plastic plant pot was filled with the reference soil and a soil thoroughly mixed with the examined ionic liquid. The quality analysis of the substrate showed that it was light loamy sand with a fine earth particle content of approx. 10%, an organic carbon content of 0.8%, and a pH(KCl) of 5.9. Exchangeable acidity, pH (KCl) refers to acidic cations associated with soil solids that are rapidly released into the soil solution using a concentrated solution of a neutral salt such as potassium chloride. During the experiment, the moisture of soil was kept at the level of 70% of water hold capacity of soil and to avoid leaking of ILs through the holes placed at the bottom of each pot, the volume of 20 ml distilled water per pot/per day has been used to irrigation of plants. Twenty identical seeds of selected plants coming from the same source were sowed into each pot (Fig. 2). Fig. 2. Digital photographs of pots with planted seeds.
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The examination of potential phytotoxicity encompassed 2 testing cycles: preliminary tests and final tests. The preliminary tests were carried out to establish the concentrations of compounds affecting the quality of the substrate on which the both plant species were grown. To this end, according to Standard [47], ionic liquids were applied in concentrations 0 mg, 1 mg, 10 mg, 100 mg and 1000 mg, respectively, per 1 kg of soil dry matter. In the final tests, concentrations were selected in a geometric progression using a coefficient of 2. Depending on the ionic liquid toxicity found from the preliminary tests, concentrations equal to, respectively, 200 mg, 400 mg and 800 mg of substance/kg soil, or 20 mg, 40 mg and 80 mg of substance per 1 kg of soil were used in the tests. All ionic liquids were introduced to the soil in the form of water solutions of a specified concentration. Growth of plants is the process by which a plant increases in the number and size of leaves and stems. Since plants have a high composition of water and the level of water in a plant will depend on the amount of water in its environment (which is very difficult to control), using dry weight as a measure of plant growth tends to be more reliable. Increasing level of dry matter indicate the good productivity of plants. Usually the water content of an organ is given as a percentage of the dry weight and is expressed as an amount of dry weight per fresh matter unit. To assess the phytotoxicity of imidazolium ionic liquids, the emergence and mass (dry and green) of control plant sprouts with the emergence and mass (dry and green) of the sprouts of plants growing on the soil, with respective amounts of the examined substances added, were determined and compared. The visual assessment of any damage to the examined plant species, mainly the degree growth inhibition, necrosis (is the symptom of death of plant 5 Page 5 of 25
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tissues or organs that results from infection, leaf and stem deformation) or chlorosis (is a condition in which leaves produce insufficient chlorophyll or in the other words this is a process of yellowing tissue due to a lack of chlorophyll), was made, as reflected by the digital photographs included in this paper. Based on the obtained investigation results, the magnitudes of the LOEC (lowest observed effect concentration) and NOEC (no observed effect concentration) were determined for all chemical compounds examined. The statistical estimation of the obtained results was done using variance analysis (the F Fisher-Snedecor test), while the values of LSD0.95 were calculated with the Tukey test. LSD0.95 means the least significance difference at the 95% confidence level determined from analysis of variance. Moreover, the mean standard deviations were determined, which were plotted as vertical lines in the diagrams presented in the paper.
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3. Results and discussion
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The results obtained in this study have proved the moderate phytotoxicity against upon both seedlings of barley and common radish. The negative impact against the plants was mainly dependent on the plant species and concentration applied, whilst the type of anion moderately affect the phytotoxicity. In the case of the spring barley, practically no differences in toxic effect were found between four of the five imidazolium ionic liquids, namely bromide [EMIM][Br], tosylate [EMIM][Ts], dimethylphosphate [EMIM][dMP] and methanesulfonate [EMIM][MS] (see digital photographs of seedlings, Fig. 1-4 in suppl. materials). The increasing concentration of the above-mentioned ionic liquids in soil led to a consistent decrease of the crop of barley fresh weight, which approached several dozen percent for the highest concentrations (1000 mg substance/kg of soil) as compared to fresh weight of control plants. The highest concentration that did not reduce the crop of barley seedlings (NOEC) was found to be 100mg/kg of dry weight of soil (Fig. 3). The analysis of the dry plant mass crop of [EMIM][Br, Ts, dMP and MS] ionic liquids, starting from the concentration of 200 mg, through 400 mg, 800 mg, to 1000 mg/kg of dry soil mass, resulted in a consistent increase in the level of the analyzed dry mass and ranged from a dozen to several dozens percent (Tab. 1). For these concentrations, a substantial inhibition of barley seedlings (the higher the soil ionic liquid concentration, the greater the seedling growth reduction) and distinct chlorotic changes could be observed. The above regularities were found for all the four ionic liquids, regardless of the anion type. The ionic liquids did not reduce the germination power of spring barley seeds (Tab. 2) Effect of various concentration of ionic liquids on plants has been also proved by Wang et al. [48]. They observed that the high concentration of tetrafluoroborate 1-buthyl-3-methylimidazolium negatively affect the growth of wheat seedlings and decreased the chlorophyll level resulting in inhibiting of photosynthesis process. Drop of germination power and significant chlorotic changes of cress leaves when exposed to increasing concentration of 1alkyl-3-methylimidazolium ionic liquids has been desribed by Studzińska et al. [37]. Growing concentaration of ionic liquids in soil may cause abnormal plants appearance (dwarfizm, 6 Page 6 of 25
chlorosis, necrosis), which differ from their phenotypic feature resulting in disorders of phytohormonal regulation of plants [38].
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Fig. 3. Comparison of the effects of ionic liquids on the yield of fresh weight of seedlings of spring barley. Data are expressed as a mean ± SD (standard deviation) of three replicate for each concentration.
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Table 1. Variations in the dry weight (mg/g f.m.) for spring barley following the introduction of specific amounts of the compound (in mg/kg of soil dry mass) to the soil (mean ± SD., n = 3, where n = number of replicates)
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Table 2. Variations in number of plants for spring barley following the introduction of specific amounts of the compound (in mg/kg of soil dry mass) to the soil (mean ± SD., n = 3)
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In the scientific literature describing the toxic effect of ionic liquids on organisms, there prevails the view that the magnitude of such an effect is dependent on the chemical structure of the substance As has been mentioned before, type of cation is one of the main factor affecting the toxicity of ionic liquids [22, 25, 39, 41, 49]. Results of the toxicity evaluation of imidazolium, pirydinum, pyrrolidinium, phosphonium and ammonium ionic liquids on the algae (Selenastrum capricornutum) obtained by Cho et al. [22] have proved that among tested ILs the 1-buthyl-1-methyl pyrrolidinium cation has the lowest toxicity. Stolte et al. [39] reported about high toxicity of ionic liquids containing aromatic structure cations like imidazolium or pirydynium against terrestrial plants and one celled organisms. Couling et al. [49] have revealed that with the increasing number of nitrogen heteroatom in ring structure of ionic liquids the toxicity of cations has ordered as follows: triazolium>imidazolium>piridynium >ammonium. The growing toxicity attributed to lengthening of the side chain of ionic liquids has been widely reported [21, 26, 41, 50-52] but it was mainly devoted to assess of ecotoxicity of aqua organisms. Cho et al. [50] have found that the growing amount of carbon atoms (4, 6, 8 BMIM, HMIM, OMIM respectively) in side chain of imidazolium ionic liquids caused the stronger toxicity impact on algae (Selenastrum capricornutum). These results coincide with those obtained by Ma et al. [51] where the strong correlation between length of side chains of imidazolium ILs ranged from to 4-12 of carbon atoms and the degree of toxicity against algae Scenedesmus obliquus and Chlorella ellipsoidea has been revealed. The growth of the side chain starting from 1-buthyl-, 1-hexyl- to 1-oktyl-3methylimidazolium ionic liquids also caused the increasing of toxicity impact on algae Scenedesmus quadricauda and Chlamydomonas reinhardtii [21]. Acccording to literature, the anions of ionic liquids do not affect the ecotoxicity, but are associated with determining of their physical and chemical properties including viscosity, solubility or melting temperature [35, 37-39, 41, 49, 53-55]. Study of phytotoxicity of imidazolium ionic liquids with various organic and inorganic anions have shown that the main 7 Page 7 of 25
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agent responsible for the plants inhibition growth was not the type of the anion, but the length of side chain [38]. Results of toxicity assessment of imidazolium ILs with various anions on the growth of algae (Selenastrum capricornutum) are ambiguous, however the degree of toxicity decreased in the following order: SbF6- > PF6- > BF4- > CF3SO3- > C8H17OSO3- > Br≈ Cl- indicating the group containing fluorine as the most toxic. It could be explained by the hydrolysis of ionic liquids containing fluoric groups resulting in the leakage of hydrogen fluoride into the environment [38, 53]. The outcomes of presented study, similarly like described above results, do not strictly elucidate the role of the anions of applied imidazolium ionic liquids as a toxic agent against upon both the mono- and dicotyledon plants. 1-Ethyl-3-methylimidazolium nitrate [EMIM][NO3] turned out to be a compound characterized by a slightly higher phytotoxicity to the spring barley compared to the remaining ionic liquid examined in this study. The NOEC in this case was determined to be at a level of 80 mg substance/kg dry soil mass. The nitrate resulted in a drop in fresh barley mass crop by as much as up to 55%, a distinct dwarfism of seedlings and extensive chlorotic changes (Fig. 4).
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Fig. 4. Digital photographs of spring barley on the 14th day after introduction to the soil 1-ethyl-3methylimidazolium nitrate [EMIM][NO3] (in mg/kg of soil dry mass).
