Esterquat herbicidal ionic liquids (HILs) with two different herbicides: Evaluation of activity and phytotoxicity

Article abstract of DOI:10.1039/c8nj01239c

Herbicidal ionic liquids (HILs) constitute a new concept in crop protection products. Their main advantage is the potential to combine the efficiency of traditional herbicides with low vapour pressure and adjustable water solubility which leads to improved environmental safety in the agricultural sector. Among many strategies to obtain new HILs, esterquats seem to be well suited for modification since both the cation and anion may be constituents of herbicides. In the framework of this study 16 new esterquat HILs were synthetized based on standard herbicides: 2,4-D, MCPA, MCPP, 4-CPA, Clopyralid and Dicamba. Germination tests performed on agricultural soil using cornflower indicated the best two HILs. Furthermore, analysis of the toxicological effects of HILs on wheat plants revealed an additional advantage of the two selected HILs. The glutathione (GSH) content and glutathione S-transferase (GST) activity showed a lower oxidative stress level in wheat plants treated with examined HILs, respectively, in comparison to a mixture of reference compounds. Finally the Ames test was applied in order to analyse the mutagenic activity of the two selected HILs.

Full text of DOI:10.1039/c8nj01239c

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. Chrzanowski, New J. Chem., 2018, DOI: 10.1039/C8NJ01239C.
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Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
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ARTICLE
Esterquat Herbicidal Ionic Liquids (HILs) with two different
herbicides: evaluation of activity and phytotoxicity
Received 00th January 20xx,
b
c
a
Anna Syguda,*a Anna Gielnik, Andrzej Borkowski, Marta Woźniak-Karczewska, Anna Parus,
aAneta Piechalak, b Anna Olejnik, d Roman Marecik, d Łukasz Ławniczak a and Łukasz Chrzanowski a
Herbicidal ionic liquids (HILs) constitute a new concept in crop protection products. Their main advantage is the potential
to combine efficiency of traditional herbicides with low vapour pressure and adjustable water solubility which leads to
improved environmental safety in the agricultural sector. Among many strategies to obtain new HILs, esterquats seem to
be well suited for modification since both the cation and anion may be composed of herbicides. In the framework of this
study 16 new esterquat HILs were synthetized based on standard herbicides: 2,4-D, MCPA, MCPP, 4-CPA, Clopyralid and
Dicamba. Germination tests performed on agricultural soil against cornflower indicated the best two HILs. Furthermore
analysis of toxicological effect of HILs towards wheat plants revealed an additional advantage of two selected HILs. The
glutathione (GSH) content and glutathione S-transferase (GST) activity showed lower oxidative stress level in wheat plants
treated with examined HILs, respectively, in comparison to the mixture of reference compounds. Finally Ames test was
applied in order to analyse the mutagenic activity of two selected HILs.
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
auxins can be divided into twelve main chemical families based on
the structure of the active ingredients. In plants, auxins are
responsible for elongation of shoot and root as well as tropism
Introduction
Ionic liquids (ILs) have gained worldwide popularity due to their
promising properties and numerous applications. Potentially the
utilization of ILs is unlimited1, however after one decade there are
some fields of exploitation which are considered most important,
such as solvents for organic syntheses2, catalysis3, extraction4 and
gas storage5. Furthermore, ILs are extensively researched for energy
storage purposes6-9 and applications in the paper industry due to
their ability to dissolve cellulose.10
functions, however at high concentrations they can be toxic for
plants due to abnormal development of organs and inhibition of
tissue growth.26
It should be noted that herbicides can be hazardous to natural
and semi-natural environments due to possible contamination of
aquatic and soil ecosystems.27 The main undesirable feature of the
most popular synthetic auxin - 2,4-D - is the high water solubility of
its ammonium and sodium salts, which results in water mobility and
further risk of ground and surface waters contamination.28 Another
adverse property of 2,4-D is its high volatility, which poses a risk
mainly to agricultural workers. To improve the safety of use, it is
desirable to develop new forms of 2,4-D.
Recently, the possible formation of herbicidal ionic liquids
(HILs) has gained increasing popularity.11-23 HILs represent an
alternative to well-known herbicides and provide an opportunity to
combine efficient crop protection with environmental safety due to
their unique physicochemical properties. Herbicides are commonly
used all over the world in agriculture and are often considered as
necessary to obtain efficient crops. One of the most popular group
of herbicides used to protect monocotyledons (mainly family
Poaceae) against dicotyledons are synthetic auxins, also known as
growth regulators.13,24 According to Forouzesh et al.25, synthetic
Furthermore, the prolonged exposure of weeds to a herbicide
may result in the development of resistance, which is currently a
crucial problem. Increased resistance is the result of the overuse of
plant protection products. There are numerous reports regarding
new weed species, which exhibit resistance to synthetic auxins. Two
hundred and twenty weed species have evolved resistance to one
or more herbicides, and there are now 404 unique cases (species,
site of action) of herbicide-resistant weeds globally29 and there is a
strong need to develop new chemical solutions which will increase
herbicidal diversity and provide future alternatives for elimination
of resistant weeds.
