Biochemical Responses of Wheat Seedlings on the Introduction of Selected Chiral Ionic Liquids to the Soils

Article abstract of DOI:10.1021/acs.jafc.8b05517

In this study, new chiral ionic liquids (CILs) were obtained from the natural-origin material (1R,2S,5R)-(-)-menthol. The physicochemical characteristics of the studied imidazolium salts were investigated. The obtained 3-ethyl-1-[(1R,2S,5R)-(-)-menthoxy-m

Full text of DOI:10.1021/acs.jafc.8b05517

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Agricultural and Environmental Chemistry
Biochemical Responses of Wheat Seedlings on the Introduction of Selected Chiral Ionic Liquids to the Soils
Barbara Paw#owska, Joanna Feder-Kubis, Arkadiusz Telesi#ski, and Robert Biczak
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05517 • Publication Date (Web): 25 Feb 2019
Downloaded from http://pubs.acs.org on March 1, 2019
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[
Im-Ethyl-Men][PF6]
-
1
-1
-1
-1
-1
0
mg kg
1 mg kg
10 mg kg
100 mg kg
of soil DW
1000 mg kg
of soil DW
of soil DW
of soil DW
of soil DW
-
1
-1
-1
-1
-1
0
mg kg
1 mg kg
10 mg kg
100 mg kg
of soil DW
1000 mg kg
of soil DW
of soil DW
of soil DW
of soil DW
[
Im-Ethyl-Men][ClO4]
-
1
-1
-1
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mg kg
1 mg kg
10 mg kg
100 mg kg
of soil DW
1000 mg kg
of soil DW
of soil DW
of soil DW
of soil DW
-
1
-1
-1
-1
-1
0
mg kg
1 mg kg
10 mg kg
100 mg kg
of soil DW
1000 mg kg
of soil DW
of soil DW
of soil DW
of soil DW
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Biochemical Responses of Wheat Seedlings on the Introduction of Selected
Chiral Ionic Liquids to the Soils
†
‡
†‡
†,*
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Barbara Pawłowska , Joanna Feder-Kubis , Arkadiusz Telesiński , Robert Biczak
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†
The Faculty of Mathematics and Natural Sciences, Jan Dlugosz University in Czestochowa,
13/15 Armii Krajowej Av., 42-200 Częstochowa, Poland; b.pawlowska@ajd.czest.pl
‡
Faculty of Chemistry, Wrocław University of Science and Technology, Wybrzeże
Wyspiańskiego 27, 50-370 Wrocław, Poland; joanna.feder-kubis@pwr.edu.pl
†
‡
The Faculty of Environmental Management and Agriculture, West Pomeranian University
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of Technology, Juliusza Słowackiego st. 17, 71-434 Szczecin, Poland;
arkadiusz.telesinski@zut.edu.pl
* Correspondence: r.biczak@ajd.czest.pl;
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ABSTRACT
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In this study, new chiral ionic liquids (CILs) were obtained from the natural-origin
material (1R,2S,5R)-(–)-menthol. The physicochemical characteristics of the studied
imidazolium salts were investigated. The obtained 3-ethyl-1-[(1R,2S,5R)-()-menthoxy-
methyl]imidazolium salts are non-volatile, non-flammable, and stable in air, in contact with
water, and in commonly used organic solvents. The influences of the obtained chiral salts on
physiological and biochemical parameters were determined for wheat (Triticum aestivum L.)
seedlings. Both salts led to changes in plant metabolism, which resulted in decreased
assimilation pigments, decreased fresh weight, and increased dry weight and proline in wheat
seedlings. Moreover, the growth of the above-ground parts and roots was inhibited.
Additionally, there was a drop in the potential and germination capacity of wheat seeds after
using the highest concentrations of the ionic liquids. The salts caused oxidative stress in wheat
seedlings, which was demonstrated by increased malondialdehyde content. In response, the
plants engaged their defensive system against free oxygen radicals, which resulted in increased
catalase and peroxidase activity and decreased H
2
2
O levels in the plants. There was no changes
in the activity of superoxide dismutase. All of the changes observed in the levels of determined
biomarkers of oxidative stress in the plants were linearly correlated with the increase in the
concentrations of the chiral ionic liquids in the soil. The salt with hexafluorphosphate anion
exhibited slightly higher toxicity towards wheat seedlings than the other salt. The CILs led to
premature aging of plants, which was demonstrated by the increase in peroxidase activity and
a decrease of chlorophyll in the seedlings. The experiment also showed good correlation
between the increase in peroxidase activity and the decrease in chlorophyll level, which proves
that the decrease in chlorophyll content resulted from not only the increase in CILs
concentration in the soil, but also the increased POD activity, which leads to the damage of
chlorophyll particles.