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When analysing the investigation results showing the phytotoxicity of imidazolium ionic liquids to the common radish, it can be inferred that the dicotyledonous plant exhibited much higher tolerance to the applied ionic liquids compared to the spring barley. In contrast to what was found for the spring barley, the anion type did influence the magnitude of the toxic effect of imidazolium ionic liquids, though the effect of the anion did not show any special regularities. Higher sensitivity of monocotyledon seedlings of barley as compared to dicotyledon species in the presence of ionic liquids has been already observed [56]. In contrast, no differences in toxicity of ILs between wheat and cress have been reported by Matzke et al. [18]. They suggest that described in various papers differences between the mono- and dicotyledon plants are probably caused by different morphological structures of their roots. Cereal with fibrous root system do not have secondary growth, whilst the dicotyledon plants with taproot system may significantly affect the rate of ionic liquids uptake from soil. From among the five salts, the highest toxicity to the common radish was found after application of 1-ethyl-3-methylimidazolium dimethylphosphate [EMIM][dMP]. The highest concentration not causing the change of the parameters, based on which phytotoxicity is described, was 100 mg substance/kg dry soil mass. The increase in the substrate concentration of [EMIM][dMP] up to 200 mg, 400 mg, 800 mg and 1000 mg substance/kg dry of soil mass led to a distinct decline in the crop of fresh common radish plant mass by, respectively, 13%, 32%, 42% and 58%, compared to the crop of the control plot plants. In the case of this substance, a steady increase in the dry mass content of radish plants and a progressive growth reduction were also observed, while at concentrations of 800 and 1000 mg/kg dry soil mass, distinct chlorotic changes occurred on the leaves (Fig. 5, Tab. 3 and Fig. 5 in suppl. materials). From the investigations presented the highest phytotoxicity against radish has been found for the ionic liquid containing dimethyl phosphate anion (dMP). The main reason of this negative impact is probably the presence of organophosphorus compound which are commonly known as a components of pesticides. Dimethyl phosphonium anions are 8 Page 8 of 25
components of herbicides containing bipyridine or any other insecticides. Zhang et al. [57] have revealed that high doses of insecticide chlorpyrifos composed of methyl phosphonium group significantly decreased the growth of roots and fresh matter of cabbage leaves. In contrast, some researchers report about no toxicity influence of ionic liquid’s halogen anions on the organisms [36, 38, 39, 53].
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Fig. 5. Comparison of the effects of ionic liquids on the yield of fresh weight of seedlings of common radish. Data are expressed as a mean ± SD of three replicate for each concentration.
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Table 3. Variations in the dry weight (mg/g f.m.) for common radish following the introduction of specific amounts of the compound (in mg/kg of soil dry mass) to the soil (mean ± SD., n = 3)
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A slight toxic effect, on the other hand, was exhibited by two other salts, namely 1ethyl-3-methylimidazolium methanesulfonate [EMIM][MS] and 1-ethyl-3-methylimidazolium bromide [EMIM][Br]. In the case of the former substance, the fresh plant mass crop decreased by over 10% compared to the controls (the limiting value decisive to the significance of the effect of any chemical substance on the phytotoxicity parameter under discussion), and only for the highest concentrations, i.e. 800mg and 1000mg/kg dry soil mass, by 18% and 23%, respectively. A similar change in the fresh radish mass crop under the influence of [EMIM][Br] was only demonstrated for the highest concentration (1000mg/kg dry soil mass). The reduction of the crop amounted to 18% compared to the controls. The above-mentioned concentrations of the both ionic liquids resulted in an increase in the crop of dry common radish plant mass relative to the controls (Fig. 4, Tab. 3 and Fig. 6, 7 in suppl. materials). The analysis of the results representing the effect of 1-ethyl-3-methylimidazolium nitrate [EMIM][NO3] showed clearly that the numerical values of all indicators considered for the determination of the phytotoxicity of this compound did not differ significantly between radish plants grown on the substrate with the addition of the ionic liquid and the control plants. The only parameter likely to have indicated a potential phytotoxicity of [EMIM][NO3] for the radish was a clearly seen leaf discolouration. In the digital photographs presented in the figures (Fig. 6), distinct chlorotic changes can be observed on the radish plants growing on the substrate with the highest ionic liquid content; in a few instances, the radish plant leaves were totally discoloured. Fig. 6. Digital photographs of common radish on the 14th day after introduction to the soil 1-ethyl-3methylimidazolium nitrate [EMIM][NO3] (in mg/kg of soil dry mass).
From the obtained results of the author’s investigations, as shown in the digital photos, it can be found that 1-ethyl-3-methylimidazolium tosylate [EMIM][Ts] does not exhibit any toxicity to the common radish (Fig. 7). Fig. 7. Digital photographs of common radish on the 14th day after introduction to the soil 1-ethyl-3methylimidazolium tosylate [EMIM][Ts] (in mg/kg of soil dry mass).
It is worth noting, at the same time, that none of the examined ionic liquids caused any reduction of the germination capacity of common radish seeds (Tab. 4) 9 Page 9 of 25
Table 4. Variations in number of plants for common radish following the introduction of specific amounts of the compound (in mg/kg of soil dry mass) to the soil (mean ± SD., n = 3)
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The results of the author’s investigations to determine the influence of imidazolium ionic liquids on the growth and development of the common radish find some confirmation in the available scientific literature. The phytotoxicity of ionic liquids, as observed in the studies of Bałczewski et al. [33], Matzke at al. [38] and Biczak et al. [15], was depended mainly on the applied concentration of these compounds and on the genetic features of the species and varieties of plants used in the experiment. In the presented experiment, the occurred differences in the level of the toxic influence of imidazolium salts on the radish plants were also influenced by the anion type; a similar dependence is also reported in other studies [36, 39, 49].
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4. Conclusion
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Based on the testing results obtained from the pot experiment it can be stated that all imidazolium ionic liquids can be regarded as chemical compounds exhibiting a potential, slight toxicity to the growth and development of the early developmental stages of higher land plants. The toxic effect of imidazolium salts was dependent primarily on the applied substance concentration and the plant species. A plant most prone to the adverse action of the examined ionic plants turned out to be the spring barley; the highest no observed effect concentration (NOEC) not causing any noticeable reduction of the emergence and crop of the plants amounted to 100mg/kg dry soil mass, practically regardless of the compound structure. In the case of the spring barley, the regularity reported in the literature that the anion type is of minor significance for the toxicity of ionic liquids clearly manifested itself. The common radish turned out to be a plant of high tolerance to the imidazolium ionic liquids used in the pot experiment; for example, 1-ethyl-3-methylimidazolium tosylate [EMIM][Ts] did not cause any symptoms of phytotoxicity in this dicotyledonous plant, while the greatest toxic effect was observed upon applying 1-ethyl-3-methylimidazolium dimethylphosphate [EMIM][dMP]. For the common radish, the anion type was crucial the occurred toxicity of the compound to this plant, though no regularity of that effect could be observed. None of the investigated ionic liquids exhibited any adverse effect on the germination capacity of the seeds of the both plants used in the experiment.