HILs offer a possibility to solve both problems of synthetic
auxins herbicides
– to improve the undesirable properties
(reduction of volatility and water solubility) and negate the
resistance of weeds. The unusual capabilities of ILs give an
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opportunity to rearrange known herbicides and present them in a two active herbicides may potentially result in an improved range of
new form, with improved physicochemical properties,30,31 which weed control, as a single HIL would exhibit activity against two
makes them a promising group of new generation herbicides in different types of weed. Additional advantages of the newly
perspective of environmental and human safety.12
designed HILs, in comparison to conventional herbicides, also
The aim of the study was to design new HILs based on include high surface activity and low contact angle of a liquid-leaf
herbicidal esterquats with herbicidal anions and to evaluate their area, which is especially important in foliar application and
herbicidal efficacy. HILs analyzed in this study are based on prevention of substance dropping from the leaf surface.13 These
following chemical groups: phenoxy-acids (2,4-D, MCPA, MCPP, 4- unique surface properties were obtained by incorporating a long
CPA),
chloropicolinic-acid
(Clopyralid),
chlorobenzoic-acid chain alkyl substituent (C10H21) in the cation of each HIL. Low
(Dicamba). They belong to synthetic auxins and are analogues of volatility of HILs is another important advantage, since crop
the natural phytohormone, indole-3-acetic acid (IAA), the most treatment requires wide-scale application of herbicides on the field.
important component in auxin composition. Germination tests The low volatility of HILs results in decreased inhalation risks for
using cornflower (Centaurea cyanus) were conducted in order to workers during spray treatments and reduced risk of unintended
establish the herbicidal activity of the studied HILs. Two transport, which relates to mitigation of the negative consequences
compounds, characterized by the highest rate of cornflower growth for non-target organisms.13,15
inhibition were selected for further studies regarding their impact
In the framework of this study 16 new herbicidal salts were
on homeostasis of crop plants. The analysis includes toxicological obtained (Table 1), which comprised a herbicide in both the cation
tests on common wheat (Triticum aestivum), with the involvement and the anion (Scheme 1). Each compound was a liquid at room
of oxidative stress markers: glutathione S-transferase (GST) and temperature, which allows to classify the synthesized salts as
glutathione in reduced (GSH) and oxidised (GSSG) form.
herbicidal ionic liquids. Overall, the HILs may be divided into a
group which contains derivatives of phenoxy-acids in both the
cation and the anion (HILs A) and the group which comprises
phenoxy-acid derivatives in the cation and other herbicides
(Dicamba or Clopyralid) as the anion (HILs B). The yield of the
obtained HILs ranged from 80% for[MCPA-DAE-C10][MCPP] to 99%
for [MCPA-DAE-C10][Dicamba]. Overall, the synthesized HILs were
characterized by high purity, as evidenced by the measured values
of surfactant content which exceeded 91% and which were
determined by a direct two-phase titration technique (EN ISO 2871-
1,2:2010). The introduction of two different herbicides in the
structure of esterquat ionic liquids is a new concept, which has not
been previously described in the literature, therefore the obtained
group of compounds introduces scientific novelty to the current
state-of-the-art.
Results and discussion
Firstly synthesized HILs comprised a quaternary ammonium cation
and synthetic auxin as the anion.11 HILs were also synthesized in the
form of esterquats, in which the herbicide center remained in the
anion.11 The transitional stage was the tests in which the herbicide
was placed in the cation as an ester substituent and the anion was
inorganic.17 These herbicidal esterquats were precursors of ionic
liquids. Introduction of an anionic herbicide to obtain a system
containing two herbicide centers was the next research stage. By
synthesizing the herbicidal esterquats, a molecule can be designed
to include two different herbicides in its structure - one in the ester
substituent of the cation and another as the anion. The presence of
R1
CH3
Cl
R2
CH3
H
Abbreviation
[MCPP]
[2,4-D]
Abbreviation
[Clopyralid]
Abbreviation
[Dicamba]
CH3
H
H
H
[MCPA]
[4-CPA]
Scheme 1 Synthesis of herbicidal esterquats with herbicidal anions (HILs).
2 | J. Name., 2012, 00, 1-3
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Table 1 Herbicidal esterquats with herbicidal anions (ionic liquids) according to Scheme 1
ARTICLE
HILs
Abbreviation of ionic liquid
R1
in the cation
R2
Anion
Surfactant content
[%]
99.5
Yield
[%]
89
92
89
91
80
81
94
95
86
89
87
85
90
99
96
95
HILs A
[2,4-D-DAE-C10][MCPA]
[2,4-D-DAE-C10][MCPP]
[2,4-D-DAE-C10][4-CPA]
[MCPA-DAE-C10][2,4-D]
[MCPA-DAE-C10][MCPP]
[MCPA-DAE-C10][4-CPA]
[MCPP-DAE-C10][2,4-D]
[MCPP-DAE-C10][MCPA]
[MCPP-DAE-C10][4-CPA]
[4-CPA-DAE-C10][2,4-D]
[4-CPA-DAE-C10][MCPA]
[4-CPA-DAE-C10][MCPP]
[MCPA-DAE-C10][Clopyralid]
[MCPA-DAE-C10][Dicamba]
[4-CPA-DAE-C10][Clopyralid]
[4-CPA-DAE-C10][Dicamba]
Cl
Cl
Cl
H
H
H
H
H
[MCPA]
[MCPP]
[4-CPA]
[2,4-D]
[MCPP]
[4-CPA]
[2,4-D]
[MCPA]
[4-CPA]
99.5
96.0
95.0
92.0
92.0
99.0
98.5
99.0
95.0
94.0
94.0
97.0
98.0
97.0
96.5
CH3
CH3
CH3
CH3
CH3
CH3
H
H
CH3
CH3
CH3
H
[2,4-D]
[MCPA]
[MCPP]
H
H
H
H
HILs B
CH3
CH3
Cl
H
H
H
H
[Clopyralid]
[Dicamba]
[Clopyralid]
[Dicamba]
Cl
The obtained results of germination tests were shown in the ESI The next parameter which was analysed is the inhibition of shoot
(Table S5). Control showed 100% germination whereas the and root growth of cornflower. The obtained results were divided
reference herbicide reduced the average germination from 77 into two parts and the data compiled for HILs A were shown in the
(4-CPA + Clopyralid) to 95% (2,4-D + MCPP) All of the tested HILs Fig. 1, whereas the effects of HILs B were shown on the Fig. 2.
