Ammonium haloacetates – An alternative to glyphosate?

Article abstract of DOI:10.1016/j.chemosphere.2017.12.055

This study shows the design, synthesis and evaluation of eco(phyto)toxic and herbicidal activities of quaternary ammonium salts (QASs), derived from haloacetic acids, in context of the search for safer alternatives to the commonly used herbicide, N-(phosphonomethyl)glycine (glyphosate). The structure of the investigated QASs refers to the heteroatom sequence in the anion of glyphosate in which the (P-C)-N nitrogen atom was replaced by one or more halogens (F, Cl). The ecotoxicity of the synthesized QASs was tested against luminescent marine bacteria Vibrio fischeri (Microtox test) and the crustaceans Heterocypris incongruens (Ostracodtoxkit F). The phytotoxic effect of QASs was also studied with respect to spring barley (Hordeum vulgare) and common radish (Raphanus sativus L. radicula Pers.), whereas herbicidal activity was investigated in relation to popular weeds species gallant soldier (Galinsoga parviflora Cav.) and common sorrel (Rumex acetosa L.). The results showed that toxicity of the synthesized QASs depends on a number of halo-substituents, especially for bioluminescent bacteria Vibrio fischeri for which EC₅₀ values were those varying the most. Phytotoxicity tests proved that the investigated QASs had a similar high, toxic effect both on monocotyledonous and dicotyledonous plants with exception of DIPA - DCA. Moreover, their herbicidal activity against common sorrel was comparable to glyphosate.

Full text of DOI:10.1016/j.chemosphere.2017.12.055

Accepted Manuscript
Ammonium haloacetates – An alternative to glyphosate?
Marika Turek, Robert Biczak, Barbara Pawłowska, Ewa Różycka-Sokołowska,
Bernard Marciniak, Małgorzata Deska, Joanna Skalik, Piotr Bałczewski
PII:
S0045-6535(17)32025-8
10.1016/j.chemosphere.2017.12.055
CHEM 20435
DOI:
Reference:
To appear in: ECSN
Received Date: 12 July 2017
Revised Date: 15 November 2017
Accepted Date: 9 December 2017
Please cite this article as: Turek, M., Biczak, R., Pawłowska, B., Różycka-Sokołowska, E., Marciniak,
B., Deska, Mał., Skalik, J., Bałczewski, P., Ammonium haloacetates – An alternative to glyphosate?,
Chemosphere (2018), doi: 10.1016/j.chemosphere.2017.12.055.
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ACCEPTED MANUSCRIPT

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Ammonium haloacetates – an alternative to glyphosate?
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Marika Turek , Robert Biczak , Barbara Pawłowska , Ewa Różycka-Sokołowska ,
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Bernard Marciniak , Małgorzata Deska , Joanna Skalik , Piotr Bałczewski*
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Institute of Chemistry, Environmental Protection and Biotechnology, The Faculty of Mathematics and Natural Sciences, Jan
Długosz University in Częstochowa, Armii Krajowej 13/15, Częstochowa, 42-201, Poland,
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Department of Heteroorganic Chemistry, Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences,
Sienkiewicza 112, Łódź, 90-363, Poland.
* Address correspondence to this author at the Department of Heteroorganic Chemistry, Centre of Molecular and
Macromolecular Studies, Polish Academy of Sciences, Box: 90-363, Łódź, Poland; Tel/Fax: +48-42-6803-216, +48-42-684-
71-26; E-mail: pbalczew@cbmm.lodz.pl;
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Highlights
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Haloacetates (QASs), structurally related to glyphosate were synthesized.
Ecotoxicity on V. fischeri and H. incongruens, as well as phytotoxicity were tested.
Toxicity of examined QASs depended on the number and kind of halo-substituents.
Monochloroacetate was the most toxic to V. fischeri and H. incongruens.
QASs: herbicidal properties comparable to glyphosate and safer for the environment.