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[4]. M. Dong, L. Zhu, S. Zhu, J. Wang, H. Xie, Z. Du, Toxic effects of 1-decyl-3methylimidazolium bromide ionic liquid on the antioxidant enzyme system and DNA in zebrafish (Danio rerio) livers, Chemosphere 91 (2013) 1107-1112. [5]. A. Strádi, M. Molnár, M. Óvári, G. Dibó, F.U. Richter, L.T. Mika, Rhodium-catalyzed hydrogenation of olefins in γ-valerolactone-based ionic liquids, Green Chem. 15 (2013) 18571862. [6]. N. Ferlin, M. Courty, S. Gatard, M. Spulak, B. Quilty, I. Beadham, M. Ghavre, A. Hai , K. Kümmerer, N. Gathergood, S. Bouquillon, Biomas derived ionic liquids: synthesis from natural organic acid, characteryzation, toxicity, biodegradation and use as solvents for catalytic hydrogenation processes, Tetrahedron 69 (2013) 6150-6161. [7]. T.V. Hoogerstraete, S. Wellens, K. Verachtert, K. Binnemans, Removal of transition metals from rare earths by solvent extraction with an undiluted phosphonium ionic liquid: separation relevant to rare-earth magnet recycling, Green Chem. 15 (2013) 919-927. [8]. D. Xiong, Z. Li, H. Wang, J. Wang, Selective separation of aliphatic and aromatic amines with CO2 switchable ionic liquids aqueous two-phase systems, Green Chem. 15 (2013) 1941-1948. [9]. N. Madria, T.A. Arunkumar, N.G. Nair, A. Vadapalli, Y.-W. Huang, S.C. Jones, V.P. Reddy, Ionic liquid electrolytes for lithium batteries: Synthesis, electrochemical, and cytotoxicity studies, J. Power Sources 234 (2013) 277-284. [10]. M. Erbeldinger, A.J. Mesiano, A.J. Russell, Enzymatic catalysis of formation of Z-aspartame in ionic liquids – an alternative to enzymatic catalysis in organic solvents, Biotechnol. Prog. 16 (2000) 1129-1131. [11]. S.C. Pereira, R. Bussamara, G. Marin, R.L.C. Giordano, J. Dupont, R. de Campos Giordano, Enzymatic synthesis of amoxicillin by penicillin G acylase in the presence of ionic liquids, Green Chem. 14 (2012) 3146-3156. [12]. L. Myles, R. Gore, M. Špulák, N. Gathergood, S.J. Connon, Highly recyclable, imidazolium derived ionic liquids of low antimicrobial and antifungal toxicity: A new strategy for acid catalysis, Green Chem. 12 (2010) 1157-1162. [13]. M.I. Hossain, M. El-Harbawi, N.B.M. Alitheen, Y.A. Noaman, J.-M. Lévěque, Ch.-Y. Yin, Synthesis and anti-microbial potencies of 1-(2-hydroxyethyl)-3-alkylimidazolium chloride ionic liquids: Microbial viabilities at different ionic liquids concentration, Ecotox. Environ. Saf. 87 (2013) 65-69. [14]. D. Coleman, M. Špulák, M.T. Garcia, N. Gathergood, Antimicrobial toxicity studies of ionic liquids leading to a ‘hit’ MRSA selective antibacterial imidazolium salt, Green Chem. 14 (2012) 1350-1356. [15]. R. Biczak, P. Bałczewski, B. Bachowska, B. Pawłowska, J. Kazmierczak-Barańska, M. Cieślak, B. Nawrot, Phytotoxicity and cytotoxicity of imidazolium ionic liquids cantaining sulfur atom, Phosphorus, Sulfur Silicon Relat. Elem. 188 (2013) 459-461. [16]. S.V. Malhorta, V. Kumar, A profile of the in vitro anti-tumor activity of imidazolium-based ionic liquids, Bioorg. Med. Chem. Lett. 20 (2010) 581-585. [17]. J.S. Terrecilla, J. García, E. Rojo, F. Rodríguez, Estimation of toxicity of ionic liquids in Leukemia Rat Cell Line and Acetylocholinesterase enzyme by principal component analysis, neural networks and multiple lineal regressions, J. Hazard. Mater. 164 (2009) 182-194. [18]. M. Matzke, S. Stolte, J. Arning, U. Uebers, J. Filser, Imidazolium based ionic liquids in soil: effects of the side chain length on wheat (Triticum aestivum) and cress (Lepidium sativum) as affected by different clays and organic matter, Green Chem. 10 (2008) 584-591. [19]. S.P.M. Ventura, A.M.M. Gonçalves, F. Gonçalves, J.A.P. Coutinho, Assessing the toxicity on [C3mim][Tf2N] to aquatic organisms of different trophic levels, Aquat. Toxicol. 96 (2010) 290297. [20]. A. Arendarczyk, A. Zgórska, E. Grabińska-Sota, Toxicity of 1-heksyl-3methylimidazolium chloride according to selected marine and freshwater organisms, Inżynieria i Ochrona Środowiska 14 (2011), 137-143 [In Pol.] [21]. K.J. Kulacki, G.A. Lamberti, Toxicity of imidazolium ionic liquids to freshwater algae, Green Chem. 10 (2010) 104-110. 11 Page 11 of 25
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13 Page 13 of 25
Sample
[EMIM][Br]
[EMIM][dMP]
[EMIM][MS]
[EMIM][Ts]
Sample
[EMIM][NO3]
0control
0.1068 ± 0.0012
0– control
0.0917 ± 0.0032
0.0985 ± 0.0008
0.0933 ± 0.0008
0.0917 ± 0.0017
1
0.0928 ± 0.0012
0.0999 ± 0.0016
0.0914 ± 0.0011
0.0937 ± 0.0021
10
0.0920 ± 0.0014
0.0992 ± 0.0009
0.0916 ± 0.0016
100
0.0965±0.0009
0.1036 ± 0.0017
1000
0.1410±0.0016
200
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Preliminary test
0.1085 ± 0.0029
0.0924 ± 0.0022
10
0.1104 ± 0.0024
0.0986 ± 0.0030
0.0927 ± 0.0015
100
0.1336 ± 0.0075
0.1388 ± 0.0013
0.1350 ± 0.0005
0.1389 ± 0.0014
1000
0.1449 ± 0.0033
0.1074 ± 0.0022
0.1200 ± 0.0056
0.1183 ± 0.0032
0.1133 ± 0.0023
400
0.1279 ± 0.0019
0.1287 ± 0.0050
0.1320 ± 0.0027
800
0.1365 ± 0.0021
0.1332 ± 0.0003 LSD0.95 – 0.0023
LSD0.95 – 0.0021
LSD0.95 - 0.0019
us
0.1350 ± 0.0016
40
0.1024 ± 0.0024
0.1373 ± 0.0021
0.1390 ± 0.0018
80
0.1075 ± 0.0033
LSD0.95 – 0.0019
an
0.1018 ± 0.0038
LSD0.95 – 0.0038
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Final test
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14 Page 14 of 25
Sample
[EMIM][Br]
[EMIM][dMP]
[EMIM][MS]
[EMIM][Ts]
Sample
[EMIM][NO3]
20
Preliminary test 20
19 ± 1
19 ± 2
20
0control
1
20 ± 1
20
18 ± 2
20 ± 1
1
19 ± 1
10
19 ± 1
20 ± 1
19 ± 1
20 ± 1
10
20 ± 1
100
20
20
20
20 ± 1
1000
18 ± 2
20
20 ± 1
19 ± 1
200
20
20
20
400
19 ± 1
19 ± 1
20
800
18 ± 2
19
19 ± 1
1000
9±2
cr
19 ± 2
20
20 ± 1
40
20
20 ± 1
80
20
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20
LSD0.95 – 1
LSD0.95 – 1
LSD0.95 – 1
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LSD0.95 – 1
100
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Final test
LSD0.95 – 1
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0– control
15 Page 15 of 25
Sample
[EMIM][Br]
[EMIM][dMP]
[EMIM][MS]
[EMIM][Ts]
[EMIM][NO3]
0 - control
0.0675 ± 0.0028
0.0738 ± 0.0020
0.0720 ± 0.0023
0.0698 ± 0.0012
0.1021 ± 0.0067
1
0.0659 ± 0.0038
0.0725 ± 0.0021
0.0745 ± 0.0070
0.0692 ± 0.0021
0.1018 ± 0.0043
10
0.0692 ± 0.0050
0.0729 ± 0.0018
0.0725 ± 0.0026
0.0671 ± 0.0011
0.0997 ± 0.0043
100
0.0712 ± 0.0040
0.0753 ± 0.0027
0.0778 ± 0.0046
0.0717 ± 0.0026
0.0956 ± 0.0076
1000
0.0770 ± 0.0078
0.1221 ± 0.0057
0.0856 ± 0.0020
0.0740 ± 0.0034
200
0.0691 ± 0.0019
0.0789 ± 0.0012
0.0751 ± 0.0022
-
0.0827 ± 0.0024
400
0.0696 ± 0.0035
0.0934 ± 0.0064
0.0786 ± 0.0022
-
0.0806 ± 0.0051
800
0.0739 ±0.0036
0.1059 ± 0.0026
0.0855 ± 0.0027
-
0.0671 ± 0.0080
cr
an
LSD0.95 – 0.0035
LSD0.95 – 0.0036
LSD0.95 – 0.0029
0.0702 ± 0.0070
LSD0.95 – 0.0079
M
LSD0.95 – 0.0044
us
Final test
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Preliminary test
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Sample
[EMIM][Br]
[EMIM][dMP]
[EMIM][MS]
[EMIM][Ts]
[EMIM][NO3]
0 - control
20 ± 1
19 ± 1
20
19 ± 2
20
1
20
20 ± 1
20
19 ± 1
20
10
19 ± 1
20
20
19 ± 1
100
20
19 ± 1
20
19 ± 1
1000
19 ± 1
20 ±1
20
200
20
19 ± 1
19 ±1
400
20 ± 1
20 ±1
20
800
20
19 ±1
cr us
20
LSD0.95 – 1
LSD0.95 – 0
20 ± 1
20 ± 1
19 ± 1
-
20 ± 1
-
19 ± 1
-
19 ± 1
LSD0.95 – 1
LSD0.95 – 1
M
LSD0.95 – 0
20
an
Final test
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Preliminary test
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Highlights 1. Phytotoxicity of 5 imidazolium ionic liquids containing various anions was studied. 2. The toxic effect of imidazolium salts was dependent primarily on the applied ionic liquids concentration.
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3. The common radish revealed the higher tolerance to the imidazolium as compared to spring barley.
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4. The anion type of ionic liquid was crucial for the toxicity against common radish.
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Fig. 1.
1-ethyl-3-methylimidazolium bromide
1-ethyl-3-methylimidazolium dimethylphosphate [EMIM][dMP]
us
cr
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[EMIM][Br]
1-ethyl-3-methylimidazolium methanesulfonate
1-ethyl-3-methylimidazolium nitrate [EMIM][NO3]
ed
M
an
[EMIM][MS]
1-ethyl-3-methylimidazolium p-toluenesulfonate (tosylate)
Ac
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[EMIM][Ts]
15 Page 19 of 25
Ac
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ed
M
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cr
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Fig. 2.
16 Page 20 of 25
Ac
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ed
M
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cr
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Fig. 3.
17 Page 21 of 25
Fig. 4.
0 mg/kg of soil
1 mg/kg of soil
10 mg/kg of soil
cr
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Preliminary test
100 mg/kg of soil
ed
M
an
us
Final test
1000 mg/kg of soil
40 mg/kg of soil
80 mg/kg of soil
Ac
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20 mg/kg of soil
18 Page 22 of 25
Ac
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ed
M
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cr
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Fig. 5.
19 Page 23 of 25
Fig. 6.
0 mg/kg of soil
1 mg/kg of soil
10 mg/kg of soil
100 mg/kg of soil
1000 mg/kg of soil
400 mg/kg of soil
800 mg/kg of soil
Ac
ce pt
ed
200 mg/kg of soil
M
an
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cr
Final test
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Preliminary test
20 Page 24 of 25
Fig. 7.
1 mg/kg of soil
10 mg/kg of soil
100 mg/kg of soil
1000 mg/kg of soil
Ac
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ed
M
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cr
0 mg/kg of soil
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Preliminary test
21 Page 25 of 25
S0304-3894(14)00197-6 http://dx.doi.org/doi:10.1016/j.jhazmat.2014.03.021 HAZMAT 15794
To appear in:
Journal of Hazardous Materials
Received date: Revised date: Accepted date:
3-12-2013 8-3-2014 10-3-2014
Please cite this article as: R. Biczak, B. Pawlowska, P. Balczewski, P. Rychter, The role of the anion in the toxicity of imidazolium ionic liquids, Journal of Hazardous Materials (2014), http://dx.doi.org/10.1016/j.jhazmat.2014.03.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The role of the anion in the toxicity of imidazolium ionic liquids Robert Biczaka, Barbara Pawłowskaa, Piotr Bałczewskia,b, Piotr Rychtera,* a
Jan Dlugosz University in Czestochowa, Institute of Chemistry, Environmental Protection and Biotechnology, 13/15 Armii Krajowej Av., 42-200 Czestochowa, Poland
b
*
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Corresponding author. Tel.: +48 343614918 int.195; fax: +48 343665322 E-mail address: [email protected] (P. Rychter).