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Keywords: raw material of natural origin, oxidative stress, growth inhibition, antioxidant
enzyme activity
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INTRODUCTION
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Ionic liquids (ILs) are a group of chemical compounds that have sparked great interest
among numerous researchers around the world. These compounds have a wealth of desirable
properties, such as non-flammability, non-volatility, high ionic conductivity, good chemical
and thermal stability, and good catalytic activity. Thus, they have been used in catalysis,
chemical preparation, extraction, the pharmaceutical industry, electrochemistry in capacitors,
and plant protection products.1-3 A very interesting group of ILs are herbicidal ionic liquids
(HILs), which have stronger herbicidal properties than previously used herbicides. Like the
traditional ILs, these compounds are made of ions with one having confirmed herbicidal
properties, such as 2,4-D, MCPA, MCPP, MCPB, dicamba, TBA, glyphosate, clopyraid, and
MSM. The latest trends in the synthesis of HILs are directing the attention of researchers
towards the synthesis of HILs from naturally occurring substances such as Pelargonium
roseum, rapeseed oil, choline, and D-glucose. he resulting compounds are called Bio-HILs
(bioherbicide ionic liquids).4,5
However, due to the wide possibilities of application of ILs, there is high risk that these
compounds may penetrate into the natural environment and contaminate it. Due to the good
solubility in water and high stability, ILs can easily enter the soil from waste water and sewage
sludge, as well as from failures of production systems or disasters during transport. These salts
may also be washed from post-production piles and enter the soils in the form of leachate or
post-production discharge. The soil is not only a natural store of plant nutrients but also a
“natural dustbin” in which all contaminants accumulate.6
Ionic liquids that enter the soil may be retained by soil colloids through sorption. The
strength of soil sorption depends on both the structure of the ILs and the physicochemical
properties of different sorbents. Furthermore, the cation exchange capacity, soil organic matter
and clay minerals content are significant factors. The most important soil colloid is organic
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matter, which consists of substances with high molecular weight, highly complex structure and
specific properties. Soil humus is a colloid with a negatively charged core that can successfully
bind the cations of ILs. The anions of ionic liquids are not adsorbed in such conditions. Soil ILs
can be retrieved by higher plants and affect their growth, development, and the size and quality
of yield.6-8
Herein the influences of two alkyl imidazolium ILs were evaluated for wheat, one of the
world’s most commonly cultivated cereals. The level of oxidative stress caused by contact with
the examined salts, was evaluated in wheat seedlings. The fresh and dry weight (DW) content
and the growth inhibition of above-ground parts of the plants and their roots were also analysed.
Oxidative stress was evaluated based on the content of malondialdehyde (MDA), hydrogen
peroxide (H
2
2
O ), proline, and photosynthetic pigments, as well as changes in the activity of the
enzymes peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD).
New ILs were selected, which contain the natural component (1R,2S,5R)-(−)-menthol
in the cationic part and hexafluorophosphate or chlorate(VII) in the counterion. It is very
important to synthesize compounds that are safe for the environment using the safest
components. Therefore, the interest in synthesizing ILs has also turned towards naturally
occurring source materials, such as pelargonic acid from the flowers of Pelargonium roseum,
coconut oil, rapeseed oil or just (–)-menthol.5,9,10
Considering the beneficial properties of (–)-menthol, such as a non-toxicity, relatively
low cost, and environmentally friendliness, it is interesting to study the phytotoxicity of IL
containing this natural terpene component. We would like to emphasize, that the Food and Drug
Administration (FDA) consider menthol as an safe and effective substance, which help in
common cold symptoms and labeled with low toxicity profile.11,12 This compound is widely
used in the food and pharmaceutical industries. Menthol has been indicated to have cooling,
analgesic, but also muscle relaxing activities.13 This well-known representative of the
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monoterpene class also enhances smooth muscle relaxation, reduces lower esophageal sphincter
tone, and the skin barrier by vasodilation, etc.14,15 The safety profile of menthol has been
demonstrated by in vitro and in vivo studies and most investigations reveal a low potential for
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toxicity in humans. It has been proven that menthol also has antioxidant and antibacterial
properties. Mahdavikia and Saharkhiz16 demonstrated that peppermint extracts containing
menthol in their composition may influence the germination of growth and development of
higher plants, including weeds. It should be stress that the natural origin of presented alcohol
means more safety to the people and the environment.
The imidazolium salts examined are chiral ionic liquids (CILs) where the chirality is
located in the cation and associated with the presence of the optically active monoterpene
substituent. Previous works have presented various quaternary salts that contain (−)-menthol
derivatives and exhibit a number of interesting properties. Some of the most encouraging
properties are their biological functions, which include antifungal activity,1117 antibacterial
activity,1218 and stabilizing lacccase in enzymatic systems.1319 Compounds with a similar
structure have also been tested for their herbicidal properties.1420 Other properties of ILs with a
menthol derivative include catalytic activity,1521 antielectrostatic activity,1622 and usefulness in
HPLC techniques.1723 There are many technological areas where these salts might be
successfully applied, such as enzymology, microbiology, and wood protection systems. Hence,
the study of ILs with this naturally occurring terpene derivative seems to be very advisable.
Furthermore, the type of anion involved in the ILs structure, is greatly important. One
of the projects, in our lab is looking for good or excellent solvents in separation science studies.
We have already shown very good results in this area, and terpene-based ILs were used as chiral
selectors in HPLC analysis.17,1823,24 In those studies only chlorides chiral salts were tested, and
we want to go further in testing other possible anions that are good for separation.
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The unique properties of ILs combined with their potential uses have already increased
the interest of researchers in the field of separation science.19-2125-27 Ionic liquids with [ClO
] anions are well known as good additives in chromatographic analysis.22,2328,29
4
]
and [PF
6
Despite the potential benefits, it is necessary to examine the toxicity of these compounds
for important cereals such as wheat. The results will enable risk assessment of environmental
contamination with these compounds and the evaluation of their influence on wheat cultivation.
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MATERIALS AND METHODS
Chemicals, Materials and General Techniques
The materials used were sourced as follows: (1R,2S,5R)-(–)-menthol (99%),
paraformaldehyde (powder, 95%), hydrochloric acid (35-38%), sulphuric acid (≥96%),
imidazole (99,5%), sodium (cubes, contains mineral oil, 99.9% trace metals basis),
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bromoethane (98%), potassium hexafluorophosphate (99%), and potassium perchlorate
(99.99%) were purchased from Sigma-Aldrich. The drying agents sodium sulphate
(anhydrous, pure) and phosphorus pentoxide (powder, anhydrous, ≥98%) were provided by
P.O.Ch (Gliwice, Poland). All reagents and solvents were dried beforehand using standard
techniques. For NMR analysis, deuterated chloroform was purchased from Merck.