Keywords
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haloacetates, eco(phyto)toxicity, glyphosate, Microtox , Ostracodtoxkit F™, quaternary
ammonium salts (QAS)
Abstract
This study shows the design, synthesis and evaluation of eco(phyto)toxic and herbicidal
activities of quaternary ammonium salts (QASs), derived from haloacetic acids, in context of
the search for safer alternatives to the commonly used herbicide, N-
(phosphonomethyl)glycine (glyphosate). The structure of the investigated QASs refers to the
heteroatom sequence in the anion of glyphosate in which the (P-C)-N nitrogen atom was
replaced by one or more halogens (F, Cl). The ecotoxicity of the synthesized QASs was tested
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against luminescent marine bacteria Vibrio fischeri (Microtox test) and the crustaceans
Heterocypris incongruens (Ostracodtoxkit F™). The phytotoxic effect of QASs was also
studied with respect to spring barley (Hordeum vulgare) and common radish (Raphanus
sativus L. radicula Pers.), whereas herbicidal activity was investigated in relation to popular
weeds species gallant soldier (Galinsoga parviflora Cav.) and common sorrel (Rumex acetosa
L.). The results showed that toxicity of the synthesized QASs depends on a number of halo-
substituents, especially for bioluminescent bacteria Vibrio fischeri for which EC values were
those varying the most. Phytotoxicity tests proved that the investigated QASs had a similar
high, toxic effect both on monocotyledonous and dicotyledonous plants with exception of
DIPA - DCA. Moreover, their herbicidal activity against common sorrel was comparable to
glyphosate.
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1. Introduction
Useful quaternary ammonium salts (QAS), most of which constitute ionic liquids (ILs)
are interesting replacements to conventional solvents due to their non-flammability, non-
volatility and biodegradability (Dupont et al., 2002). Another useful property of QASs is their
"designability" because by selecting appropriate types of anions and cations, QASs with
desirable properties can be obtained. All these advantages determine considerable application
potential of QASs in various areas, such as agriculture (Pernak et al., 2013), electrochemistry
(Sun et al., 2014), chemistry (Liu et al., 2010), biotechnology (Fister et al., 2015), chemical
engineering (Vidal et al., 2012) and pharmaceutical industry (Ferraz et al., 2011). Due to the
increasing importance of QASs in agriculture and their unavoidable persistence in water and
soil, it is essential to investigate the ecotoxicity of these potential, environment contaminants
(Bubalo et al., 2014).
Haloacetic acids (HAAs) are compounds which may be found in aquatic systems. For
example, trichloroacetic acid (TCA) is formed as a result of atmospheric degradation of
chlorofluorohydrocarbons, whereas chloroacetic acid (MCA) is
a
product of
photodegradation of herbicides (Hanson and Solomon, 2004). From the beginning of the 21st
century, a number of studies concerning the impact of HAAs on aquatic organisms has been
conducted (Hanson et al., 2001; Hanson and Solomon, 2002a; Hanson et al., 2002b, 2002c,
2002d, 2003; Hanson and Solomon, 2004). The QASs derived from HAAs have not been
widely studied. There is one patent which characterizes diisopropylammonium and
isopropylammonium salts of TCA as herbicides (Kelly, 1951). In 1948, the potential
application of quaternary ammonium salts of TCA against Agropyron repens and Kentucky
bluegrass was described (Carlson and Moulton, 1948).
The main objective of this work is to synthesize and evaluate the eco(phyto)toxic and
herbicidal activities of ammonium salts derived from haloacetic acids, with hope that they
may be applied as environmentally friendly herbicides of improved properties, and for
instance, they may replace broad spectrum herbicides, such as commonly used N-
(phosphonomethyl)glycine (glyphosate). All the compounds possess melting points above 100
°C, therefore they should not be qualified as ionic liquids but as quaternary ammonium salts
(QASs). The designed QASs refer to ionic structure of glyphosate, which possesses the
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herbicidal anion of a general formula (P-C)-N-C-COO and which should be regarded as a
derivative of aminoacetic acid (glycine) (Fig. 1). The characteristic sequence of heteroatoms
in the carbon chain of glyphosate determines its biological activity. The synthesized QASs are
composed of the diisopropylammonium cation and modified acetic acid anion of the general
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structure Y -C-COO , in which the (P-C)-N nitrogen has been replaced by one or more
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halogens (Y = Cl, F). The present studies will determine, how a different arrangement of
atoms in glyphosate anion influences the selectivity and modulation of herbicidal properties
of the new compounds. Despite the possible application of the QASs as new, environmentally
safer herbicides to glyphosate, their eco(phyto)toxic and herbicidal activities have never been
investigated. Therefore, this paper presents first, a determination of eco(phyto)toxicity of the
synthesized QASs in order to evaluate their effect and selectivity on plants, and then applied
investigations of their herbicidal activity.
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Fig. 1. Chemical structures of the widely used herbicide (glyphosate) and investigated QASs as heteroatom
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derivatives of acetic acid.