exhibited a potent ability to inhibit the development of cornflower. Strong inhibition of seed germination and growth of cornflower was
The values of GI oscillated from 33% [4-CPA-DAE-C10][Dicamba] to recorded in the soil which contained [MCPP-DAE-C10][2,4-D],
0% [2,4-D-DAE-C10][MCPP]. And only [2,4-D-DAE-C10][MCPP] caused however it was comparable with the results obtained for the soil
complete inhibition of germination and growth of cornflower and containing the mixture of commercial herbicide (MCPP + 2,4-D). On
exhibited the strongest toxic effect among all analyses herbicides. the other hand, the presence of [2,4-D-DAE-C10][MCPP] in the soil
0 – control, 1 – 2,4-D + MCPA, 2 – [2,4-D-
DAE-C10][MCPA], 3 – [MCPA-DAE-C10][2,4-
D], 4 – 2,4-D + MCPP, 5 – [2,4-D-DAE-
C10][MCPP],
7 – 2,4-D
C10][4-CPA],
6
–
[MCPP-DAE-C10][2,4-D],
4-CPA, [2,4-D-DAE-
[4-CPA-DAE-C10][2,4-D],
+
8 –
9
–
10 – MCPA + MCPP, 11 – [MCPA-DAE-
C10][MCPP], 12 – [MCPP-DAE-C10][MCPA],
13 – MCPA + 4-CPA, 14 – [MCPA-DAE-
C10][4-CPA], 15 – [4-CPA-DAE-C10][MCPA],
16 – MCPP + 4-CPA, 17 – [MCPP-DAE-
C10][4-CPA], 18 – [4-CPA-DAE-C10][MCPP].
Fig. 1 Average length of root and shoot for seedlings in soil with addition of HILs A and reference herbicides.
0
– control, 1 – MCPA + Clopyralid,
2 – [MCPA-DAE-C10][Clopyralid], 3 – MCPA
+ Dicamba, 4 – [MCPA-DAE-C10][Dicamba],
5 – 4-CPA + Clopyralid, 6 – [4-CPA-DAE-
C10][Clopyralid],
7 – 4CPA + Dicamba,
8 – [4-CPA-DAE-C10][Dicamba].
Fig. 2 Average length of root and shoot for seedlings in soil with addition of HILs B and reference herbicides.
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led to a complete inhibition of germination of cornflower seeds. The + MCPP and MCPA + Clopyralid), and vegetation changes were
greatest inhibitory effect on seed germination among HILs B was observed after application of these substances after 7 days from
noted for the [MCPA-DAE-C10][Clopyralid], however it was similar to herbicide spraying. Only cornflower showed a significant reduction
the results obtained for the mixture of commercial herbicides in the development of shoot parts, which was illustrated by the
(MCPA + Clopyralid).
wilting and curling of plant leaves. Such changes have not been
The obtained results stand in good agreement with the previous observed for wheat treated by [2,4-D-DAE-C10][MCPP] and
reports regarding the herbicidal activity of HILs. In a recent study [MCPA-DAE-C10][Clopyralid].
regarding the herbicidal activity of ionic liquids based on betaine, it
The analysed HILs did not exhibit toxic effects towards wheat
was established that HILs comprising MCPP and MCPA as the anions seedlings and did not cause growth and vegetation inhibition.
exhibited a high efficacy (95-100%) towards cornflower.32 Another Because this type of compounds can penetrate inside the plant and
study reported that the fresh weight reduction of cornflower was 4 cause oxidative stress through overproduction of reactive oxygen
times higher compared to control when HILs based on 2,4-D were species (ROS), the evaluation of the activity of plant markers of the
used.33 The study of Zhu et al. regarding HILs based on Clopyralid defence system, GST, SOD, APX, CAT enzymes, as well as
also indicates that the introduction of the ionic liquids structure quantitative analysis of non-enzymatic factors, GSH and chlorophyll
contributes to a more efficient and more rapid herbicidal effect a and b, was carried out.