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The structure and purity of all of the synthesized substances were confirmed by spectral
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analysis. The H NMR and C NMR spectra were recorded on a Bruker DRX instrument with
tetramethylsilane as the standard (at 400 and 150 MHz, respectively). Elemental analyses were
carried out for all of the synthesized substances using a VARIO EL-III. Melting points were
determined using an electrothermal digital-melting-point apparatus (model JA 9100,
temperature resolution ± 0.1 °C; accuracy ± 1%; ramp rate 1.0 °C/min). The cationic active
content was assayed by direct two-phase titration according the EN ISO 2871-2 procedure.2430
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General Methods for Synthesis
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1-Ethylimidazole was prepared according to a published method2531 and purified by
vacuum distillation. Chloromethyl (1R,2S,5R)-(–)-menthyl ether was obtained by passing HCl
through a mixture of paraformaldehyde and (1R,2S,5R)-(–)-menthol, similar to the method by
Pernak et al.2632 The precursor of the ILs, 3-ethyl-1-[(1R,2S,5R)-(–)-menthoxy-
methyl]imidazolium chloride, was synthesized according to previously described
procedures.2531
Synthesis of new chiral ionic liquids containing (1R,2S,5R)-(–)-menthol derivative
Next, 0.031 mol of the solid inorganic salts potassium hexafluorphosphate, and
potassium perchlorate were dissolved in water at room temperature. Then, the prepared solution
was
added
to
saturated
water
containing
3-ethyl-1-[(1R,2S,5R)-(–)-
menthoxymethyl]imidazolium chloride (0.03 mol). The reaction mixture was stirred at room
temperature and the product precipitated from it. After 24h, the crude product was separated
and washed with distilled water until chloride ions were no longer detected using AgNO
3
solution. Prior to any measurements, 1-alkyl-3-[(1R,2S,5R-(–)-menthoxymethyl]imidazolium
salt samples were dried at 40 °C for 48h under vacuum.
3-Ethyl-1-[(1R,2S,5R)-(–)-menthoxymethyl]imidazolium hexafluorophosphate, [Im-
1
Ethyl-Men][PF
6
]. H NMR (CDCl
3
, 400 MHz, 25°C) δ [ppm] = 0.48 (d, J = 7.1 Hz, 3H); 0.88
(m, 9H); 1.22 (m, 1H); 1.40 (m, 1H); 1.58 (m, 5H); 1.92 (sept d, J1,3 = 6.9 Hz, J1,2 = 2.5 Hz,
1,3
1,2
1H); 2.10 (d, J = 12.0 Hz, 1H); 3.38 (td, J = 10.6 Hz, J = 4.4 Hz, 1H); 4.45 (kw, J = 7.1 Hz,
1
,2
2H); 5.48 and 5.72 (d, J = 10.4 Hz, 2H, AB system); 7.31 (t, J = 1.9 Hz, 1H); 7.20 (t, J = 1.9
Hz, J2,1’ = 1.6 Hz, 1H); 9.01 (t, J = 1.1 Hz, 1H); C NMR (CDCl
13
, 150 MHz, 25°C) δ [ppm] =
3
15.4; 15.5; 20.7; 22.0; 22.6; 25.1; 31.0; 33.9; 40.2; 45.1; 47.5; 76.6; 79.4; 120.6; 121.5; 136.2.
Elemental analysis calc (%) for C16
46.90, H 7.23, N 6.72
H29ON
2
PF
6
(410.38): C 46.83, H 7.12, N 6.83; found: C
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3-Ethyl-1-[(1R,2S,5R)-(–)-menthoxymethyl]imidazolium chlorate(VII), [Im-Ethyl-
1
Men][ClO
4
]. H NMR (CDCl
3
, 400 MHz, 25°C) δ [ppm] = 0.48 (d, J = 7.1 Hz, 3H); 0.87 (m,
9H); 1.22 (m, 1H); 1.40 (m, 1H); 1.58 (m, 5H); 1.90 (sept d, J1,3 = 6.9 Hz, J1,2 = 2.5 Hz, 1H);
1,3
1,2
2.11 (d, J = 12.0 Hz, 1H); 3.37 (td, J = 10.6 Hz, J = 4.4 Hz, 1H); 4.45 (kw, J = 7.1 Hz, 2H);
1
,2
5.52 and 5.79 (d, J = 10.4 Hz, 2H, AB system); 7.41 (t, J = 1.9 Hz, 1H); 7.25 (t, J = 1.9 Hz,
J2,1’ = 1.6 Hz, 1H); 9.37 (t, J = 1.1 Hz, 1H); C NMR (CDCl
13
, 150 MHz, 25°C)δ [ppm] = 15.2;
3
15.4; 20.7; 22.1; 22.6; 25.0; 31.0; 33.9; 40.0; 45.1; 47.5; 76.6; 79.3; 120.6; 121.5; 136.5.
Elemental analysis calc (%) for C16
52.73, H 8.11, N 7.61
H
29
O
5
ClN (364.86): C 52.67, H 8.01, N 7.68; found: C
2
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Phytotoxicity Study
Growth Conditions and Treatment
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The phytotoxicity of the ILs was determined according to the OECD/OCDE 208
Guidelines for the testing of chemical. Terrestrial plant: seedling test: seedlings emergence and
seedling growth test.2733 Based on these guidelines, the following concentrations of CILs were
used: 0, 1, 10, 100, and 1000 mg of compound per 1 kg of DW of soil. When preparing these
concentrations, appropriate amounts of CILs were dissolved in acetone, followed by mixing
with quartz sand. After the overhead evaporation of acetone, quartz sand containing
imidazolium salts was mixed with the soil. Three independent samples were prepared for each
concentration of CILs. Control samples were prepared using the same procedure by adding
acetone to the sand but not CILs.