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There are several biotests for ecotoxicity assessment utilizing bacteria, crustaceans,
earthworms, algae and plants. The most common bacterial bioassay which may be applied to
evaluate the ecotoxicity of QASs is the Microtox test based on the inhibition of
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bioluminescence of Vibrio fischeri (Romero et al., 2008; Arendarczyk et al., 2011; Pretti et
al., 2011; Ventura et al., 2012; Peric et al., 2013; Ventura et al., 2014; Hernandez-Fernandez
et al., 2015; Montalban et al., 2016; Oliveira et al., 2016). During the exposure of Vibrio
fischeri to a toxic substance, respiration processes are disrupted, which results in a reduced
intensity of bioluminescence. In addition to disturbing respiratory processes, toxic substances
may also interact with the bacterial cell receptors, disrupt the cell membrane and affect
enzymatic pathways (Wilk and Szalińska, 2011). Another frequently used mortality and
growth inhibition assay which allows to examine the chronic toxicity of freshwater sediments
to crustaceans Heterocypris incongruens is Ostracodtoxkit F™ (Oleszczuk, 2008; Marmonier
et al., 2013). These two biotests were chosen in view of migration of QASs to water and soil
(aquatic bacteria Vibrio fischeri) and further possible accumulation in the bottom sediments
(benthic ostracods Heterocypris incongruens).
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2. Materials and methods
2.1. Tested compounds
The following QASs were tested in this work, namely diisopropylammonium
chloroacetate (DIPA-MCA), diisopropylammonium dichloroacetate (DIPA-DCA),
diisopropylammonium trichloroacetate (DIPA-TCA), diisopropylammonium difluoroacetate
(DIPA-DFA),
diisopropylammonium
trifluoroacetate
(DIPA-TFA)
and
diisopropylammonium chlorodifluoroacetate (DIPA-CDFA), whose chemical structures are
presented in Scheme 1. These compounds have been synthesized in the reaction of haloacetic
acids with 10% excess of diisopropyl amine without solvent (Scheme 1). In case of DIPA-
MCA, DIPA-DCA, DIPA-TCA and DIPA-CDFA, the reactions were conducted at room
temperature, whereas in the case of DIPA-DFA and DIPA-TFA, the reactions were carried
out in a cooling bath. The haloacetic acids were divided into 3 groups containing in the α
position: a) only chlorine atoms (Y = Cl; n = 1-3), b) only fluorine atoms (Y = F; n = 2,3), c)
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mixed chlorine (Y = Cl; n = 1) and fluorine (Y = F; n = 2) atoms. H, C and F NMR
spectra have been collected in Supplementary material (Fig. A1-A15). Ammonium and
potassium fluorides (>98%) were purchased from POCh S.A. All organic solvents were used
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as received from commercial sources and/or dried using standard procedures. The H NMR,
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C NMR and F NMR spectra were measured on a Brucker AV 200, AV 500 spectrometer
in CDCl with chemical shifts (δ) given in ppm relative to TMS as an internal standard. The
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mass spectra of pure compounds were obtained using a Finnigan Mat 95 spectrometer.
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Melting points were measured using Boetius apparatus. Elemental analysis was determined
with an EuroVector analyser model 301.
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Scheme 1. Synthesis of QASs derived from haloacetic acids.
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2.2. Microtox test
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The Microtox bioassay utilizes bioluminescent marine bacteria Vibrio fischeri (strain
NRRL B-11177) to evaluate the acute and chronic toxicity of water, sewage sludge and soil.
The inhibition of bioluminescence was measured using the Microtox Model 500 Analyzer
(MODERNWATER Inc., New Castle, USA).
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In this test, diluted aqueous solutions of the synthesized QASs and two inorganic fluorides
were used. Two additional QASs (DIPA-DFA and DIPA-TFA) were employed in order to
determine the influence of fluorine atom on the bioluminescence of Vibrio fischeri. As a result
of this test, the EC values (EC - effective concentration) have been obtained, which refers to
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the concentration of a toxicant resulting in the 50% inhibition of bioluminescence of the
bacteria population. The range of applied concentrations was established by using the 81.9%
Screening Test, while the EC50 values were established using the 81.9% Basic Test, in
accordance with the manufacturer procedures. The bioluminescence inhibition of Vibrio
fischeri was measured after 5 and 15 minutes of exposure to each QAS and compared with the
light output of a control sample. EC values (mg/L) after 5 and 15 minutes of exposure and
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the corresponding 95% confidence intervals were estimated for each QAS using the Microtox
Omni Software™.
2.3. Ostracodtoxkit F™ test
The Ostracodtoxkit F™ is the 6-day “direct contact” toxicity test for freshwater sediments
and soil. It is based on the investigation of two effects: mortality (%) and growth inhibition
(%) of crustaceans Heterocypris incongruens after 6 days of contact with a given sample.