compared to the commercial formulation.13 This corresponds well
Analysis of the effects of HILs and reference herbicide mixtures
with the results presented in the framework of this study, as HILs on the content of wheat assimilation pigments clearly
comprising a combination of 2,4-D + MCPP and MCPA + Clopyralid demonstrated the inhibitory effect of RH on chlorophyll b level,
displayed a potent ability to inhibit the germination (germination at whereas in plants treated with HILs a slight increase in chlorophyll a
0 and 3%, respectively) as well as to reduce the shoot and root and b level was observed (Table 2). The next analysed parameter
growth (by 100 and 85%, respectively) of cornflower. Other studies was the ratio of chlorophyll a to chlorophyll b (Chla/Chlb). An
regarding oligomeric HILs incorporating two different herbicides as increase in the Chla/Chlb ratio was observed for plants treated with
anions indicate that such a solution may result in high efficacy the reference mixture (MCPP + 2,4-D and MCPA + Clopyralid)
towards different weed species (e.g. common lambsquarters, resulting from the decrease in chlorophyll b in these plants. In
oilseed rape, black bindweed or white mustard).16,34 This allows for contrast, the Chla/Chlb ratio in plants sprayed with HILs was similar
a significant increase of the herbicidal activity by 10-20% compared to that of the control plants.
to commercial formulations34 with subsequent reduction of the
herbicide dose per hectare.16 It is plausible that the effect of
The activity of superoxide dismutase in the tested samples
combinations of two different herbicides used as either cations or remained at a similar level in all crop variants (Table 2). The highest
anions may differ depending on the species and possibly result in SOD activity was observed in plants treated with a solution of
the introduction of an additional pesticidal functionality35, which [2,4-D-DAE-C10][MCPP] and [MCPA-DAE-C10][Clopyralid] in which
opens a new pathway for future studies.
the increase in enzymatic activity was approx. higher 10% relative
On the basis of the inhibition growth of root, two of the most to the control. The level of catalase activity (CAT) in plants treated
efficient compounds ([2,4-D-DAE-C10][MCPP] and [MCPA-DAE- with a mixture of reference herbicides was comparable to the level
C10][Clopyralid]) were selected for further studies, which included recorded in control samples (Table 2). On the other hand, in plants
the comparison of their activity with reference herbicides as well as treated with HILs, catalase activity was increased by 30% in the case
the assessment of their effect on the physiological equilibrium of of [2,4-D-DAE-C10][MCPP], while a three-fold increase was noted in
wheat leaf cells.
the case of [MCPA-DAE-C10][Clopyralid]. Ascorbic peroxidase was
In order to evaluate the impact of HILs on wheat and another analysed antioxidant enzyme (Table 2). An increase in APX
cornflower, the plants were treated with [2,4-D-DAE-C10][MCPP] and levels by 36% was observed in case of plants treated [MCPA-DAE-
[MCPA-DAE-C10][Clopyralid] and reference herbicide mixtures (2,4-D
C10][Clopyralid], but in the case of plants treated with reference
Fig. 3 Changes in the activity of GST in wheat plants, seven
days after treatment with HILs or reference herbicides.
4 | J. Name., 2012, 00, 1-3
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Chla
Chla
Chlb
Chla/Chlb
SOD
CAT
APX
+Chlb
3.180
2.710
3.280
2.020
1.200
Control
1.450 ± 0.101 1.730 ± 0.201
1.510 ± 0.102 1.200 ± 0.106
1.470 ± 0.109 1.810 ± 0.106
1.420 ± 0.203 0.600 ± 0.105
0.839
1.258
0.812
2.367
0.810
4.13 ± 0.34
4.23 ± 0.19
4.62 ± 0.22
4.35 ± 0.38
4.58 ± 0.11
0.45 ± 0.0014
0.41 ± 0.0017
0.59 ± 0.0013
0.42 ± 0.0023
1.30 ± 0.0016
0.22 ± 0.032
0.26 ± 0.022
0.25 ± 0.010
0.31 ± 0.016
0.33 ± 0.013
2,4-D + MCPP
[2,4-D-DAE-C10][MCPP]
MCPA + Clopyralid
[MCPA-DAE-C10][Clopyralid] 1.500 ± 0.106 1.250 ± 0.103
herbicide (MCPA + Clopyralid). A similar increase in APX was aromatic nitroso derivatives of amine carcinogens.34 In addition,
observed (approx. 30%) compared to control plants. [2,4-D-DAE-C10][MCPP], induced significant increase in the
a
GST is a detoxification enzyme, responsible for deactivation of number of revertant colonies in TA100 strain, so that the frequency
toxins, by catalysing the reaction of the conjugate formation of mutations increased 2-fold (MI = 2.0). The strain TA100 carrying
between GSH and xenobiotics.32 The activity of GST in plant cells hisG46 mutation was sensitive to the treatment with [2,4-D-DAE-
provides useful information regarding the toxicological state of the C10][MCPP] that caused base-pair substitution mutations probably
plant. Changes in the activity of GST were observed depending on at one of the GC pairs. The hisG46 marker in strains TA100 and
the applied treatment (Fig. 3). The lowest activity of GST has been TA1535 results from the substitution of a leucine (GAG/CTC) by a
observed in plants treated with reference herbicides. The obtained proline (GGG/CCC).35 In addition, the potential promutagenic
values for solution of 2,4-D + MCPP and MCPA + Clopyralid activity of [2,4-D-DAE-C10][MCPP] was detected in the strains
corresponded to 71% and 60% of their ionic equivalent. The activity TA1535 and TA1537, for which the frequency of mutations induced
of GST in plants exposed to [2,4-D-DAE-C10][MCPP], and [MCPA- by [2,4-D-DAE-C10][MCPP] treated with microsomal fraction
DAE-C10][Clopyralid] did not significantly differ in relation to the increased to 1.7 and 1.9, respectively. Strain TA1537 which carries
activity observed for the control. These results correspond well with the hisC3076 mutation appears to have a +1 frameshift mutation
the obtained GSH values.