Granulometric analysis showed that the loam used in this study as a soil contained about
−
1
11% content with a diameter of <0.02 mm, 9.5 g kg of organic carbon, and pH 6.1. Plastic
pots were filled with the prepared medium and then seeded with 20 seeds of wheat (Triticum
aestivum L.), which were derived from the same source. Seed germination and plant growth
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were carried out for 14 days under strictly controlled conditions: soil moisture at 70% ppw,
−2 −1
temperature of 20 ± 2 °C, and constant illumination equal to 170 μmol m s within a system
of 16 h day/8 h night. Stable growth and development conditions were assured by a vegetation
hall that is part of the Department of Biotechnology, Biochemistry and Ecotoxicology at Jan
Dlugosz University in Czestochowa.
The phytotoxicity of the CILs for wheat was estimated based on the fresh weight yield
of the plants, dry weight content, and the length of the aerial parts of the plants and their roots.
The inhibition factor of fresh weight, plant length (shoot length), and root lengths were
calculated according to a study by Wang et al.2834 Based on the calculated inhibition, non-linear
regression analysis was used to estimate EC50 with GraphPad Prism software (GraphPad
Software, Inc., La Jolla, CA, USA). The germination potential (GP) and germination rate (GR)
of the wheat seeds were also determined.
푁푢푚푏푒푟 표푓 푔푒푟푚ꢀ푛푎푡푒푑 푠푒푒푑푠 표푛 푡ℎ푒 3ꢁꢂ 푑푎푦
2
2
10
11
퐺푃 =
퐺푅 =
× 100%
× 100%
푆푒푒푑푠 푛푢푚푏푒푟 푓표푟 푡ℎ푒 푡푒푠푡
푁푢푚푏푒푟 표푓 푔푒푟푚ꢀ푛푎푡푒푑 푠푒푒푑푠 표푛 푡ℎ푒 7ꢁꢂ 푑푎푦
푆푒푒푑푠 푛푢푚푏푒푟 푓표푟 푡ℎ푒 푡푒푠푡
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Seeds with a germ more than 2 mm long were considered germinated.2935
The content of all assimilation pigments, proline, malondialdehyde, H
2
2
O , and enzyme
activity were determined in fresh plant material. Samples treated with high concentrations of
CILs were not included in some analyses. For some of the samples investigated, the inhibition
of growth of the aerial parts of wheat was so large that we failed to collect plant material for
research purposes.
Determination of Pigments Content
The content of assimilation pigments was determined by spectrophotometry according
to the methodology presented by Oren et al.3036 A weighed portion (200 mg) of fresh leaves
was homogenized with the addition of 80% acetone solution that had been cooled to 4 °C. The
resulting extract was transferred into centrifuge tubes and left in the dark for 24 h. The extract
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was then centrifuged, and in the filtrate obtained, the content of assimilation pigments was
determined by measuring the absorbance at 470, 647, and 664 nm. The content of chlorophylls
-1
and carotenoids was expressed in mg g of plant DW.
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Determination of Lipid Peroxidation and Hydrogen Peroxide
A weighed 500-mg portion of fresh leaves was homogenized with the addition of 0.1%
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trichloroacetic acid solution that had been cooled to 4 °C. After centrifugation, MDA and H
2
O
2
content was determined in the supernatant according to procedures described by Hodges et
al.3137 and Singh et al.3238 respectively. Thiobarbituric acid was used as a substrate to determine
MDA. The MDA content was determined by measuring the absorbance at 532 nm and 600 nm.
To determine the content of H
2
2
O , the absorbance was measured at a wavelength of 390 nm for
a reaction mixture consisting of supernatant, phosphate buffer pH 7.0, and potassium iodide.
The content of MDA and H
2
O
2
was calculated using an extinction coefficient of 155 mM−1
−
1
−1
cm and expressed in μmol g fresh weight (FW).
2
36
Determination of Free Proline
2
2
2
2
2
2
2
37
38
39
40
41
42
43
The free proline content was determined according to the method reported by Bates.3339
A fresh sample (0.5 g) was homogenized with 5 ml of 3% sulphosalicylic acid. Next, 2 ml of
the supernatant was added to a mixture of 2 ml of glacial acetic and 2 ml of 2.5% (w/v) acidic
ninhydrin before boiling at 100 °C for 1 hour. The reaction was terminated in an ice bath. After
the solution was cooled, 5 ml of toluene was added for extraction prior to reading the
absorbance of the upper red layer (toluene) at 520 nm and calculating the content of proline
-1
(mg g FW).
2
44
Antioxidant Enzyme Activity Determination
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52
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Enzymatic extracts for the determination of antioxidant enzymes activity were obtained
by homogenizing a weighed portion (500 mg) of fresh leaves with the addition of cooled (4 °C)
extraction mixture. The mixture contained phosphate buffer pH 7.4, 1 mM EDTA solution, and
0.1% polyvinylpyrrolidone solution. After centrifugation, the obtained supernatant was also
used to determine the protein content.
The activity of superoxide dismutase (SOD) [EC 1.15.1.1] was determined
spectrophotometrically by measuring the degree of reduction of nitroblue-tetrazolium (NBT)
by superoxide anions that formed as a result of the photochemical reduction of riboflavin in the
presence of light.3440 NBT reduction is inhibited by SOD. To determine the activity of SOD,
the absorbance of the reaction mixture was measured at a wavelength of 560 nm. Superoxide
−
1
dismutase activity was expressed as U mg protein, where 1[U] is the amount of enzyme that
induces 50% inhibition of NBT reduction.
Catalase (CAT) activity [EC 1.11.1.6] was determined by the decomposition of H
by this enzyme for 15 min, and the H remaining in the reaction mixture was titrated with
0.01 N KMnO
.3541 Catalase activity was expressed as U mg protein min .
The activity of peroxidase (POD) [1.11.1.7] was determined spectrophotometrically by
2
O
2
2
O
2
-1
-1
4
the rate of guaiacol oxidation in the presence of H
2
O
2
via enzymes occurring in a given volume
of extract for 1 min.3642 To measure the activity of POD, the absorbance of the reaction mixture
-1
-
was measured at a wavelength of 470 nm. POD activity was expressed as U mg protein min
1.