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Center of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363 Lodz, Poland
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ABSTRACT
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From the environmental protection point of view, the growing interest of ionic liquids in various industrial branches has raised concerns for the toxicity assessment of these compounds. The paper discusses the effect of salts containing the shared 1-ethyl-3methylimidazolium [EMIM] cation as coupled with five different anions: bromide [Br], nitrate [NO3], p-toluenesulfonate (tosylate) [Ts], dimethylphosphate [dMP] and methanesulfonate [MS] on the growth and development of higher land plants – spring barley and common radish. The experiment was done according to the ISO Standard 11269-2:1995 and the OECD/OCDE Guide 208/2006. As the indications of phytotoxicity, the percentage of sprouts and the level of dry and fresh plant mass were used; in addition, the visual assessment of any signs of damage to the examined plant species, such as growth inhibition and chlorotic changes, was also made. Results of our study has proved the negative impact of ILs on the tested plants and the toxic effect of imidazolium salts was dependent primarily on the applied ionic liquids concentration. The common radish revealed the higher tolerance to the imidazolium as compared to spring barley. The anion type of ionic liquid was crucial for the toxicity against common radish. Keywords: ionic liquids, phytotoxicity, terrestrial plants, yield, dry weight 1. Introduction
The principle of “Green Chemistry” is the design of chemical products and processes that reduce or eliminate the use or generation of hazardous substances. Ionic liquids have been generating increasing interest over the last decade as an alternative for classic but often toxic reagents or solvents. Ionic liquids (ILs) make a very interesting class of chemicals, which principally have their melting point below 100 °C, which can be achieved by combining an asymmetric large-size cation with an anion of weak coordination properties. Thanks to their physicochemical properties, such as low vapour pressure, incombustibility, high thermal stability, high ionic conductivity and good catalytic properties, as well as almost perfect ability to dissolve organic and inorganic materials, ionic liquids have been finally used on a large scale in industry in recent years [1-4]. These unique properties of ionic liquids compared to classic solvents have enabled these compounds to be used in many branches of chemical industry, such as catalysis and 1 Page 1 of 25
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biocatalysis [5,6], extraction and separation [7,8] and electrochemistry [9], as well in the pharmaceutical and food industries and biotechnology [10,11]. Currently, research on ILs are also mostly focused on the possibility of using ionic liquids for controlling the growth of bacteria and fungi as well as for eliminating pathogenic microbes resistant to antibiotics and other medicines [12, 13]. These substances have been already used as additives for delayed-action drugs, medical products, including dressing materials, as well as disinfectant detergents [14]. ILs have found a great interest in medicine for controlling neoplastic cells in tumor therapy and the preliminary results of such research are promising [15-17]. Nevertheless, the most important reason for that wide use of ionic liquids in laboratories and industry is the possibility of modifying their individual physicochemical properties to meet the requirements of specific chemical reactions or entire technological processes. This can be achieved thanks to their virtually unlimited capabilities to exchange cations or anions – hence, these salts has been named designer solvents [18-20]. It seems highly probable that this feature will allow also the toxicity of ionic liquids to be reduced, which will result in such technologies that, on the one hand, will assure the optimization of industrial processes and, on the other hand, will eliminate or reduce to a minimum the undesirable effects of these chemical compounds on the natural environment [1]. However, at present, the increasing interest of the industry in ionic liquids leads to a rapid increase in their production and practical use, which in turn results in these substances getting into the environment. The low pressure of ionic liquid vapours was initially equated (wrongly, as it turned out later) with the lack of toxicity of these salts. Now it is known above all doubt that ionic liquids do exhibit toxicity and thus they may pose hazards to human health and the quality of the natural environment [2, 18-20]. Initial research on the ecotoxicity of ionic liquids was focused on the water environment due to the fact that the majority of ionic liquids exhibits almost perfect solubility in water. The obtained results have proved negative impact of the examined substances on many groups of aquatic organisms, including algae, water microorganisms, water crustaceans, mollusks and some vertebrates. In this reason the term of “ecological or green solvents” assigned to ionic liquids started to become doubtful [19-26]. The common use of ionic liquids in many branches of industry may create a huge amount of ILs wastes leading finally to their accumulation in soil, therefore behaviour of these substances especially in agriculture field seems to be crucial from the environmental protection and public health point of view. At present, thanks to their antistatic, antibacterial and fungicidal properties, ionic liquids are gradually becoming substitutes for traditional pesticides (bactericides, fungicides) or compounds used for wood preservation [14, 20, 27, 28]. Comprehensive studies are also conducted on the possibility of using ionic liquids as herbicidal substances and plant growth regulators, which would make it possible to eliminate many high-toxicity herbicidal compounds from use, which long linger in the soil [29-31]. Therefore, ionic liquids that will pass to the soil environment may become absorbed by soil colloids, which will not only have an impact on the development of the edaphon, but will also influence on the crops and the quality of plants grown on these soils, because the phytotoxicity may serve a good indicator of the hazardous nature of the ionic liquids [32]. With this respect, if for some reason ionic liquids contaminate the soil, they may be adsorbed by soil colloids affecting the development and growth and development upon both the plants and all living organisms of the soil surface [15, 18, 33-41]. The toxic impact of ionic liquids on environmental organisms is dependent on many factors such as their structure, alkyl chain length, concentration in environment or plant resistance. According to literature the structure 2 Page 2 of 25
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of cation of ionic liquids is the most important agent affecting their toxicity [34, 38, 39, 41]. The toxicity assessment of ILs with dimethylpiridinium, pyridinium, imidazolium, piperidinum, pyrrolidinium, morpholinium and ammonium cations containing the same side chain and halogens as a counterions against marine bacteria (Vibrio fischeri), algae (Scenedesmus vacuolatus) and duckweed (Lemna minor) has been reported by Stolte et al. [39]. They have noticed that the most toxic were ionic liquids containing aromatic cations as compared to quaternary ammonium salts and morpholinium cations. Toxicity results of ionic liquids containing aromatic cations were ambiguous, however, the highest toxic effect has been observed for the ILs with dimethylpyridinium cations. Toxicity of these compounds is also strongly correlated with the length of side chain and the more the carbon atoms in chain, the higher toxicity impact is observed [34, 37-41]. Matzke et al. [38] has assessed the acute toxicity of 1-alkyl-3-methylimidazolium bonded with six organic and inorganic anions against marine and land organisms living on various level of food chain including marine bacteria (Vibrio fischeri), algae (Scenedesmus vacuolatus), duckweed (Lemna minor), wheat (Triticum aestivum) and cress (Lepidium sativum). They have proved that regardless the bonded anion, the lengthening of alkyl chain negatively affect the viability of organisms. Results of ecotoxicity evaluation of 1-ethyl-, 1-buthyl- and 1-hexyl-3-methylimidazolium against cress conducted by Studzińska et al. [37] confirm fact, that the extent of toxicity of used ILs was dependent on the length of alkyl side chain and hydrophobic behavior of cations. However the phenomena of growing toxic impact with lengthening the side chain is still unclear. It is generally considered that the toxicity mechanism of ionic liquids due to their structure is similar to detergents, antibiotics and pesticides resulting in dysfunction of double lipid bilayer. Long, hydrophobic alkyl chains of ionic liquids easily interact with double phospholipid bilayer containing hydrophobic protein domains which may cause the damage and final lysis of cells [39, 41]. Described till now toxicity mechanisms of ionic liquids take a place mostly in water environment. However in soil, the degree of ecotoxicity of any chemical substances including ionic liquids is strongly dependent on soil sorption capacity and the longer alkyl chain of ILs the higher value of soil sorption is observed [40, 42]. Stepnowski et al. [42] proposed the model of double layer sorption with two partition coefficients where the first one is assigned to layer of ionic liquid directly adhered to surface of soil mineral via electrostatic forces and the second is assigned to outer layer already adhered via hydrophobic interactions. These results do not coincide with those obtained by Matzke et al. [18]. The phytotoxicity evaluation of 1-ethyl-, 1-buthyl-, 1-oktyl-3-imidazolium ionic liquids on the growth and development of cress and wheat has revealed that the degree of ILs toxicity increased with lengthening of side chain but only up to 4 carbon atoms. They state, that this is due to high mobility of ionic liquids with short side chain when compare to ILs with alkyl chain consisted of more than few carbon atoms. The mobility of solutes in soil facilitate the contamination of different layer of soil and ground water. This process is controlled by the flow velocity of the water and by interaction processes in the soil, as cation exchange reactions, solutionprecipitation reactions, content of organic matter, complex formation, biochemical reactions as well as amount of mineral colloids including kaolinite, illite or montmorillonite. 3 Page 3 of 25
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Organic matter is the most important fraction of the soil colloids composed of high molecular weight substances with intricate structure and specific properties. Since the humus is negatively charged colloid may easily bind imidazolium or piridynium cations of ionic liquids inhibiting their toxicity impact [18, 40, 43]. However till now there is a pure information in literature related to sorption of ammonium, phosphonium or pyrrolidynium quaternary salt and ILs anions are not absorbed by soil colloids in such conditions. As could be expected, increasing level or organic carbon in soil results in lowering toxicity of ionic liquids, but the outcomes related to relationship between the soil sorption capacity and the ILs colloids adsorption is still ambiguous [18, 36, 39]. Additional factors influencing on ionic liquids toxicity against terrestrial plants are specimen resistant, presence of other xenobiotics and as could be expected concentration of ILs in soil [15, 18, 33, 34, 37]. In phytotoxicity assessment of imidazolium ionic liquids against algae (Scenedesmus vacuolatus) and wheat seedlings (Triticum aestivum) Matzke et al. [44] have proved synergistic interaction of these ILs with cadmium cations. These results are in accordance with those obtained by Zhang et al. [45] where the synergistic influence of mixture consisted of tetrafluoroborate 1-benzyl-3-methylimidazolium ionic liquids with carbamate pesticides have been revealed. Since the soil is a living, dynamic ecosystem, the fate of chemical substances including the more and more popular newly synthesized ionic liquids must be constantly monitored. The literature concerned with comprehensive assessment of ionic liquids ecotoxicity is soil is scarced, therefore the near future study on relationship between toxicity of ILs and such soil parameters like acidity, moisture, colloids amount, granulometric composition or soil sorption capacity is required. The purpose of the presented study was to assess the toxic action of 5 imidazolium ionic liquids on higher land plants. The effect of the anion type on the phytotoxicity of the examined substances was also determined in the experiment.