near the site of a repetitive –C–C–C– sequence and is reverted to
Glutathione (GSH) is the most important part of antioxidative the wild-type state by frameshift mutagensthat are not readily
system among non-enzymatic low molecular weight antioxidants. detected by the hisD3052 marker.34 In contrast, [MCPA-DAE-
The level of GSH and GSSG in the plant cell, expressed as GSH/GSSG C10][Clopyralid] compound did not cause a significant increase in
ratio, is an important tool in monitoring of plant homeostasis.33 the frequency of mutations in any of the standard tester strains
Under conditions of physiological stress, the ratio decreases, as GSH both in the presence and absence of microsomal fraction. The MI
is involved in binding of ROS generated during action of stressor and values for [MCPA-DAE-C10][Clopyralid] were calculated at the level
in reparation of oxidated cellular elements, such as peptides, which of 1.6 or less.
results in disruption of the redox state of the cell. The level of GSH
in wheat (Triticum aestivum) treated with [2,4-D-DAE-C10][MCPP]
Conclusions
and [MCPA-DAE-C10][Clopyralid], remained at a high level, but
significantly differ in relation to control plants (Fig. 4). The lowest
level of GSH was observed in plants treated with mixtures of
reference herbicides (MCPP + 2,4-D and MCPA + Clopyralid).
Significant reduction in the amount of GSH resulted in decrease of
GSH/GSSG ratio, which is a sign of disordered redox state of the
cells.
In conclusions, 16 new esterquat HILs with two different herbicides
were synthesized. Germination testes, indicated that the studied
HILs exhibited
a high efficacy of inhibition of cornflower
development. Additionally, performed analysis of the activity of
detoxification enzyme GST and assessment of the redox state of the
wheat cells indicated a lower level of stress in crop plants sprayed
with two HILs: [2,4-D-DAE-C10][MCPP] and [MCPA-DAE-
C10][Clopyralid] in comparison with the reference herbicide
mixtures (2,4-D + MCPP and MCPA + Clopyralid). Esterquat HILs
with two different herbicides can be considered as an alternative to
conventional herbicides.
The obtained results of mutagenic activity indicated that
reference
mutagens
(2-aminofluorene,
sodium
azide,
2-aminoacridine, 2-aminoanthracene) used as positive controls
caused a significant response in the tester strains of S. typhimurium,
as reflected by the increased number of revertant colonies and MI
exceeding 2.0. A positive mutagenic response, attributed to the
doubling of revertant colonies, was also observed in the TA98 strain
following exposure to [2,4-D-DAE-C10][MCPP] without metabolic
activation. The obtained results suggest that [2,4-D-DAE-C10][MCPP]
is able to revert the hisD3052 mutation carried by strain TA98 and
can produce genetic damage by frameshift mutation. Reversion of
the hisD3052 mutation back to the wild-type state is induced by
various frameshift mutagens such as 2-nitrofluorene and various
Experimental
Materials
2-Dimetyhaminoetanol (DAE) 99.5%, 1-bromodecane 98%, (2,4-
dichlorophenoxy)acetic acid (2,4-D) 98%, triethylamine 99.5%,
tionyl chloride 97% and active carbon were purchased from Sigma-
Aldrich. (4-Chlorophenoxy)acetic acid (4-CPA) 98% was purchased
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mL of acetone then slowly added in a dropwise manner. The flask
was equipped with a reflux condenser and heated to 55 °C for 24
hours, then placed in a cooler in order to obtain the crystalline
product. The product was filtered under vacuum, rinsed with
hexane and dried. The reaction was presented in Scheme S4 and
the product yields were given in Table S4, which can be found in the
ESI.
from
Koch-Light
Laboratories
Ltd.
(4-Chloro-2-
methylphenoxy)acetic acid (MCPA) 97% and (±)-2-(4-chloro-2-
methylphenoxy)propionic acid (MCPP) 96% (3,6-dichloro-2-
methoxy)benzoic acid (Dicamba) 95%, (3,6-dichloro)-2-picolinic acid
97% (Clopyralid) were purchased from Organika-Sarzyna (Poland).
All solvents, acetone, isopropanol, ethyl acetate, chloroform,
toluene, hexane, and NaOH were purchased from Sigma-Aldrich
(Poland) and used without further purification. Herbicides used in
synthesis (2,4-D; MCPA; MCPP; 4-CPA; Clopyralid and Dicamba)
were first purified by dissolution in the hot toluene, addition
activated carbon, filtration of the hot solution to remove colored
impurities absorbed on the activated carbon and crystallization
from the cold toluene.