The total protein content necessary to calculate the activity of SOD, CAT, and POD was
determined using the Bradford assay.3743
Statistical Analysis
2
2
68
69
The results obtained from three independent measurements were subjected to statistical
analysis using one-way analysis of variance (ANOVA). Tukey’s test with p<0.05
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(STATISTICA 12.5) was used to determine the significance of reported differences. The data
presented in tables and figures are expressed as the mean ± standard error (SE) obtained from
3 measurement replicates.
2
73
RESULTS AND DISCUSSION
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
74
75
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79
80
81
82
83
84
85
86
87
88
89
90
Synthesis and the Characterization of New Chiral Ionic Liquids Containing (1R,2S,5R)-
(–)-Menthol Derivative
Two CILs were prepared with a natural-origin material in the cation, (1R,2S,5R)-(–)-
menthol, and hexafluorophosphate and chlorate(VII) as the counterion (Scheme 1) using
previously described methods.25,2631,32 In the first step, chloromethyl (1R,2S,5R)-(–)-menthyl
ether was obtained by chloromethylation of (1R,2S,5R)-(–)-menthol under isothermal
conditions at 10 °C. Then, 3-ethyl-1-[(1R,2S,5R)-(–)-menthoxymethyl]imidazolium chloride
was prepared using a published method.2531 Chiral imidazolium chloride containing a
monoterpene moiety was prepared by the Menschutkin reaction. Quaternization was achieved
using distilled 1-ethylimidazole and freshly distilled chloromethyl (1R,2S,5R)-(–)-menthyl
ether under strictly anhydrous conditions. The reaction was carried out in anhydrous hexane at
room temperature, and the product precipitated from the solution. Quaternization takes place
immediately and proceeds readily at room temperature. The chiral chloride with (1R,2S,5R)-(–
)-menthol derivative was used as a precursor for the new CILs, which were obtained via
metathesis reaction. The anion exchange processes progressed smoothly at room temperature,
and the final products were obtained in very good yields (98.6% and 98.2%). The total three-
step synthesis process is energy efficient and very effective.25,2631,32
2
91
Scheme 1. The synthesis of ionic liquids using (1R,2S,5R)-(–)-menthol as a substrate.
2
2
92
93
Table 1 shows a list of the 3-ethyl-1-[(1R,2S,5R)-(–)-menthoxymethyl]imidazolium
salts, which have not been described previously in the literature. The are hydrophobic and stable
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2
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2
2
94
95
96
97
98
99
in both air and aqueous solutions. The purities of the hexafluorophosphate and chlorate(VII)
salts were determined by a direct two-phase titration technique (EN ISO 2871-2: 2010)2430 with
a range of 99.9% for the hexafluorphosphate salt and 99.8% for the perchlorate salt. The rest
consists of water. The determination could be performed due to the structure of the cation, and
the obtained salts were titrated using this method.
Table 1. Structures and physicochemical characteristics of ionic liquids.
Structure and name
Formula
M
r
Yielda;b Melting
Surfactant
content
(%)
-1
c
d
(g·mol ) (%)
point
°C)
(
C
16
H
29
N
2
OPF
6
410.44
98.6
99.2–99.5 99.9
3
-ethyl-1-[(1R,2S,5R)-()-
menthoxymethyl]imidazolium
hexafluorophosphate
[
Im-Ethyl-Men][PF ]
6
C
16
H
29
N
2
O
5
Cl 364.92
98.2
96.3–96.8 99.8
3
-ethyl-1-[(1R,2S,5R)-()-
menthoxymethyl]imidazolium
chlorate(VII)
[
Im-Ethyl-Men][ClO ]
4
a
b
c
d
3
3
3
3
00
01
02
03
Isolated yield after purification and drying; Accuracy ± 0.1%; Accuracy ± 0.1 °C;
Accuracy ± 0.1%.
o
The obtained salts are solid, and their melting point does not exceed 100 C. Therefore,
they should be considered as ILs according to the definition.3844
3
3
04
05
3
06
Phytotoxicity Assay of CILs
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The chiral ILs exhibited a phytotoxic effect towards wheat seedlings. After sowing the
seeds in soil with the addition of [Im-Ethyl-Men][PF
6
] and [Im-Ethyl-Men][ClO ], there was a
4
drop in the potential and germination capacity of the seeds in comparison to the controls. These
changes were higher with increasing CIL concentrations (Table 2).
3
3
3
11
12
13
Table 2. Effects of CILs on the germination potential (GP) and germination rate (GR) of wheat.
Data are expressed as a mean ± SE of three replicates for each concentration. Values denoted
by the same letters in the columns do not differ statistically at p<0.05.
CILs
[
Im-Ethyl-Men][PF
6
]
[Im-Ethyl-Men][ClO ]
4
concentration
-1
(
mg kg soil
GP (%)
GR (%)
GP (%)
GR (%)
DW)
0
1
a
a
a
a
a
a
a
a
91.67 ± 1.67
86.67 ± 4.41
96.67 ± 1.67
96.67 ± 1.67
91.67 ± 1.67
96.67 ± 3.33
96.67 ± 1.67
81.67 ± 7.39
7
1
3.33 ±
1.61
1
0
81.67 ± 4.41a 95.00 ± 2.89a 96.67 ± 1.67a
a
b
a
a
ab
1
00
6.67 ± 4.41
81.67 ± 4.41
3.33 ± 1.67b
93.33 ± 1.67 70.00 ± 5.00
b
a
5.00 ± 2.89b
1
000
0
66.67 ± 1.67
3
3
3
3
3
3
3
3
3
3
3
3
14
15
16
17
18
19
20
21
22
23
24
25
GP – germination potential; GR – germination rate.
th
Figure 1. Digital photographs of wheat seedlings and roots on the 14 day after introduction to
-
1
the soil [Im-Ethyl-Men][PF
6
] and [Im-Ethyl-Men][ClO ] (in mg kg of soil DW).