Studies were conducted in accordance with the manufacturer's instructions by using the
Ostracodtoxkit F™ (MicroBioTests Inc., Belgium). For this assay, four of the six synthesized
QASs were used.
The mortality assessment - the total number of the living organisms was counted for three
wells of the test plate (A). The number of organisms which did not survive the test (B) was: B
= 30-A. Mortality (%) was calculated from the equation: Mortality (%) = B/30 · 100.
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The growth inhibition assessment as an average length of surviving organisms in reference
soil (A) and tested soil containing QASs (B) was made. The growth inhibition (%) of
crustaceans was calculated according to the formula: Growth inhibition (%) = 100 - [(A/B) ·
100].
The inhibitory concentrations (IC ) were estimated by the non-linear regression using
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GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA, USA).
2.4. Phytotoxicity test
Examination of phytotoxicity of QASs was carried out according to the OECD/OCDE
208 guidelines (OECD/OCDE 208, 2006). Studies were performed in a vegetation hall under
controlled conditions. Throughout the testing period, a constant substrate moisture content at
the optimum level required for the plants (70% of field water capacity), a constant
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temperature of 20 ± 2 C and a constant illumination of 160 ꢀmol m s were maintained for
16 h/day and 8 h/night. The source of light included the high pressure 600W sodium lamp
(OSRAM, E40/ES, CCG).
For this purpose, seeds of the spring barley (Hordeum vulgare) and common radish
(Raphanus sativus L. radicula Pers.) were used. Barley grains were sourced from the Plant
Breeding Station in Nieznanice (Silesian province, Poland), and radish seeds were purchased
from PlantiCo - Garden breeding and seeding, Zielonka Co. Ltd. (Mazowieckie province,
Poland). The soil used contained about 10% of the fraction with a diameter of <0.02 mm, 1%
of organic carbon and pH 5.9. Pots were filled with 250 g of soil with 25 mL of water (control
sample) or 25 mL of aqueous solution of the QAS in an appropriate concentration.
For each QAS, the following concentrations were used in the preliminary test: 0, 1, 10, 100,
1000 mg per 1 kg of soil dry weight (DW). For the final testing, the concentrations were the
following: 20, 40, 80 mg per 1 kg of soil DW, except of DIPA-TCA (test for spring barley)
for which concentrations of 2, 4, 8 mg per 1 kg of soil DW were used and DIPA-DCA (test
for spring barley and common radish) with concentrations of 200, 400, 800 mg per 1 kg of
soil DW. Each pot was seeded with 20 seeds of spring barley or common radish. The seed
germination and plant growth were continued for 14 days in vegetation hall under conditions
described above. After 14 days, the germination, the fresh and dry mass contents were
compared. Based on these results, the values of LOEC (lowest observed effect concentration)
and NOEC (no observed effect concentration) were established.
To evaluate the significance of the test results, analysis of variance (Fisher–Snedecor F-test)
was used. The values of LSD0.95 (Least Significant Difference at the confidence level of 95%)
were calculated using the Tukey's range test. The results are presented as an average of three
replicates.
2.5. Herbicidal activity
Greenhouse
experiments
concerning
the
herbicidal
properties
of
diisopropylammonium chloroacetates, chlorofluoroacetate and fluoroacetates were carried out
in the vegetation hall at the conditions described above for phytotoxicity test (Subsection 2.4).
For this purpose, plastic flowerpots with a diameter of 90 mm, containing 250 g of soil were
seeded with the same amount (determined by weight) of gallant soldier (Galinsoga parviflora
Cav.) and common sorrel (Rumex acetosa L.). The soil used was light, loamy sand containing
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about 10% of the fraction with a diameter of <0.02 mm, 0.9% of organic carbon and pH 6.0.
Three weeks after germination, weeds were sprayed with aqueous solutions of investigated
compounds having the concentrations of 0.5%, 1.0% and 2.0%. For glyphosate,
concentrations of 0.5% and 1.0% were applied - this is the maximum dose recommended by
the manufacturer for spray application. The control sample was also prepared in an analogous
way, where weeds were sprayed with distilled water without the addition of compounds.
Further studies were conducted for 21 days after spraying. Herbicidal activity of examined
compounds was determined by visual assessment of growth inhibition, damages and their
gradual drying out, what was documented on digital photographs presented in Supplementary
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material. Roundup 360 SL with active substance content (glyphosate) in the form of
isopropylammonium salts, 360 g in 1 litre of water, was used.