Esterquats with herbicidal anions (HILs). Solutions were
prepared by dissolving 6.5 g of the appropriate esterquat bromide
in 100 mL of deionized water and 50 mL of isopropanol. The
appropriate acidic form of a herbicide selected as the anion (2,4-D;
MCPA; MCPP; 4-CPA; Clopyralid or Dicamba) was introduced into a
separate flask containing equimolar amount of 10% water solution
of NaOH and 30 mL of water in order to obtain a stoichiometric
amount of herbicidal sodium salt. The mixture was heated until the
solution became clear. Afterwards, the solution containing the
herbicidal sodium salt was carefully introduced into the flask
containing the water-isopropanol solution of the precursor and the
combined solution was stirred with a spatula. Next, the combined
solution was introduced into a separator and 50 mL of chloroform
were added. After shaking and separation, the bottom organic
phase (which contained the ionic liquid) was rinsed five times with
deionized water (50 mL) in order to remove the residual bromides.
The organic phase was then introduced into a round bottom flask
(100 mL) and the solvent was removed using a rotary evaporator.
Finally, the flask containing the product was placed in a vacuum
dryer and dried until the mass was stable. An alternative method
for the isolation of the product from the reaction mixture includes a
total evaporation of water and isopropanol, followed by the
extraction of the ionic liquid with anhydrous acetone. However, in
this case the foaming of compounds during the evaporation was a
problem as it resulted in notable losses of the product and was time
consuming. The reaction was presented in Scheme 1 and the
product yields were given in Table 1.
Synthesis
Phenoxy-acid chlorides. A 100 g portion of appropriate phenoxy-
acid (2,4-D, MCPA, MCPP or 4-CPA) was introduced into a round
bottom flask (500 mL) equipped with a funnel and magnetic stirrer,
and thionyl chloride (at threefold excess) was slowly added
dropwise into the flask. The reaction was conducted for 2 hours at
75 °C. Afterwards, the excessive amount of thionyl chloride was
removed by distillation (the fraction was collected at 76 – 78 °C)
and the obtained phenoxy-acid chloride was distilled two times
under vacuum. The reaction was presented in Scheme S1 and the
product yields were given in Table S1, which can be found in the
Electronic Supplementary Information (ESI).
Aminoester hydrochlorides. Chloroform solutions of phenoxy-
acid chlorides and 2-dimethylaminoethanol (DAE) were prepared by
dissolving 80 g of appropriate phenoxy-acid chloride in 100 mL of
chloroform and an equimolar amount of DAE in 100 mL of
chloroform. Appropriate chloroform solution of phenoxy-acid
chloride was slowly added in a dropwise manner into a round
bottom flask (500 mL) filled with the chloroform solution of DAE,
which was equipped with a magnetic stirrer and an ice-water bath.
The product emerged in the whole volume of the flask. Next, the
product was filtered under vacuum, rinsed two times with hexane
and dried. The reaction was presented in Scheme S2 and the
product yields were given in Table S2, which can be found in the
ESI.
Soil samples
Agricultural soil was used in the experiment. The subsoil samples
from the depth of 10-20 cm were collected separately in sterile
plastic jars according to the procedure described by Alef and
Nannipieri36 and stored at 4 °C until use. In the samples, organic
matter (OM) and ash content was determined after drying at
550 °C.37 Soil pH was determined in a suspension in distilled water
and KCl (with a solid to liquid phase ratio of 1.0:2.5) by an
electrometric method (pH-meter Elmetron CPI-551). CaCO3 content
was determined by Scheibler method.38 Granulometric analysis
showed that in this study, loam was used as a soil and was
characterized by the following particle size profile: 1 – 0.1 mm =
95%, 0.1 – 0.02 mm = 5%, < 0.02 mm 0%. For the experiment, the
soil with the following elemental composition was used: P 81 mg
kg-1 of soil, K 88 mg kg-1 of soil, Mg 69 mg kg-1 of soil, pH 5.92 (in
KCl), C organic content of 1.01% (10.1 g kg-1 of soil), 2.4 mg N-
NH4/kg of soil and 9.2 mg N-NO3/kg of soil.
Aminoesters. A 100 g suspension of an appropriate aminoester
hydrochloride in 300 mL of chloroform was introduced into a round
bottom flask (1000 mL) and an equimolar amount of triethylamine
was added. The flask was heated in order to achieve
homogenisation of the mixture. After removal of chloroform using a
rotary evaporator, the residues were suspended in 200 mL of
hexane, filtered under vacuum and rinsed with hexane. The filtrate
was introduced into a separator and rinsed three times with 100 mL
of deionized water. Afterwards, the product was obtained by
removal of hexane using a rotary evaporator. The reaction was
presented in Scheme S3 and the product yields were given in Table
S3, which can be found in the ESI.
Esterquat bromides. Acetone solutions of aminoesters were
prepared by dissolving 25 g of an appropriate aminoester in 25 mL
of acetone. The solutions were introduced into round bottom flasks
(250 mL) and then an equimolar solution of 1-bromodecane in 25
Analysis
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1H NMR spectra were recorded on a Varian VNMR-S spectrometer extract was centrifuged for 30 min at 4 °C and 12500 rpm (25000 ×
operating at 300 MHz with tetramethylsilane as the internal g).
standard. 13C NMR spectra were obtained with the same instrument
Antioxidant enzymes activity. The activity of superoxide
at 75 MHz. CHN elemental analyses were performed at Adam dismutase (SOD) was assayed by measuring its ability to inhibit the
Mickiewicz University, Poznan (Poland).
photochemical reduction of nitrobluetetrazolium (NBT), according
to the method of Beauchamp and Fridovich.40 The reaction mixture
contained 13 μM of riboflavin, 13 mM of methionine, 63 μM of NBT,
and 50 mM of potassium phosphate buffer (pH 7.4). Absorbance
was then measured at 560 nm. One unit of SOD activity has been
defined as the amount of enzyme, which causes a 50% decrease of
the inhibition of NBT reduction.