4
The growth of the plants and their roots was inhibited as the compounds concentrations
increased (Figure 1). Inhibition of the root pattern is considered by some authors to be one of
the most significant phytotoxic biomarkers.3945 This is connected with the functions of these
bodies, which include keeping the plant in the substrate, as well as the collection and transport
of water and nutrients. Therefore, proper development and condition of the roots determine the
optimal growth and development of the plant, which in turn translates into the size and quality
of the crop. The wheat seedling growth inhibition was over 90% in comparison to the control
when using [Im-Ethyl-Men][PF ] at concentrations of 100 and 1000 mg/kg of soil DW and [Im-
6
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26
27
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29
30
31
32
33
34
Ethyl-Men][ClO ] at 1000 mg kg of soil DW. The direct effect of the plant growth and their
4
roots consisted of a drop in fresh weight yield of the wheat seedlings in comparison to the
controls, which is positively correlated with the increase of the CILs in soil (Figure 2). The
EC50 values were estimated from the inhibition levels in reference to the root length, above-
ground part length, and fresh weight yield. The confidence intervals for the individual EC50
values are given in parentheses. In the case of hexafluorphosphate, the values were 75 (41.7-
−
1
138), 8 (4.75-12.8), and 6 (5.24-6.82) mg kg of soil DW, respectively. The EC50 values after
−
1
the use of perchlorate were 171 (70.3-426), 10 (8.33-12.2), and 7 (5.60-8.13) mg kg of soil
DW.
3
3
3
35
36
37
Figure 2. The inhibition rate for shoot length (a); root length (b); and fresh weight (c) of wheat
after exposure to CILs. Data are expressed as a mean ± SE of three replicates for each
concentration.
3
3
3
3
3
38
39
40
41
42
A significant element that determines the influence of the compounds on the plants is
the changes in dry weight of the plants. A significant increase in dry weight of the wheat
seedlings was observed with the increase of the concentration of CILs in soil (Figure 3). The
CIL concentration was a very important biomarker, that had a decisive impact on the growth
and development of the wheat seedlings.
-
1
3
3
3
43
44
45
Figure 3. Variations in the dry weight (g g FW) for wheat seedlings following the introduction
-1
of specific amounts of the compound (in mg kg of soil DW) to the soil (mean ± SE; n = 3).
Values denoted by the same letters in the concentrations do not differ statistically at p<0.05.
8
3
3
3
3
46
47
48
49
The results are confirmed by the literature. Studzińska and Buszewki studied the
influence of increasing doses of imidazolium ILs on the growth and development of the
watercress. They observed a drop in the germination potential of watercress seeds and clear
discoloration on the leaves under the influence of these compounds. In previous studies on the
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50
51
52
53
54
55
56
57
58
59
60
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66
67
determination of phytotoxicity of ILs with iodide anions, we also observed a small effect of
these salts on seed germination, which was only visible when compounds were used at the
highest concentrations.4046 However, other studies show a lack of influence of some
imidazolium ILs on the germination potential of seeds of common radish and spring barley.4147
These results are corroborated by Liu et al.2935 Cvjetko Bubalo et al. and Wang et al.
1
2834
These
authors also stated that the level of phytotoxicity of chemical compounds introduced to a
substrate depends on the concentration. Low IL concentrations may even stimulate plant
growth, acting similarly to growth hormones, whereas high concentrations lead to an almost
linear drop in yield, sprout length, and root length. [Im-Ethyl-Men][PF ] had a slightly stronger
6
phytotoxic effect, which may be related to the fact that it contains fluorine. This element has a
negative effect on assimilation and photosynthesis, which results in plant growth inhibition.42-
4
448-50
Similar conclusions were also drawn by Matzke et al.4551 when studying the effect of ILs
-
with a CF
3
SO
3
anion on the growth and development of wheat.
A very important indicator of phytotoxicity is the changes in the dry matter level of the
plants. The growth of dry matter content in both mono- and dicotyledonous plants that have had
7
contact with chemical compounds in the substrate has also been reported by Biczak et al. and
Biczak et al.4046 A higher increase in dry matter usually correlated with a greater the decrease
in the fresh weight of plants.
3
68
Effect of CILs on Pigment Content
3
3
3
3
3
3
69
70
71
72
73
74
The yield size is inseparably linked to the efficiency of photosynthesis, in which the
content of photosynthetic pigments (i.e. chlorophylls and carotenoids) plays a very important
role.46,4751,52 Numerous researchers report an almost linear correlation between the
concentration of the chemical substance in the environment and the drop of photosynthetic
pigment content in plants.29,48-5135,54-57 The results of our study demonstrate a drop in Chla,
Chlb, Chl(a+b), and carotenoid content, which was positively correlated with an increase of
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the content of the examined CILs in the substrate. However, no significant changes were
observed in the ratio of chlorophyll a to chlorophyll b and the ratio of total chlorophyll to
carotenoids (Table 3).
3
3
3
3
78
79
80
81
Table 3. Effects of [Im-Ethyl-Men][PF
6
] and [Im-Ethyl-Men][ClO ] on photosynthetic
4
pigment in wheat seedlings. Data are expressed as a mean ± SE of three replicates for each
concentration. Values denoted by the same letters in the columns do not differ statistically at
p<0.05.