3. Results and discussion
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3.1. Microtox test
The obtained 81.9% Basic Test results indicate that examined QASs exhibit a
differentiated toxicity (Table 1). According to the European Classification (EU, 2011) all of
the examined QASs can be considered as completely non-toxic. The most toxic QAS to
Vibrio fischeri is DIPA-MCA, while the least toxic is DIPA-TFA. It can be concluded that the
tested bacteria are most sensitive to QASs with one chlorine atom or/and two fluorine atoms
(DIPA-MCA, DIPA-DFA and DIPA-CDFA). The most striking were huge differences in
toxicity for diisopropylammonium chloroacetates: EC (TCA : DCA : MCA) = 55(37) :
5,(15)
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5 times less toxic than analogous trichloroacetate: EC (TFA : TCA) = 3.4(5.3) : 1(1)
5(15)
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which is rather an unexpected result, considering stronger biological interactions of fluorine
containing compounds, which is due to a stronger hydrogen bonding which creates fluorine in
comparison to chlorine. It is interesting that only in case of DIPA-CDFA, the EC value after
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15 minutes of exposure is higher that after 5 minutes. There are several described ILs, whose
toxicity to Vibrio fischeri decreases after 5 minutes (Ventura et al. 2012; Ventura et al. 2014;
Oliveira et al. 2016) but the differences between EC50, 5 min and EC50, 15 min values are not as
noticeable as in the case of DIPA-CDFA. This result prompted us to investigate the
ecotoxicity of ammonium and potassium fluorides to find out how inorganic fluorine atoms
may affect the bioluminescence of Vibrio fischeri. The examined potassium fluoride belongs
to the Category: Acute III, whereas ammonium fluoride is non-toxic (Table 1) which was also
evidenced in the results of their toxicity for Vibrio fischeri: EC (NH F : KF) = 240 : 1. In
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the case of inorganic fluorides, there was no increased bioluminescence after 15 minutes of
exposure, so it may be assumed that the above mentioned effect is specific only for DIPA-
CDFA. Extending the exposure time over 15 min could clarify the inconsistencies noticed for
DIPA-CDFA.
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The Microtox test is commonly used to evaluate toxicity of ILs as a subgroup of
QASs. It has been proven that the toxicity of imidazolium ILs depends on the length of the
imidazolium carbon chain in substituents (Romero et al. 2008). The same conjunction has
been
found
for
N-alkyl-N-methylmorpholinium
and
N-alkyl-DABCO
(1,4-
diazabicyclo[2.2.2]octane) based ILs (Pretti et al. 2011), protic ILs (Oliveira et al. 2016) and
others (Montalban et al. 2016). Similar results were also obtained for guanidinium-,
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phosphonium- and imidazolium-based ILs (Ventura et al. 2012). It was proven that aprotic
ILs are more toxic than protic ones (Peric et al. 2013) and that inorganic chlorine, in the form
of chloride anion is the least toxic, whereas the bis(trifluoromethylsulfonyl)amide anion is the
most toxic to Vibrio fischeri (Montalban et al. 2016). However, the effect of organic halogen
on Vibrio fischeri has not been widely explored in the literature.
Because the examined QASs are structurally-related to the commonly used herbicide
glyphosate, it is important to compare their ecotoxicity with this compound. For example, the
toxicity of various glyphosate formulations to Vibrio fischeri has been evaluated and the
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IC50(15min) values for the Roundup , isopropylamine salt of glyphosate and glyphosate acid on
the level of 24.9, 162 and 17.5 mg/L were obtained, respectively (Tsui and Chu, 2003). Thus,
it may be stated that the synthesized QASs are less toxic to Vibrio fischeri than glyphosate
(except for DIPA-MCA, which is more toxic than isopropylamine salt of glyphosate) (Table
1).
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Table 1
The EC50 values (mg/L) estimated after 5 and 15 min. exposure of luminescent bacteria Vibrio fischeri to the
synthesized QASs, with the corresponding 95% confidence interval (81.9% Basic Test).