Germination tests
Germination tests were performed on vertical plastic Phytotoxkit
containers (Phytotoxkit, Tigret company, Belgium). The seeds of
cornflower (Centaurea cyanus) were purchased from a commercial
seed producer, PNOS (Ożarów Mazowiecki, Poland). A 100 g portion
of soil was applied on the plates and 20 mL of appropriate HILs
solution in water with isopropanol (1 mL isopropanol in 1 L water)
was added so that their effective concentrations were equal to
0.2∙10-3 mmol/100 g of soil. Control samples were treated with 20
ml of a reference solution (water with isopropanol). Additional
reference samples were prepared as mixture of appropriate
commercial herbicides (0.2∙10-3 mmol herbicide 1 + 0.2∙10-3 mmol
herbicide 2 /100 g of soil). The commercial herbicides were in the
form of sodium salts. Spiking was applied directly before preparing
the germination plates. Phytotoxkit plastic containers were placed
in the dark and constant parameters such as temperature (25 ± 1
°C) as well as humidity (25%) were maintained. Soil prepared in
such a manner was used to plant 10 seeds of cornflower (3 plates
for each sample). After the end of the experiment (6 days) the
number of germinated seeds was counted and measurements of
root and shoot length were conducted.
The activity of catalase (CAT) was determined by directly measuring
the decomposition of H2O2 at 240 nm for 3 min as described by
Aebi41 in 50 mM of phosphate buffer (pH 7.0) containing 5 mM of
H2O2 and 40 μl of the enzyme extract. CAT activity was determined
using the molar extinction coefficient of 36 mM-1cm-1 for H2O2.
Ascorbate peroxidase (APX) activity was assayed, 5mM of
ascorbate was added to the extracted buffer and homogenized. The
homogenate was centrifuged at 14 000 g for 30 min, then the
supernatant was stored in separate aliquots at -80 °C to be used for
enzyme assays. APX was measured by following the decline in
absorbance of the oxidized ascorbate at 290 nm, according to Chen
and Asada42. Enzyme activity was calculated using the molar
extinction coefficient of 2.8 mM-1 cm-1 for ascorbate calculated
using the coefficient of absorbance of 2.8 mM-1 cm-1 for ascorbate.
Assessment of the activity of GST. The activity of glutathione
S-transferase (GST) was assessed according to the method of Mauch
and Dudler43 with some modifications. GST catalyses the reaction
between GSH and 1-chloro-2,4-dinitrobenzene (CDNB). The product
of the reaction is coloured 2,4-dinitrophenyl-S-glutathione. The rate
of product formation corresponds to the activity of GST.
After termination of germination tests, the impact of
herbicides on root growth inhibition of cornflower was assessed.
Root length in control and treated plants were measured with a
ruler. Based on obtained data, the germination index (GI) was
calculated according to the equation39 (1):
The reaction mixture consisted of: 850 μL of 100 mM of
KH2PO4/K2HPO4 buffer (pH 6.5), 50 μL of 20 mM GSH, 50 μL of 20
mM CDNB (dissolved in ethanol), 50 μL of the extract. In a blank
sample the extract was replaced with 50 μL of buffer. Measurement
was performed spectrophotometrically at 340 nm within 60 s.
Assessment of the level of GSH and GSSG. Assessment of the
level of GSH and GSSG was conducted based on the method
described by Griffith44 with some modifications. The measurements
were based on the oxidation of glutathione present in the sample
by 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) which was reduced
to a coloured product – 5-thio-2-nitrobenzoic acid (TNB). The
amount of reduced DTNB corresponds to the content of total
glutathione (GSH + GSSG) in the sample.
GI = Gs/Gc ∙ Ls/Lc ∙ 100 [%]
(1)
where: Gs and Gc are numbers of seeds germinated in the sample
and control, respectively, whereas Ls and Lc are the radicle lengths
in the sample and control, respectively.
Oxidative stress factors
Preparation of plant extracts. Seeds of common wheat (Triticum
aestivum), were obtained from a homestead in Poznan, Poland.
Wheat and cornflower were cultivated in a growth chamber for two
weeks. Next, the plants were sprayed with
a solution of
[2,4-D-DAE-C10][MCPP] and [MCPA-DAE-C10][Clopyralid] or mixture
classical herbicides in an amount of 5 ml of solution at a
concentration of 10-3 mol L-1. Leaves were collected after 7 days and
stored at -60 °C. In order to prepare an extract for measuring the
activity of antioxidant enzymes (SOD, CAT, APX), glutathione S-
transferase (GST) and glutathione in reduced (GSH) and oxidised
(GSSG) form, 0.5 g of plant material was homogenized with 50 mM
of phosphate buffer (pH 7.0), 7 mM of 2-mercaptoethanol, 5 mM of
EDTA, 1mM of ascorbate acid, 1% of Trition X-100, and 1% of PVP
(polyvinylpyrolidone) using a mortar and pestle. The obtained
The assessment of GSSG was realized by incubation of sample with
2-vinylpyridine (2-VP) which blocks thiol groups of GSH.43 The
difference between the amount of total glutathione and GSSG
allowed to calculate the level of GSH.