2. Experimental 2.1.
Ionic liquids
The ionic liquids was purchased from Sigma-Aldrich Chemical Co. and was used as received without any additional purification: -1-ethyl-3-methylimidazolium bromide [EMIM][Br] ≥ 97.0% (T) ≥ 98.5% (HPLC/T), (impurities: ≤ 200 ppm water); 1-ethyl-3-methylimidazolium nitrate [EMIM][NO3] ≥ 99.0% (NT), (impurities: ≤ 1.0% water); -1-ethyl-3methylimidazolium tosylate [EMIM][Ts] ≥ 98.0% (HPLC), (impurities: ≤ 1.0% water); -1ethyl-3-methylimidazolium dimethyl phosphate [EMIM][dMP] ≥ 98.0% (HPLC), (impurities: ≤ 0,5% water); -1-ethyl-3-methylimidazolium methanesulfonate [EMIM][MS] ≥ 95.0%, (impurities: ≤ 0,5% water). All tested ionic liquids are easily soluble in a water. The presented study has determined the effect of five mentioned above commercial imidazolium ionic liquids on the sprouting and growth of higher plants. The structures of analyzed substances are shown in the Fig. 1. 4 Page 4 of 25
Fig. 1. Structures and abbreviations of the analyzed imidazolium ionic liquids.
2.2. Plant growth inhibition test
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A pot experiment for the determination of the potential phytotoxicity of ionic liquids and the roles of the anion in the magnitude of this adverse effect was carried out in the vegetation hall of the Department of Biochemistry and Bioproduct Technology of Jan Dlugosz University in Czestochowa, based on the OECD/OCDE Guide 208/2006 [46] and the ISO Standard 112692:1995 [47]. A monocotyledonous plant – the spring barley (Hordeum vulgare) and a dicotyledonous plant – the common radish (Raphanus sativus L. subvar. radicula Pers.) were used in the experiment. 90 mm-diameter plastic plant pot was filled with the reference soil and a soil thoroughly mixed with the examined ionic liquid. The quality analysis of the substrate showed that it was light loamy sand with a fine earth particle content of approx. 10%, an organic carbon content of 0.8%, and a pH(KCl) of 5.9. Exchangeable acidity, pH (KCl) refers to acidic cations associated with soil solids that are rapidly released into the soil solution using a concentrated solution of a neutral salt such as potassium chloride. During the experiment, the moisture of soil was kept at the level of 70% of water hold capacity of soil and to avoid leaking of ILs through the holes placed at the bottom of each pot, the volume of 20 ml distilled water per pot/per day has been used to irrigation of plants. Twenty identical seeds of selected plants coming from the same source were sowed into each pot (Fig. 2). Fig. 2. Digital photographs of pots with planted seeds.
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The examination of potential phytotoxicity encompassed 2 testing cycles: preliminary tests and final tests. The preliminary tests were carried out to establish the concentrations of compounds affecting the quality of the substrate on which the both plant species were grown. To this end, according to Standard [47], ionic liquids were applied in concentrations 0 mg, 1 mg, 10 mg, 100 mg and 1000 mg, respectively, per 1 kg of soil dry matter. In the final tests, concentrations were selected in a geometric progression using a coefficient of 2. Depending on the ionic liquid toxicity found from the preliminary tests, concentrations equal to, respectively, 200 mg, 400 mg and 800 mg of substance/kg soil, or 20 mg, 40 mg and 80 mg of substance per 1 kg of soil were used in the tests. All ionic liquids were introduced to the soil in the form of water solutions of a specified concentration. Growth of plants is the process by which a plant increases in the number and size of leaves and stems. Since plants have a high composition of water and the level of water in a plant will depend on the amount of water in its environment (which is very difficult to control), using dry weight as a measure of plant growth tends to be more reliable. Increasing level of dry matter indicate the good productivity of plants. Usually the water content of an organ is given as a percentage of the dry weight and is expressed as an amount of dry weight per fresh matter unit. To assess the phytotoxicity of imidazolium ionic liquids, the emergence and mass (dry and green) of control plant sprouts with the emergence and mass (dry and green) of the sprouts of plants growing on the soil, with respective amounts of the examined substances added, were determined and compared. The visual assessment of any damage to the examined plant species, mainly the degree growth inhibition, necrosis (is the symptom of death of plant 5 Page 5 of 25
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tissues or organs that results from infection, leaf and stem deformation) or chlorosis (is a condition in which leaves produce insufficient chlorophyll or in the other words this is a process of yellowing tissue due to a lack of chlorophyll), was made, as reflected by the digital photographs included in this paper. Based on the obtained investigation results, the magnitudes of the LOEC (lowest observed effect concentration) and NOEC (no observed effect concentration) were determined for all chemical compounds examined. The statistical estimation of the obtained results was done using variance analysis (the F Fisher-Snedecor test), while the values of LSD0.95 were calculated with the Tukey test. LSD0.95 means the least significance difference at the 95% confidence level determined from analysis of variance. Moreover, the mean standard deviations were determined, which were plotted as vertical lines in the diagrams presented in the paper.
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3. Results and discussion
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The results obtained in this study have proved the moderate phytotoxicity against upon both seedlings of barley and common radish. The negative impact against the plants was mainly dependent on the plant species and concentration applied, whilst the type of anion moderately affect the phytotoxicity. In the case of the spring barley, practically no differences in toxic effect were found between four of the five imidazolium ionic liquids, namely bromide [EMIM][Br], tosylate [EMIM][Ts], dimethylphosphate [EMIM][dMP] and methanesulfonate [EMIM][MS] (see digital photographs of seedlings, Fig. 1-4 in suppl. materials). The increasing concentration of the above-mentioned ionic liquids in soil led to a consistent decrease of the crop of barley fresh weight, which approached several dozen percent for the highest concentrations (1000 mg substance/kg of soil) as compared to fresh weight of control plants. The highest concentration that did not reduce the crop of barley seedlings (NOEC) was found to be 100mg/kg of dry weight of soil (Fig. 3). The analysis of the dry plant mass crop of [EMIM][Br, Ts, dMP and MS] ionic liquids, starting from the concentration of 200 mg, through 400 mg, 800 mg, to 1000 mg/kg of dry soil mass, resulted in a consistent increase in the level of the analyzed dry mass and ranged from a dozen to several dozens percent (Tab. 1). For these concentrations, a substantial inhibition of barley seedlings (the higher the soil ionic liquid concentration, the greater the seedling growth reduction) and distinct chlorotic changes could be observed. The above regularities were found for all the four ionic liquids, regardless of the anion type. The ionic liquids did not reduce the germination power of spring barley seeds (Tab. 2) Effect of various concentration of ionic liquids on plants has been also proved by Wang et al. [48]. They observed that the high concentration of tetrafluoroborate 1-buthyl-3-methylimidazolium negatively affect the growth of wheat seedlings and decreased the chlorophyll level resulting in inhibiting of photosynthesis process. Drop of germination power and significant chlorotic changes of cress leaves when exposed to increasing concentration of 1alkyl-3-methylimidazolium ionic liquids has been desribed by Studzińska et al. [37]. Growing concentaration of ionic liquids in soil may cause abnormal plants appearance (dwarfizm, 6 Page 6 of 25
chlorosis, necrosis), which differ from their phenotypic feature resulting in disorders of phytohormonal regulation of plants [38].
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Fig. 3. Comparison of the effects of ionic liquids on the yield of fresh weight of seedlings of spring barley. Data are expressed as a mean ± SD (standard deviation) of three replicate for each concentration.
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Table 1. Variations in the dry weight (mg/g f.m.) for spring barley following the introduction of specific amounts of the compound (in mg/kg of soil dry mass) to the soil (mean ± SD., n = 3, where n = number of replicates)
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Table 2. Variations in number of plants for spring barley following the introduction of specific amounts of the compound (in mg/kg of soil dry mass) to the soil (mean ± SD., n = 3)
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In the scientific literature describing the toxic effect of ionic liquids on organisms, there prevails the view that the magnitude of such an effect is dependent on the chemical structure of the substance As has been mentioned before, type of cation is one of the main factor affecting the toxicity of ionic liquids [22, 25, 39, 41, 49]. Results of the toxicity evaluation of imidazolium, pirydinum, pyrrolidinium, phosphonium and ammonium ionic liquids on the algae (Selenastrum capricornutum) obtained by Cho et al. [22] have proved that among tested ILs the 1-buthyl-1-methyl pyrrolidinium cation has the lowest toxicity. Stolte et al. [39] reported about high toxicity of ionic liquids containing aromatic structure cations like imidazolium or pirydynium against terrestrial plants and one celled organisms. Couling et al. [49] have revealed that with the increasing number of nitrogen heteroatom in ring structure of ionic liquids the toxicity of cations has ordered as follows: triazolium>imidazolium>piridynium >ammonium. The growing toxicity attributed to lengthening of the side chain of ionic liquids has been widely reported [21, 26, 41, 50-52] but it was mainly devoted to assess of ecotoxicity of aqua organisms. Cho et al. [50] have found that the growing amount of carbon atoms (4, 6, 8 BMIM, HMIM, OMIM respectively) in side chain of imidazolium ionic liquids caused the stronger toxicity impact on algae (Selenastrum capricornutum). These results coincide with those obtained by Ma et al. [51] where the strong correlation between length of side chains of imidazolium ILs ranged from to 4-12 of carbon atoms and the degree of toxicity against algae Scenedesmus obliquus and Chlorella ellipsoidea has been revealed. The growth of the side chain starting from 1-buthyl-, 1-hexyl- to 1-oktyl-3methylimidazolium ionic liquids also caused the increasing of toxicity impact on algae Scenedesmus quadricauda and Chlamydomonas reinhardtii [21]. Acccording to literature, the anions of ionic liquids do not affect the ecotoxicity, but are associated with determining of their physical and chemical properties including viscosity, solubility or melting temperature [35, 37-39, 41, 49, 53-55]. Study of phytotoxicity of imidazolium ionic liquids with various organic and inorganic anions have shown that the main 7 Page 7 of 25
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agent responsible for the plants inhibition growth was not the type of the anion, but the length of side chain [38]. Results of toxicity assessment of imidazolium ILs with various anions on the growth of algae (Selenastrum capricornutum) are ambiguous, however the degree of toxicity decreased in the following order: SbF6- > PF6- > BF4- > CF3SO3- > C8H17OSO3- > Br≈ Cl- indicating the group containing fluorine as the most toxic. It could be explained by the hydrolysis of ionic liquids containing fluoric groups resulting in the leakage of hydrogen fluoride into the environment [38, 53]. The outcomes of presented study, similarly like described above results, do not strictly elucidate the role of the anions of applied imidazolium ionic liquids as a toxic agent against upon both the mono- and dicotyledon plants. 1-Ethyl-3-methylimidazolium nitrate [EMIM][NO3] turned out to be a compound characterized by a slightly higher phytotoxicity to the spring barley compared to the remaining ionic liquid examined in this study. The NOEC in this case was determined to be at a level of 80 mg substance/kg dry soil mass. The nitrate resulted in a drop in fresh barley mass crop by as much as up to 55%, a distinct dwarfism of seedlings and extensive chlorotic changes (Fig. 4).