EC (mg/L) 5 min
EC (mg/L) 15 min
(Confidence interval)
147.5
Toxicity category
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QAS
(
Confidence interval)
69.4
38.59 - 469.6)
1950.5
7187 - 95992)
273.1
529.9 - 173551)
56.8
80.45 - 819.7)
1666
10827 - 92617)
33.6
266.2 - 2373)
298
(2184 - 4980)
3.80
4.062 - 46.86)
(EU, 2011)
1
DIPA-MCA
DIPA-DCA
DIPA-TCA
DIPA-DFA
DIPA-TFA
DIPA-CDFA
(
(
(15.12 - 1445)
12691
(10130 - 15032)
5426.4
(305.1 - 38171)
230.9
(62.91 - 847.2)
28525
(18332 - 44387)
2715
(798.1 - 11657)
3126
(1706 - 5728)
12.95
(4.014 - 41.76)
2
9
(
2
No acute toxicity
(
3
(
7
(
3
Ammonium
fluoride
1
Potassium fluoride
Acute III
(
2
2
2
2
2
2
2
2
2
2
2
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3.2. Ostracodtoxkit F™ test
The percentage of mortality and growth inhibition of ostracods in soils with increasing
concentrations of the examined QASs are presented in Figure 2. Analysis of the obtained
results shows that the synthesized QASs derived from haloacetic acids exhibit a low toxicity
to Heterocypris incongruens. The examined compounds with one chlorine atom (DIPA-MCA
and DIPA-CDFA) were more toxic than those with two (DIPA-DCA) or three (DIPA-TCA)
chlorine atoms. A similar correlation has been found for these compounds in the Microtox
test.
The ostracods are useful bioindicators to evaluate toxicity of the sewage sludges
(Oleszczuk, 2008, 2009), sediments (Belgis et al., 2003; Casado-Martinez et al., 2016),
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petroleum contaminated soils (Płaza et al., 2005), however there are still no data related to the
ILs and QASs.
1
00
IC = 590.8
IC = 887.2
50
IC = 881.4
50
IC = 671.1
50
5
0
9
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
0
-
10
DIPA-MCA
DIPA-DCA
DIPA-TCA
DIPA-CDFA
Concentration of QAS [mg/kg of soil]
Mortality (%) Growth inhibition (%)
2
2
2
2
2
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82
Fig. 2. Mortality (%) and growth inhibition (%) of Heterocypris incongruens in soils depending on concentration
of QASs. The IC50 values for crustacean following exposure to QASs. Data are means from 3 independent
experiments.
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3.3. Phytotoxicity test
The obtained test results referring to the influence of examined QASs on germination
and growth of spring barley and common radish are presented in Table 2. The studies show
that QASs derived from haloacetic acids affect plant growth. A visual evaluation of growth
inhibition, chlorotic and necrotic damages upon treatment of plants seedlings in soil with
DIPA-MCA, DIPA-DCA, DIPA-TCA and DIPA-CDFA, was used as an indicator of toxicity.
These changes were documented in the form of digital photographs presented in
Supplementary material (Fig. A16-A19). The most toxic compound was found to be DIPA-
TCA, whereas the least toxic was DIPA-DCA (Table 3). The tested compounds exhibited
similar toxic effect in respect to both monocotyledonous (spring barley) and dicotyledonous
plants (common radish).
It has been found that dry weight increased and fresh weight decreased with increasing
concentration of QASs, both for spring barley and common radish. Similar relationships have
been found in other studies (Biczak et al., 2010; Biczak et al., 2014; Matusiak et al., 2013).
A number of studies concerned the effect of the chain length on the ILs phytotoxicity
(Studzieńska, 2009; Bubalo et al., 2014; Liu et al., 2015; Biczak, 2016), whereas there is no
data regarding the influence of halogen substituents. The obtained results suggest that there is
no strict relationship between the type and number of halogen substituents on the
phytotoxicity of the synthesized QASs. However, it can be assumed that the QASs composed
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Table 2
Variations in the yield of fresh weight (g/pot), dry weight (g/g f.m.) and germination for spring barley and common radish as the effect of increasing concentration of QAS in
soil (in mg/kg of soil d.w.). Data are means ± SD from 3 independent experiments.
Concentration of QAS [mg/kg of soil d.w.]