In order to determine the level of the total glutathione, 1 mL of the
extract was neutralized with 1.5 mL of 50 mM potassium buffer (pH
7.5). To determine the level of GSSG, the same step was repeated,
after 0.2 mL of 2-VP was added and the solution was incubated for
one hour at room temperature. Afterwards, the samples were
purified by double extraction with 5 mL of diethyl ether to remove
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2-VP.
they were added to the top agar supplemented with traces of
The obtained extracts were used for measurements, performed histidine and biotin, and poured onto the plates with minimum
on Shimadzu UV-1800 UV-Vis spectrophotometer. The measured glucose agar. The plates were incubated for 48 hours at 37 °C, and
reaction mixture contained 100 μL of the neutralized extract, 500 μL the number of revertant (His+) colonies per plate was counted
of 0.1 M Na/P buffer with EDTA, 100 μL of NADPH, 100 μL of manually. The mutagenic index (MI), which reflects the number of
glutathione reductase and 200 μL of DTNB. In the blank, 100 μL of revertants with the mutagen (Ri) divided by the number of
SSA (5%) was used instead of the extract. Analyses were performed spontaneous revertants (Rs) was used to express mutagenic
within 180 s at 412 nm at 25 °C.
activity. A mutagenic potential of a test compound was considered
Protein Quantification. Total soluble protein contents were when MI ≥ 2.0, a possible mutagenic effect for MI between 1.7-1.9,
determined according to the method of Bradford45 using the Bio- and non-mutagenic potential for M ≤ 1.6. A doubling of the MI was
Rad assay kit with bovine serum albumin as a calibration standard.
Chlorophyll level. The chlorophyll content was determined by
spectrophotometric method.45 The fresh leaves were cut to small
pieces and placed in the Falcon tubes, then 5 mL of DMSO were
added and the sample was incubated at 65 °C for 2 h. The solution
was separated from the leaves by centrifugation and filtrated. The
obtained supernatant was used to the measure chlorophylls a and
assumed to be significant for HILs mutagenicity.
Statistical analysis
The obtained data were analysed for significant mean differences
using a one-way analysis of variance (ANOVA) at p < 0.05. Post hoc
tests for pair-wise differences and the identification of
homogeneous subgroups were conducted using Tukey’s honestly
significant difference procedure. Homogenous subgroups were
indicated by diagrams marked with the same lowercase letters.
ANOVA was computed with Statistica 10 software (StatSoft. Inc.,
Tulsa, OK, USA).
b. The maximum absorbance was measured on
a Jasco
spectrophotometer, in a 1ml quartz cuvette at 665 nm for
chlorophyll a and 649 nm for chlorophyll b. The amount of
chlorophylls was calculated from the formulas (2) and (3):
Chla = (12.9 ∙ A665 – 3.45 ∙ A649) ∙ 50 [μg/g fresh weight]
(2)
Acknowledgements
Chlb = (21.99 ∙ A649 – 5.32 ∙ A665) ∙ 50 [μg/g fresh weight] (3)
This research was founded by the National Science Centre in Poland
conferred on the basis of the decision DEC-2011/03/B/NZ9/00731.
Mutagenic activity
Determination of mutagenicity of HILs was carried out using the
Salmonella reversion assay (Ames test) with Salmonella
typhimurium tester strains TA98, TA100, TA1535, TA1537 derived
from the Polish Collection of Microorganisms of the Institute of
Immunology and Experimental Therapy of the Polish Academy of
Sciences in Wroclaw. Auxotrophic histidine-requiring Salmonella
typhimurium mutants (His-) TA98 (hisD3052) and 1537 (hisC3076)
were used for determining frameshift mutations, and TA100
(hisG46) and TA1535 (hisG46, plasmid pKM101) served to detect
base pair exchange (substitution mutations).35 Investigations on
HILs mutagenicity were performed in the liquid pre-incubation
assay, with and without in vitro metabolic activation by applying the
S9 liver microsomal fraction including a cofactor mixture, according
to the procedure described by Mortelmans and Zeiger.34 The
reaction mixture comprised the following components: 100 μLof
bacterial suspension originating from 12−14-hour culture in Oxoid
Nutrient Broth No. 2 (Oxoid Thermo Scientific, UK), reference
mutagen or analyzed HILs, 500 μ of rat microsomal fraction (S9,
Sigma−Aldrich) mixture/soluꢀon and 0.2 M phosphate buffer pH 7.4
(Sigma−Aldrich) in the amount filling up to the total volume of 1 mL.
The mutagens: 2-aminofluorene (100 μg/plate), sodium azide (1
μg/plate) and 2-aminoacridine (30 μg/plate) (all supplied by
Sigma−Aldrich) were used to reverse the mutaꢀon in the TA98,
TA100, TA1535 and TA1537 strain, respectively. The mutagen:
2-aminoanthracene (5 μg/plate) (Sigma−Aldrich) served as posiꢀve
control in promutagenicity experiments for all strains tested. HILs
were analyzed at a concentration of 10 μg/plate. The prepared
reaction mixtures were pre-incubated at 37 °C for 20 min. Next,
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Novel weed control agents in the form of herbicidal ionic liquids comprising two different
herbicides as a cation and anion.