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Fig. 4. Digital photographs of spring barley on the 14th day after introduction to the soil 1-ethyl-3methylimidazolium nitrate [EMIM][NO3] (in mg/kg of soil dry mass).
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When analysing the investigation results showing the phytotoxicity of imidazolium ionic liquids to the common radish, it can be inferred that the dicotyledonous plant exhibited much higher tolerance to the applied ionic liquids compared to the spring barley. In contrast to what was found for the spring barley, the anion type did influence the magnitude of the toxic effect of imidazolium ionic liquids, though the effect of the anion did not show any special regularities. Higher sensitivity of monocotyledon seedlings of barley as compared to dicotyledon species in the presence of ionic liquids has been already observed [56]. In contrast, no differences in toxicity of ILs between wheat and cress have been reported by Matzke et al. [18]. They suggest that described in various papers differences between the mono- and dicotyledon plants are probably caused by different morphological structures of their roots. Cereal with fibrous root system do not have secondary growth, whilst the dicotyledon plants with taproot system may significantly affect the rate of ionic liquids uptake from soil. From among the five salts, the highest toxicity to the common radish was found after application of 1-ethyl-3-methylimidazolium dimethylphosphate [EMIM][dMP]. The highest concentration not causing the change of the parameters, based on which phytotoxicity is described, was 100 mg substance/kg dry soil mass. The increase in the substrate concentration of [EMIM][dMP] up to 200 mg, 400 mg, 800 mg and 1000 mg substance/kg dry of soil mass led to a distinct decline in the crop of fresh common radish plant mass by, respectively, 13%, 32%, 42% and 58%, compared to the crop of the control plot plants. In the case of this substance, a steady increase in the dry mass content of radish plants and a progressive growth reduction were also observed, while at concentrations of 800 and 1000 mg/kg dry soil mass, distinct chlorotic changes occurred on the leaves (Fig. 5, Tab. 3 and Fig. 5 in suppl. materials). From the investigations presented the highest phytotoxicity against radish has been found for the ionic liquid containing dimethyl phosphate anion (dMP). The main reason of this negative impact is probably the presence of organophosphorus compound which are commonly known as a components of pesticides. Dimethyl phosphonium anions are 8 Page 8 of 25
components of herbicides containing bipyridine or any other insecticides. Zhang et al. [57] have revealed that high doses of insecticide chlorpyrifos composed of methyl phosphonium group significantly decreased the growth of roots and fresh matter of cabbage leaves. In contrast, some researchers report about no toxicity influence of ionic liquid’s halogen anions on the organisms [36, 38, 39, 53].
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Fig. 5. Comparison of the effects of ionic liquids on the yield of fresh weight of seedlings of common radish. Data are expressed as a mean ± SD of three replicate for each concentration.
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Table 3. Variations in the dry weight (mg/g f.m.) for common radish following the introduction of specific amounts of the compound (in mg/kg of soil dry mass) to the soil (mean ± SD., n = 3)
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A slight toxic effect, on the other hand, was exhibited by two other salts, namely 1ethyl-3-methylimidazolium methanesulfonate [EMIM][MS] and 1-ethyl-3-methylimidazolium bromide [EMIM][Br]. In the case of the former substance, the fresh plant mass crop decreased by over 10% compared to the controls (the limiting value decisive to the significance of the effect of any chemical substance on the phytotoxicity parameter under discussion), and only for the highest concentrations, i.e. 800mg and 1000mg/kg dry soil mass, by 18% and 23%, respectively. A similar change in the fresh radish mass crop under the influence of [EMIM][Br] was only demonstrated for the highest concentration (1000mg/kg dry soil mass). The reduction of the crop amounted to 18% compared to the controls. The above-mentioned concentrations of the both ionic liquids resulted in an increase in the crop of dry common radish plant mass relative to the controls (Fig. 4, Tab. 3 and Fig. 6, 7 in suppl. materials). The analysis of the results representing the effect of 1-ethyl-3-methylimidazolium nitrate [EMIM][NO3] showed clearly that the numerical values of all indicators considered for the determination of the phytotoxicity of this compound did not differ significantly between radish plants grown on the substrate with the addition of the ionic liquid and the control plants. The only parameter likely to have indicated a potential phytotoxicity of [EMIM][NO3] for the radish was a clearly seen leaf discolouration. In the digital photographs presented in the figures (Fig. 6), distinct chlorotic changes can be observed on the radish plants growing on the substrate with the highest ionic liquid content; in a few instances, the radish plant leaves were totally discoloured. Fig. 6. Digital photographs of common radish on the 14th day after introduction to the soil 1-ethyl-3methylimidazolium nitrate [EMIM][NO3] (in mg/kg of soil dry mass).
From the obtained results of the author’s investigations, as shown in the digital photos, it can be found that 1-ethyl-3-methylimidazolium tosylate [EMIM][Ts] does not exhibit any toxicity to the common radish (Fig. 7). Fig. 7. Digital photographs of common radish on the 14th day after introduction to the soil 1-ethyl-3methylimidazolium tosylate [EMIM][Ts] (in mg/kg of soil dry mass).
It is worth noting, at the same time, that none of the examined ionic liquids caused any reduction of the germination capacity of common radish seeds (Tab. 4) 9 Page 9 of 25
Table 4. Variations in number of plants for common radish following the introduction of specific amounts of the compound (in mg/kg of soil dry mass) to the soil (mean ± SD., n = 3)
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The results of the author’s investigations to determine the influence of imidazolium ionic liquids on the growth and development of the common radish find some confirmation in the available scientific literature. The phytotoxicity of ionic liquids, as observed in the studies of Bałczewski et al. [33], Matzke at al. [38] and Biczak et al. [15], was depended mainly on the applied concentration of these compounds and on the genetic features of the species and varieties of plants used in the experiment. In the presented experiment, the occurred differences in the level of the toxic influence of imidazolium salts on the radish plants were also influenced by the anion type; a similar dependence is also reported in other studies [36, 39, 49].
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4. Conclusion
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Based on the testing results obtained from the pot experiment it can be stated that all imidazolium ionic liquids can be regarded as chemical compounds exhibiting a potential, slight toxicity to the growth and development of the early developmental stages of higher land plants. The toxic effect of imidazolium salts was dependent primarily on the applied substance concentration and the plant species. A plant most prone to the adverse action of the examined ionic plants turned out to be the spring barley; the highest no observed effect concentration (NOEC) not causing any noticeable reduction of the emergence and crop of the plants amounted to 100mg/kg dry soil mass, practically regardless of the compound structure. In the case of the spring barley, the regularity reported in the literature that the anion type is of minor significance for the toxicity of ionic liquids clearly manifested itself. The common radish turned out to be a plant of high tolerance to the imidazolium ionic liquids used in the pot experiment; for example, 1-ethyl-3-methylimidazolium tosylate [EMIM][Ts] did not cause any symptoms of phytotoxicity in this dicotyledonous plant, while the greatest toxic effect was observed upon applying 1-ethyl-3-methylimidazolium dimethylphosphate [EMIM][dMP]. For the common radish, the anion type was crucial the occurred toxicity of the compound to this plant, though no regularity of that effect could be observed. None of the investigated ionic liquids exhibited any adverse effect on the germination capacity of the seeds of the both plants used in the experiment.