Concentration
-
1
Pigments (mg g DW)
-
of CILs (mg kg
1
soil DW)
Chla
Chlb
Car Chla+Chlb
Chla/Chlb Chl(a+b)/Car
0
13.684±0.014a 3.752±0.010a 2.958±0.004a 17.436±0.013a 3.647±0.012a 5.894±0.007
12.489±0.015b 3.461±0.008b 2.689±0.006b 15.950±0.014b 3.608±0.010a 5.912±0.007a
11.889±0.255b 3.345±0.006c 2.602±0.003c 15.234±0.270c 3.554±0.073a 5.854±0.101a
a
1
1
0
1
00
-
-
-
-
-
-
-
-
-
-
-
-
1
000
0
13.684±0.014a 3.752±0.010a 2.958±0.004a 17.436±0.013a 3.647±0.012a 5.894±0.007c
11.475±0.064b 3.227±0.025b 2.253±0.009b 14.702±0.088b 3.556±0.011b 6.525±0.031a
9.814±0.009c 2.666±0.017c 1.950±0.006c 12.481±0.024c 3.681±0.022b 6.399±0.02b
6.692±0.053d 1.723±0.040d 1.287±0.021d 8.415±0.090d 3.886±0.065a 6.541±0.040a
1
1
0
1
00
1
000
-
-
-
-
-
-
3
3
82
83
Chl a – chlorophyll a, Chl b – chlorophyll b, chl a+b – chlorophyll a + chlorophyll b, car – carotenoides, a/b – chlorophyll
a/chlorophyll b, chl/car –(chlorophyll a+ chlorophyl b)/carotenoides
3
3
3
3
3
3
3
84
85
86
87
88
89
90
Chloroplasts are very susceptible to the production of ROS. The decrease of the level of
photosynthetic pigments in plants is related to the fact that the ROS oxidize and damaged
proteins contained in the PSII photosystem. This leads to a decrease in photosynthesis
efficiency, thus restricting the appropriate growth and development of plants. The harmful
influence of the CILs on wheat can also be demonstrated by the drop of the carotenoid content,
which constitutes a basic line of defense against ROS.50,5156,57
3
91
Effects of CILs on Free Proline Content
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92
93
94
95
96
97
98
99
00
01
02
Changes in proline content constitute an important biomarker of the influence of
chemical compounds on a plant, as it regulates the osmotic potential of plant cells, which
decreases during oxidative stress. Proline protects the subcellular structure and enzymes and
increase cellular osmolarity, which provides the elasticity necessary to multiply cells under
stress conditions.29,4635,51 Various studies observed higher levels of proline accumulation under
salt-stress conditions for wheat, Sorghum bicolor, and green gram.46,52-5451,58-60 However,
Sánchez-Rodríguez et al.5561 believe that the increase of the level of proline is not important for
antioxidants and only serves as a marker of stress in plants. In the present study, an increase in
the content of this amino acid was observed in wheat seedlings and was positively correlated
with the increase of CILs in the soil (Table 4). The increase of proline content in wheat seedlings
subjected to imidazolium IL was also observed by Liu et al.2935
4
4
4
4
03
04
05
06
Table 4. Effects of a [Im-Ethyl-Men][PF
6
] and [Im-Ethyl-Men][ClO
4
] on the content of proline
-1
-1
-1
(mg g FW); malondialdehyde (μmol g FW) and hydrogen peroxide (μmol g FW) in wheat
seedlings. Data are expressed as a mean ± SE of three replicates for each concentration. Values
denoted by the same letters in the columns do not differ statistically at p<0.05.
Concentration
Contents
MDA
-
of ILs (mg kg
1
Proline
mg g FW]
H
2
O
2
-1
soil DW)
-1
-1
[
[μmol g FW]
[μmol g FW]
23.661 ± 0.214a
22.532 ± 0.069b
16.611 ± 0.293c
b
35.757 ± 0.395b
34.922 ± 0.329b
40.272 ± 0.426a
-
0
1
22.549 ± 1.628
27.308 ± 0.030b
1
0
40.549 ± 1.166a
1
00
-
-
1
000
0
-
-
-
22.549 ± 1.628b
28.643 ± 1.170b
75.958 ± 1.842a
81.242 ± 1.479a
-
35.757 ± 0.395c
40.407 ± 0.290b
45.706 ± 0.074a
46.402 ± 0.291a
-
23.661 ± 0.214a
21.677 ± 0.353b
19.344 ± 0.037c
17.525 ± 0.128d
-
1
1
0
1
00
1
000
4
4
07
08
4
09
Effects of CILs on the Generation Rate of H
2
2
O and Lipid Peroxidation
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10
11
12
13
14
15
16
17
To determine the level of oxidative stress in plants exposed to CILs, the content of MDA
was also determined. MDA is a decomposition product of polyunsaturated fatty acids present
in the protein-lipid membranes (mainly linoleic acid). The increase in MDA content in plant
cells is an indisputable indicator of prevailing oxidative stress and also indirectly reflects the
degree of cell damage. An increase of MDA in wheat seedlings was observed with the increase
of the content of CILs (Table 4). This increase is explained by the ILs causing strong oxidative
stress that prevents the plants from removing ROS.1,29,561,35,62 This results in increased
peroxidation of the protein-lipid membranes that surround different cellular organelles.
4
4
4
4
4
4
4
4
4
4
4
4
4
18
19
20
21
22
23
24
25
26
27
28
29
30
The amount of H
2
O in plant cells is another very important marker of oxidative stress
2
in plants. The accumulation of H
2
O
2
in plants is always observed when an intensive
detoxication of the superoxide anion radical (O −

2
) is catalyzed by superoxide dismutase and
when the plant’s defensive mechanisms against H
2
O
2
fail.49,51,5755,57,63
H
2
O is also the most
2
stable chemical molecule of all ROS, capable of rapidly penetrating all cell membranes.5864
A
decrease in H
2
2
O content was observed with the increase of CIL concentration (Table 4), which
may be related to the increased effect of hydrogen peroxide decomposing the enzymes CAT
and POD. However, it appears that oxidative stress in plants is not always manifested by a
decrease in H
2
O
2
content according to the literature. Zhang et al.4955 and Biczak4147 studied
content in these plants under the
duckweed and radish and indicated an increase in H
2
O
2
influence of 1-octyl-3-methylimidazolium bromide and selected quaternary ammonium salts
with hexafluorphosphate anion, respectively. The situation may be related to the various
activities of H
2
2
O detoxifying enzymes in various plants.