IL
0
1
10
20
40
80
100
1000
LSD0.95
Spring barley
FW
DW
sprouts
FW
DW
sprouts
2.501 ± 0.172
0.1074 ± 0.0030
20
3.661 ± 0.442
0.0845 ± 0.0013
19
2.529 ± 0.190
0.1038 ± 0.0013
19
3.817 ± 0.270
0.0826 ± 0.0012
19
2.582 ± 0.086
0.1044 ± 0.0019
20
3.746 ± 0.233
0.0837 ± 0.0021
19
2.472 ± 0.142
0.1056 ± 0.0018
20
1.521 ± 0.198
0.1152 ± 0.0036
19
2.577 ± 0.204
0.1074 ± 0.0032
20
0.758 ± 0.033
0.1357 ± 0.0077
18
2.283 ± 0.248
0.1127 ± 0.0021
1.555 ± 0.260
0.1191 ± 0.0034
0.240
0.1557
6
-
-
0.187
0.0024
0
0.363
0.0052
1
DIPA-MCA
DIPA-CDFA
19
-
-
18
-
-
19
19
15
Common radish
FW
DW
sprouts
FW
DW
sprouts
FW
3.838 ± 0.242
0.1035 ± 0.0038
19
4.389 ± 0.055
0.0544 ± 0.0021
18
4.989 ± 0.007
0.0608 ± 0.0057
18
3.726 ± 0.207
0.1011 ± 0.0074
19
4.901 ± 0.018
0.0541 ± 0.0017
17
5.076 ± 0.345
0.0584 ± 0.0027
17
4.042 ± 0.275
0.0990 ± 0.0041
19
4.367 ± 0.136
0.0585 ± 0.0042
18
4.592 ± 0.854
0.0639 ± 0.0025
18
3.694 ± 0.210
0.1005 ± 0.0060
20
3.771 ± 0.158
0.0702 ± 0.0039
17
4.310 ± 0.283
0.0683 ± 0.0034
17
4.076 ± 0.317
0.0947 ± 0.0057
20
3.364 ± 0.165
0.0804 ± 0.0054
17
4.274 ± 0.346
0.0728 ± 0.0022
19
2.288 ± 0.264
0.0934 ± 0.0068
11
2.527 ± 0.445
0.0825 ± 0.0060
18
2.842 ± 0.385
0.0860 ± 0.0025
17
1.852 ± 0.181
0.0987 ± 0.0001
9
2.266 ± 0.163
0.0890 ± 0.0025
15
2.650 ± 0.122
0.0882 ± 0.0105
15
-
-
-
0.274
0.0058
1
0.251
0.0075
2
0.408
0.0050
1
DIPA-MCA
DIPA-TCA
DIPA-CDFA
0.933 ± 0.416
0.1530 ± 0.0177
10
0.366 ± 0.202
0.1792 ± 0.0025
6
DW
sprouts
Concentration of QAS [mg/kg of soil d.w.]
0
1
10
100
200
400
800
1000
LSD0.95
Spring barley
FW
DW
sprouts
3.769 ± 0.090
0.1064 ± 0.0024
20
3.963 ± 0.187
0.1051 ± 0.0020
20
3.914 ± 0.138
0.0989 ± 0.0024
20
4.311 ± 0.060
0.0996 ± 0.0015
20
4.187 ± 0.265
0.0926 ± 0.0030
19
4.231 ± 0.197
0.0919 ± 0.0026
20
3.997 ± 0.112
0.0934 ± 0.0030
20
2.259 ± 0.287
0.1120 ± 0.0107
19
0.190
0.0044
1
DIPA-DCA
DIPA-DCA
Common radish
FW
DW
sprouts
4.458 ± 0.516
0.0726 ± 0.0034
17
4.458 ± 0.221
0.0751 ± 0.0041
19
4.919 ± 0.423
0.0738 ± 0.0054
19
4.018 ± 0.179
0.0747 ± 0.0031
16
3.792 ± 0.231
0.0676 ± 0.0031
16
3.826 ± 0.346
0.0729 ± 0.0019
17
2.832 ± 0.530
0.0821 ± 0.0093
15
1.246 ± 0.203
0.0838 ± 0.0085
10
0.369
0.0055
1
Concentration of QAS [mg/kg of soil d.w.]
0
1
2
4
8
10
100
1000
LSD0.95
Spring barley
FW
DW
sprouts
4.043 ± 0.381
0.0804 ± 0.0022
20
4.011 ± 0.141
0.0808 ± 0.0018
20
3.812 ± 0.237
0.0850 ± 0.0014
20
3.656 ± 0.219
0.0858 ± 0.0013
19
2.547 ± 0.152
0.0870 ± 0.0006
19
2.398 ± 0.157
0.0863 ± 0.0015
18
-
-
15
-
-
15
0.279
0.0018
2
DIPA-TCA
FW - fresh weight [g/pot]
DW - dry weight [g/g fresh mass]
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of haloacetic acid with three halo-substituents [DIPA-TCA (3xCl) and DIPA-CDFA (Cl,
2xF)] are most toxic, whereas those with two halo-substituents (DIPA-DCA) are least toxic.
Three of the synthesized QASs (DIPA-MCA, DIPA-TCA and DIPA-CDFA) exhibited a
higher phytotoxicity than previously reported ILs (Bałczewski et al., 2007; Biczak et al.;
2010; Pawłowska et al.; 2013; Biczak et al., 2012).