References
[1]. S.P.M. Ventura, A.M.M. Gonçalves, T. Sintra, J.L. Pereira, F. Gonçalves, J.A.P. Coutinho, Designing ionic liquids: the chemical structure role in the toxicity, Ecotoxicology, 22 (2013) 112. [2]. P. Izadiyan, M.H. Fatemi, M. Izadiyan, Elicitation of the most important structural properties of ionic liquids affecting ecotoxicology in limnic green algae; a QSAR approach, Ecotox. Environ. Saf. 87 (2013) 42-48. [3]. J. Pernak, J. Feder-Kubis, A. Cieniecka-Rosłonkiewicz, C. Fischmeister, S.T. Griffin, R.D. Rogers, Synthesis and properties of chiral imidazolium ionic liquids with a (1R,2S,5R)-(−)menthoxymethyl substituent, New J. Chem. 31 (2007) 879-892. 10 Page 10 of 25
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[4]. M. Dong, L. Zhu, S. Zhu, J. Wang, H. Xie, Z. Du, Toxic effects of 1-decyl-3methylimidazolium bromide ionic liquid on the antioxidant enzyme system and DNA in zebrafish (Danio rerio) livers, Chemosphere 91 (2013) 1107-1112. [5]. A. Strádi, M. Molnár, M. Óvári, G. Dibó, F.U. Richter, L.T. Mika, Rhodium-catalyzed hydrogenation of olefins in γ-valerolactone-based ionic liquids, Green Chem. 15 (2013) 18571862. [6]. N. Ferlin, M. Courty, S. Gatard, M. Spulak, B. Quilty, I. Beadham, M. Ghavre, A. Hai , K. Kümmerer, N. Gathergood, S. Bouquillon, Biomas derived ionic liquids: synthesis from natural organic acid, characteryzation, toxicity, biodegradation and use as solvents for catalytic hydrogenation processes, Tetrahedron 69 (2013) 6150-6161. [7]. T.V. Hoogerstraete, S. Wellens, K. Verachtert, K. Binnemans, Removal of transition metals from rare earths by solvent extraction with an undiluted phosphonium ionic liquid: separation relevant to rare-earth magnet recycling, Green Chem. 15 (2013) 919-927. [8]. D. Xiong, Z. Li, H. Wang, J. Wang, Selective separation of aliphatic and aromatic amines with CO2 switchable ionic liquids aqueous two-phase systems, Green Chem. 15 (2013) 1941-1948. [9]. N. Madria, T.A. Arunkumar, N.G. Nair, A. Vadapalli, Y.-W. Huang, S.C. Jones, V.P. Reddy, Ionic liquid electrolytes for lithium batteries: Synthesis, electrochemical, and cytotoxicity studies, J. Power Sources 234 (2013) 277-284. [10]. M. Erbeldinger, A.J. Mesiano, A.J. Russell, Enzymatic catalysis of formation of Z-aspartame in ionic liquids – an alternative to enzymatic catalysis in organic solvents, Biotechnol. Prog. 16 (2000) 1129-1131. [11]. S.C. Pereira, R. Bussamara, G. Marin, R.L.C. Giordano, J. Dupont, R. de Campos Giordano, Enzymatic synthesis of amoxicillin by penicillin G acylase in the presence of ionic liquids, Green Chem. 14 (2012) 3146-3156. [12]. L. Myles, R. Gore, M. Špulák, N. Gathergood, S.J. Connon, Highly recyclable, imidazolium derived ionic liquids of low antimicrobial and antifungal toxicity: A new strategy for acid catalysis, Green Chem. 12 (2010) 1157-1162. [13]. M.I. Hossain, M. El-Harbawi, N.B.M. Alitheen, Y.A. Noaman, J.-M. Lévěque, Ch.-Y. Yin, Synthesis and anti-microbial potencies of 1-(2-hydroxyethyl)-3-alkylimidazolium chloride ionic liquids: Microbial viabilities at different ionic liquids concentration, Ecotox. Environ. Saf. 87 (2013) 65-69. [14]. D. Coleman, M. Špulák, M.T. Garcia, N. Gathergood, Antimicrobial toxicity studies of ionic liquids leading to a ‘hit’ MRSA selective antibacterial imidazolium salt, Green Chem. 14 (2012) 1350-1356. [15]. R. Biczak, P. Bałczewski, B. Bachowska, B. Pawłowska, J. Kazmierczak-Barańska, M. Cieślak, B. Nawrot, Phytotoxicity and cytotoxicity of imidazolium ionic liquids cantaining sulfur atom, Phosphorus, Sulfur Silicon Relat. Elem. 188 (2013) 459-461. [16]. S.V. Malhorta, V. Kumar, A profile of the in vitro anti-tumor activity of imidazolium-based ionic liquids, Bioorg. Med. Chem. Lett. 20 (2010) 581-585. [17]. J.S. Terrecilla, J. García, E. Rojo, F. Rodríguez, Estimation of toxicity of ionic liquids in Leukemia Rat Cell Line and Acetylocholinesterase enzyme by principal component analysis, neural networks and multiple lineal regressions, J. Hazard. Mater. 164 (2009) 182-194. [18]. M. Matzke, S. Stolte, J. Arning, U. Uebers, J. Filser, Imidazolium based ionic liquids in soil: effects of the side chain length on wheat (Triticum aestivum) and cress (Lepidium sativum) as affected by different clays and organic matter, Green Chem. 10 (2008) 584-591. [19]. S.P.M. Ventura, A.M.M. Gonçalves, F. Gonçalves, J.A.P. Coutinho, Assessing the toxicity on [C3mim][Tf2N] to aquatic organisms of different trophic levels, Aquat. Toxicol. 96 (2010) 290297. [20]. A. Arendarczyk, A. Zgórska, E. Grabińska-Sota, Toxicity of 1-heksyl-3methylimidazolium chloride according to selected marine and freshwater organisms, Inżynieria i Ochrona Środowiska 14 (2011), 137-143 [In Pol.] [21]. K.J. Kulacki, G.A. Lamberti, Toxicity of imidazolium ionic liquids to freshwater algae, Green Chem. 10 (2010) 104-110. 11 Page 11 of 25
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[22]. Ch.-W. Cho, Y.-Ch. Jeon, T.P.T. Pham, K. Vijayaraghavan, Y.-S. Yun, The ecotoxicity of ionic liquids and traditional organic solvents on microalga Selenastrum capricornutum, Ecotox. Environ. Saf. 71 (2008) 166-171. [23]. A. Latała, M. Nędzi, P. Stepnowski, Toxicity of imidazolium and pyridinium based ionic liquids towards algae. Bacillaria paxillifer (a microphytobenthic diatom) and Geitlerinema amphibium (a microphytobenthic blue green alga), Green Chem. 11 (2009) 1371-1376. [24]. C. Pretti, C. Chiappe, I. Baldetti, S. Brunini, G. Monni, L. Intorre, Acute toxicity of ionic liquids for three freshwater organisms: Pseudokirchneriella subcapitata, Daphnia magna and Danio rerio, Ecotox. Environ. Saf. 72 (2009) 1170-1176. [25]. C. Pretti, C. Chappe, D. Pieraccini, M. Gregori, F. Abramo, G. Monni, L. Intorre, Acute toxicity of ionic liquids to the zebrafish (Danio rerio), Green Chem. 8 (2006) 238-240. [26]. D.M. Costello, L.M. Brown, G.A. Lamberti, Acute toxic effects of ionic liquids on zebra mussel (Dreissena polymorpha) survival and feeding, Green Chem. 11 (2009) 548-553. [27]. A. Fojutowski, A. Noskowiak, M. Kot, A. Kropacz, A. Stangierska, The assessment of mechanical properties of wood treated with ionic liquids, Drewno 53 (2010) 21-37 [In Pol.]. [28]. K. Bica, L.R. Cooke, P. Nugent, C. Rijksen, R.D. Rogers, Toxic on purpose: ionic liquids fungicides as combinatorial, crop protecting agents, Green Chem. 13 (2011) 2344-2346. [29]. J. Pernak, A. Syguda, D. Janiszewska, K. Materna, T. Praczyk, Ionic liquids with herbicidal anions, Tetrahedron, 67 (2011) 4838-4844. [30]. J. Pernak, M. Niemczak, K. Materna, K. Marcinkowska, T. Praczyk, Ionic liquids as herbicides and plant growth regulators, Tetrahedron 69 (2013) 4665-4669. [31]. O.A. Cojocaru, J.L. Shamshina, G. Gurau, A. Syguda, T. Praczyk, J. Pernak, R.D. Rogers, Ionic liquid rorms of the herbicide dicamba with increased efficacy and reduced volatility, Green Chem. 15 (2013) 2110-2120. [32]. N.D. Das, K. Roy, Advances in QSPR/QSTR models of ionic liquids for the design of greener solvents of the future, Mol Divers, 17 (2013) 151-196. [33]. P. Bałczewski, B. Bachowska, T. Białas, R. Biczak, W.M. Wieczorek, A. Balińska, Synthesis and phytotoxicity of new ionic liquids incorporating chiral cations and/or chiral anions. J. Agric. Food Chem. 55 (2007) 1881-1892. [34]. T.P.T. Pham, Ch.-W. Cho, Y.-S. Yun, Environmental fate and toxicity of ionic liquids: A review. Water Res. 44 (2010) 352-372. [35]. A. Arendarczyk, E. Grabińska-Sota, A. Zgórska, Assessing toxicity of selected ionic liquid to representatives of flora and fauna. Inżynieria i Ochrona Środowiska 15 (2012), 225-236 [In Pol.] [36]. M. Matzke, S. Stolte, J. Arning, U. Uebers, J. Filser, Ionic liquids in soil: effects of different anion species of imidazolium based ionic liquids on wheat (Triticum aestivum) as affected by different clay minerals and clay concentrations. Ecotoxicology 18 (2009) 197-203. [37]. S. Studzińska, B. Buszewski, Study of toxicity of imidazolium ionic liquids to watercress (Lepidium sativum L.). Anal. Bioanal. Chem. 393 (2009) 983-990. [38]. M. Matzke, S. Stolte, K. Thiele, T. Juffernholz, J. Arning, J. Ranke, U. Welz-Biermann, B. Jastorff, The influence of anion species on the toxicity of 1-alkyl-3-methylimidazolium ionic liquids observed in an (eco)toxicological test battery. Green Chem. 9 (2007) 1198-1207. [39]. S. Stolte, M. Matzke, J. Arning, A Böschen, W.-R. Pitner, U. Welz-Biermann, B. Jastorff, J. Ranke, Effects of different head groups and functionalised side chains on the aquatic toxicity of ionic liquids. Green Chem. 9 (2007) 1170-1179. [40]. S. Studzińska, T. Kowalkowski, B. Buszewski, Stydy of ionic liquid cation transport in soil. J. Hazard. Mater. 168 (2009) 1542-1547. [41]. M.C. Bubalo, K. Radošević, I.R. Redovniković, J. Halambek V.G. Srček, A brie overview of the potential environment al hazarrds of ionic liquids, Ecotox. Environ. Saf. 99 (2014) 1-12. [42]. P. Stepnowski, W. Mrozil, J. Nichthauser, Adsorption of alkylimidazolium and alkylpyridinium ionic liquids onto natural soil. Environ. Sci. Tech. 41 (2007) 511-516. [43]. W. Mrozik, Ch. Jungnickel, M. Paszkiewicz, P. Stepnowski, Interaction of novel ionic liquids with soils. Water Air Soil Pollut. 224 (2013) 1759-1765. 12 Page 12 of 25
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[44]. M. Matzke, S. Stolte, A. Böschen, J. Filser, Mixture effects and predictability of combination effects of imidazolium based ionic liquids as well as imidazolium based ionic liquids and cadmium on terrestrial plants (Triticum aestivum) and limnic green algae (Scenedesmus vacuolatus). Green Chem. 10 (2008) 784-792. [45]. J. Zhang S.-S. Liu, J. Zhang, L.-T. Qin, H.-P. Deng, Two novel indices for quantitatively characterizing the toxicity interaction between ionic liquid and carbamate pesticides. J. Hazard. Mater. 239-240 (2012) 102-109. [46]. OECD/OCDE, Guidelines for the testing of chemicals. Terrestrial plant test: Seedling emergence and seedling growth test. 208/2006. [47]. ISO-11269-2: Soil quality – determination of the effect of pollutans of the soil flota – Part 2. Effects of chemicals on the mergence and growth of higher plants. International Organization for Standarization, Geneva, 1995. [48]. L.-S. Wang, L. Wang, L. Wang, G. Wang, Z.-H. Li, J.-J. Wang, Effect of 1-butyl-3methylimidazolium tetrafluoroborate on the wheat (Triticum aestivum L.) seedlings. Environ. Toxicol. 24 (2009) 296-303. [49]. D.J. Couling, R.J. Bernot, K.M. Docherty, JN.K. Dixon, E.J. Maginn, Assesing the factors responsible for ionic liquid toxicity to aquatic organisms via quantitative structure-property relationship modeling. Green Chem. 8 (2006) 82-90. [50]. Ch.-W. Cho, T.P.T. Pham, Y.-Ch. Jeon, K. Vijayaraghavan, W.-S. Choe, Y.-S. Yun, Toxicity of imidazolium salt with anion bromide to a phytoplankton Selenastrum capricornutum: Effect of alkyl-chain lenght. Chemosphere 69 (2007) 1003-1007. [51]. J.-M. Ma, L.-L. Cai, B.-J. Zhang, L.-W. Hu, X.-Y. Li, J.-J. Wang, Acute toxicity and effects of 1-alkyl-3-methylimidazolium bromide ionic liquids on green algae. Ecotox. Environ. Saf. 73 (2010) 1465-1469. [52]. M. Petkovic, D.O. Hartmann, G. Adamova, K.R. Seddon, L.N. Rebelo, C.S. Pereira, Unravelling the mechanism of toxicity of alkyltributylphosphonium chlorides in Aspergillus nidulans conidia. New. J. Chem. 26 (2012) 56-63. [53]. Ch.-W. Cho, T.P.T. Pham, Y.-Ch. Jeon, Y.-S. Yun, Influence of anions on the toxic of ionic liquids to a phytoplankton Selenastrum capricornutum. Green Chem. 10 (2008) 67-72. [54]. A. Romero, A. Santos, J. Tojo, A. Rodríguez, Toxicity and biodegrability of imidazolium ionic liquids, J. Hazard. Mater. 151 (2008) 268-273. [55]. S. Stolte, J. Arning, U. Bottin-Weber, M. Matzke, F. Stock, K. Thiele, M. Uerdingen, U. WelzBiermann, B. Jastorff, J. Ranke, Anion effects on the cytotoxicity of ionic liquids. Green Chem. 8 (2006) 621-629. [56]. J. Pernak, K. Sobaszkiewicz, J. Foksowicz-Flaczyk, Ionic liquids with symmetrical dialkoxymethyl-substituted imidazolium cations. Chem. Eur. J. 10 (2004) 3479-3485. [57]. Z.-Y., Zhang W.-L., Shan, W.-Ch. Song, Y. Gong, X.-J. Liu, Phytotoxicity and uptake of chlorpyrifos in cabbage. Environ. Chem. Lett. 9 (2011) 547-552.