4
4
4
4
31
32
33
34
Effects of CILs on antioxidant enzymes activities
To overcome oxidative stress, plants have developed an enzymatic defense system that
includes SOD, CAT, POD, and GR. The first line of defense against ROS is SOD, which
•-
decomposes the superoxide anion radical into H
2
O
2
and O
2
. The elimination of O
2
by SOD
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40
41
42
43
44
prevents lipid peroxidation, which is why the activity of this enzyme is one of the first to be
determined in studies on the determination of the impact of oxidative stress on biochemical and
physiological changes in plants.4652 However, the literature does not allow for an unambiguous
determination of the direction of changes of SOD activity in plants exposed to oxidative stress.
Increases in SOD activity under the influence of ILs in the substrate were observed by
Liu et al.5662 for wheat seedlings, Cvjetko Bubalo et al. for barley, and Biczak
1
4147
for common
radish. The present study did not reveal any significant changes in SOD activity in wheat
seedlings under the influence of the CILs (Table 5). A similar lack of SOD activity was also
observed by Rozpądek et al.5965 in cabbage subjected to oxidative stress caused by ozone
fumigation.
4
4
4
4
45
46
47
48
Table 5. Enzymatic activities of SOD; CAT and POD in wheat seedlings treated with [Im-
Ethyl-Men][PF
6
] and [Im-Ethyl-Men][ClO ]. Data are expressed as a mean ± SE of three
4
replicates for each concentration. Values denoted by the same letters in the columns do not
differ statistically at p<0.05.
Concentration
of CILs (mg
kg soil DW)
The activity of enzymes
Superoxide dismutase
Catalase
Peroxidase
-
1
-1
-1
-1
-1
-1
-1
-1
-1
[
U mg protein ]
11.723 ± 0.293a
11.504 ± 0.303a
11.796 ± 0.124a
-
[U mg protein min ] [U mg protein min ]
0
1
0.0520 ± 0.0020b
0.0528 ± 0.0016b
0.0778 ± 0.0000a
-
10.928 ± 0.196c
13.968 ± 0.136b
16.480 ± 0.323a
-
1
0
1
00
1
000
0
-
-
-
11.723 ± 0.293a
10.165 ± 0.177b
10.020 ± 0.064b
9.377 ± 0.208b
-
0.0520 ± 0.0020c
0.0536 ± 0.0008c
0.0712 ± 0.0014b
0.0807 ± 0.0012a
-
10.928 ± 0.16d
12.604 ± 0.526c
14.538 ± 0.183b
16.737 ± 0,180a
-
1
1
0
1
00
1
000
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4
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As a result of O −

dismutation, a very strong H
O
particle is formed, which has the
2
2
2
ability to penetrate all cellular membranes. To protect their cells, plants have developed two
oxidative enzymes, CAT and POD, which carry out H detoxification. Catalase is mainly
2
O
2
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located in peroxisomes and glyoxysomes and breaks down H
2
O
2
directly into H
2
O and O
2
.
Peroxidase occurs in many cell organelles and is responsible for the degradation of H
2
O
2
with
the participation of phenolic compounds (pyrogallol, benzidine and guaiacol) or some
antioxidants (ascorbic acid).4652 CAT and POD activity increased with the increase of CILs in
soil (Table 5). This is corroborated by previous studies that observed an increase of the activity
of these antioxidant enzymes in plants subjected to stress factors.46,5052,56
A largely typical marker of oxidative stress is the increase of POD activity, which
always increases when the plant is subjected to stress factors and as long as the plant is able to
vegetate in such conditions.2834 It is also believed that an increase in peroxidase activity and
simultaneous decrease in chlorophyll content is a clear indication of premature aging of a
plant.4147 Furthermore, according to Wang et al.2834 Liu et al.2935 and Biczak4147 peroxidase
activity is the most reliable biomarker of the presence of oxidative stress in plants.
In summary, new quaternary imidazolium salts containing a natural-origin material,
(1R,2S,5R)-(–)-menthol, were successfully prepared very high efficiency. The synthesized
compounds are solid and stable in air, water, and commonly used organic solvents. The salts
o
are ILs since their melting points do not exceed 100 C, and they are also CILs since they
possess three chiral centres in the cation counterpart. The anion exchange processes proceeded
smoothly with efficiency exceeding 98% in each case. The general properties such as the
melting point, solubility, and surfactant content of the chiral ILs were studied.
The biological results clearly demonstrate that [Im-Ethyl-Men][PF
6
] and [Im-Ethyl-
Men][ClO ] exhibit phytotoxicity toward wheat seedlings. The level of this influence primarily
4
depended on the compound concentration. The CILs with hexafluorphosphate anion exhibited
slightly stronger phytotoxic properties. The decrease in the level of fresh weight of the plants
resulted from the decrease of the content of photosynthetic pigments, which are responsible for
the primary production of green plants.
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The CILs used in the pot experiment produced oxidative stress in the plants, which was
demonstrated by the increased proline content, MDA content, and activity of CAT and POD.
This led to a decrease in the level of H
2
2
O in the wheat seedlings. The data obtained could be
used to evaluate the risk that these chemical compounds pose to the natural environment and
could be applied for their industrial use.
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Acknowledgments:
The work was financed by a statutory activity subsidy from the Polish Ministry of Science and Higher
Education for The Faculty of Mathematics and Natural Sciences of Jan Dlugosz University in
Czestochowa, Faculty of Chemistry of Wrocław University of Science and Technology and The Faculty
of Environmental Management and Agriculture, West Pomeranian University of Technology in
Szczecin.
Conflicts of Interest:
The authors declare no conflict of interest.
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