13 Page 13 of 25
Sample
[EMIM][Br]
[EMIM][dMP]
[EMIM][MS]
[EMIM][Ts]
Sample
[EMIM][NO3]
0control
0.1068 ± 0.0012
0– control
0.0917 ± 0.0032
0.0985 ± 0.0008
0.0933 ± 0.0008
0.0917 ± 0.0017
1
0.0928 ± 0.0012
0.0999 ± 0.0016
0.0914 ± 0.0011
0.0937 ± 0.0021
10
0.0920 ± 0.0014
0.0992 ± 0.0009
0.0916 ± 0.0016
100
0.0965±0.0009
0.1036 ± 0.0017
1000
0.1410±0.0016
200
ip t
Preliminary test
0.1085 ± 0.0029
0.0924 ± 0.0022
10
0.1104 ± 0.0024
0.0986 ± 0.0030
0.0927 ± 0.0015
100
0.1336 ± 0.0075
0.1388 ± 0.0013
0.1350 ± 0.0005
0.1389 ± 0.0014
1000
0.1449 ± 0.0033
0.1074 ± 0.0022
0.1200 ± 0.0056
0.1183 ± 0.0032
0.1133 ± 0.0023
400
0.1279 ± 0.0019
0.1287 ± 0.0050
0.1320 ± 0.0027
800
0.1365 ± 0.0021
0.1332 ± 0.0003 LSD0.95 – 0.0023
LSD0.95 – 0.0021
LSD0.95 - 0.0019
us
0.1350 ± 0.0016
40
0.1024 ± 0.0024
0.1373 ± 0.0021
0.1390 ± 0.0018
80
0.1075 ± 0.0033
LSD0.95 – 0.0019
an
0.1018 ± 0.0038
LSD0.95 – 0.0038
Ac ce p
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20
M
Final test
cr
1
14 Page 14 of 25
Sample
[EMIM][Br]
[EMIM][dMP]
[EMIM][MS]
[EMIM][Ts]
Sample
[EMIM][NO3]
20
Preliminary test 20
19 ± 1
19 ± 2
20
0control
1
20 ± 1
20
18 ± 2
20 ± 1
1
19 ± 1
10
19 ± 1
20 ± 1
19 ± 1
20 ± 1
10
20 ± 1
100
20
20
20
20 ± 1
1000
18 ± 2
20
20 ± 1
19 ± 1
200
20
20
20
400
19 ± 1
19 ± 1
20
800
18 ± 2
19
19 ± 1
1000
9±2
cr
19 ± 2
20
20 ± 1
40
20
20 ± 1
80
20
an
20
LSD0.95 – 1
LSD0.95 – 1
LSD0.95 – 1
Ac ce p
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d
19 ± 1
M
LSD0.95 – 1
100
us
Final test
LSD0.95 – 1
ip t
0– control
15 Page 15 of 25
Sample
[EMIM][Br]
[EMIM][dMP]
[EMIM][MS]
[EMIM][Ts]
[EMIM][NO3]
0 - control
0.0675 ± 0.0028
0.0738 ± 0.0020
0.0720 ± 0.0023
0.0698 ± 0.0012
0.1021 ± 0.0067
1
0.0659 ± 0.0038
0.0725 ± 0.0021
0.0745 ± 0.0070
0.0692 ± 0.0021
0.1018 ± 0.0043
10
0.0692 ± 0.0050
0.0729 ± 0.0018
0.0725 ± 0.0026
0.0671 ± 0.0011
0.0997 ± 0.0043
100
0.0712 ± 0.0040
0.0753 ± 0.0027
0.0778 ± 0.0046
0.0717 ± 0.0026
0.0956 ± 0.0076
1000
0.0770 ± 0.0078
0.1221 ± 0.0057
0.0856 ± 0.0020
0.0740 ± 0.0034
200
0.0691 ± 0.0019
0.0789 ± 0.0012
0.0751 ± 0.0022
-
0.0827 ± 0.0024
400
0.0696 ± 0.0035
0.0934 ± 0.0064
0.0786 ± 0.0022
-
0.0806 ± 0.0051
800
0.0739 ±0.0036
0.1059 ± 0.0026
0.0855 ± 0.0027
-
0.0671 ± 0.0080
cr
an
LSD0.95 – 0.0035
LSD0.95 – 0.0036
LSD0.95 – 0.0029
0.0702 ± 0.0070
LSD0.95 – 0.0079
M
LSD0.95 – 0.0044
us
Final test
ip t
Preliminary test
Ac ce p
te
d
16 Page 16 of 25
Sample
[EMIM][Br]
[EMIM][dMP]
[EMIM][MS]
[EMIM][Ts]
[EMIM][NO3]
0 - control
20 ± 1
19 ± 1
20
19 ± 2
20
1
20
20 ± 1
20
19 ± 1
20
10
19 ± 1
20
20
19 ± 1
100
20
19 ± 1
20
19 ± 1
1000
19 ± 1
20 ±1
20
200
20
19 ± 1
19 ±1
400
20 ± 1
20 ±1
20
800
20
19 ±1
cr us
20
LSD0.95 – 1
LSD0.95 – 0
20 ± 1
20 ± 1
19 ± 1
-
20 ± 1
-
19 ± 1
-
19 ± 1
LSD0.95 – 1
LSD0.95 – 1
M
LSD0.95 – 0
20
an
Final test
ip t
Preliminary test
Ac ce p
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d
17 Page 17 of 25
Highlights 1. Phytotoxicity of 5 imidazolium ionic liquids containing various anions was studied. 2. The toxic effect of imidazolium salts was dependent primarily on the applied ionic liquids concentration.
ip t
3. The common radish revealed the higher tolerance to the imidazolium as compared to spring barley.
cr
4. The anion type of ionic liquid was crucial for the toxicity against common radish.
Ac ce p
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M
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us
18 Page 18 of 25
Fig. 1.
1-ethyl-3-methylimidazolium bromide
1-ethyl-3-methylimidazolium dimethylphosphate [EMIM][dMP]
us
cr
ip t
[EMIM][Br]
1-ethyl-3-methylimidazolium methanesulfonate
1-ethyl-3-methylimidazolium nitrate [EMIM][NO3]
ed
M
an
[EMIM][MS]
1-ethyl-3-methylimidazolium p-toluenesulfonate (tosylate)
Ac
ce pt
[EMIM][Ts]
15 Page 19 of 25
Ac
ce pt
ed
M
an
us
cr
ip t
Fig. 2.
16 Page 20 of 25
Ac
ce pt
ed
M
an
us
cr
ip t
Fig. 3.
17 Page 21 of 25
Fig. 4.
0 mg/kg of soil
1 mg/kg of soil
10 mg/kg of soil
cr
ip t
Preliminary test
100 mg/kg of soil
ed
M
an
us
Final test
1000 mg/kg of soil
40 mg/kg of soil
80 mg/kg of soil
Ac
ce pt
20 mg/kg of soil
18 Page 22 of 25
Ac
ce pt
ed
M
an
us
cr
ip t
Fig. 5.
19 Page 23 of 25
Fig. 6.
0 mg/kg of soil
1 mg/kg of soil
10 mg/kg of soil
100 mg/kg of soil
1000 mg/kg of soil
400 mg/kg of soil
800 mg/kg of soil
Ac
ce pt
ed
200 mg/kg of soil
M
an
us
cr
Final test
ip t
Preliminary test
20 Page 24 of 25
Fig. 7.
1 mg/kg of soil
10 mg/kg of soil
100 mg/kg of soil
1000 mg/kg of soil
Ac
ce pt
ed
M
an
us
cr
0 mg/kg of soil
ip t
Preliminary test
21 Page 25 of 25