In summary, the investigated DIPA - QASs (MCA, TCA and CDFA) show a similar and
rather high toxic effect both on monocotyledonous (spring barley, LOEC = 8 – 80 mg/kg of
soil) and dicotyledonous plants (common radish, LOEC = 20 – 80 mg/kg of soil) with
exception of DIPA – DCA for which LOEC = 800 – 1000 mg/kg of soil for the two groups of
plants, has been reported. The most phytotoxic was DIPA-TCA with LOEC = 8 – 20 mg/kg of
soil both for spring barley and common radish.
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Table 3
The values of NOEC and LOEC (mg/kg of soil d.w.) determined with respect to the spring barley and common
radish yields of fresh weight.
Spring barley
Common radish
NOEC
40
400
10
QAS
NOEC
40
800
4
LOEC
80
1000
8
LOEC
80
800
20
DIPA-MCA
DIPA-DCA
DIPA-TCA
DIPA-CDFA
10
20
40
80
NOEC - no observed effect concentration [mg/kg of soil dry weight]
LOEC - lowest observed effect concentration [mg/kg of soil dry weight]
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3.4. Herbicidal activity
Digital photographs of common sorrel and gallant-soldier, after spraying with 0.5, 1
and 2% aqueous solutions of the QASs obtained, are presented in Supplementary material
(Fig. A.20-A.33). All investigated compounds applied in the form of spraying exhibit
herbicidal activity against gallant soldier (Galinsoga parviflora Cav.) and common sorrel
(Rumex acetosa L.). The effectiveness of their action mainly depends on the type of
compound, the concentration used and the species of plant tested. DIPA-MCA and DIPA-
DCA showed the strongest herbicidal properties, both in relation to gallant soldier and
common sorrel. The weakest herbicidal properties exhibited DIPA-TCA, which at the highest
concentration (2.0%) inhibited only the growth of gallant soldier and common sorrel.
Fluorine-containing compounds showed very strong herbicidal properties in relation to
common sorrel, while gallant soldier was relatively resistant to their action. DIPA-CDFA,
DIPA-DFA and DIPA-TFA applied at higher concentrations (1.0% and 2.0%) resulted in a
complete wither of common sorrel. A similar outcome for both weeds was observed after
application of glyphosate, the main constituent of the popular herbicide - Roundup . Two
days after spraying with this herbicide, all plants, both common sorrel and gallant soldier
withered.
Application of the compounds in the form of spray had a visible effect on the tested
weeds, which may suggest that investigated diisopropylammonium haloacetates can be
absorbed, in the same way as foliar active herbicides, by leaf surface or by stomata (Praczyk
and Skrzypczak, 2004). Gallant soldier turned out to be the most resistant to the applied
treatments. A similar effect of QASs on gallant soldier, goosefoot and common sorrel was
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also reported by Biczak and Pawłowska (2016). In the studies presented by these authors, it
has also been shown that the most resistant plant to tetraethylammonium halides is gallant
soldier. The high sensitivity of common sorrel to the sprayed chemicals has also been
reported in previous studies on the effect of 2,2’-thiodiacetic acid relative to selected higher
plants (Pawłowska et al., 2013).
4. Conclusions
In this study, the eco(phyto)toxic and herbicidal activities of ammonium salts derived
from halo(F,Cl)acetic acids have been explored in order to find a safer alternative for
glyphosate. When the glyphosate (P-C)-N moiety was replaced with one or more halogens
(Cl, F) in the acetic acid, these activities were not changing harmoniously. Thus, the
unexpected QAS toxicity rows of MCA > TCA > DCA both for H. incongruens and V.
fischeri were observed for aquatic and benthic environments. These biological responses
contradicted the literature data and an experience, according to which the compounds
containing more chlorine atoms are more toxic. Further investigations on the cell level also
require QASs with fluorine atoms which were less toxic to V. fischeri than analogous QASs
with chlorine atoms which stands opposite to the fact that compounds with fluorine atoms
have stronger biological/pharmacological activity due to creation stronger hydrogen bondings.
The obtained results showed in all kinds of tests no strict relationship between the type
(chlorine or fluorine) and number of halogen substituents.
The synthesized QAS are generally less (phyto)toxic than glyphosate, while their herbicidal
activity is stronger or almost equal against some weeds, and therefore these compounds may
cause a less risk to the aquatic and benthic environments. Further research on the herbicidal
properties of ammonium halo(Cl, F)acetates is continued, taking into account the wider weed
spectrum.
Acknowledgements
The project was financed by the National Science Centre (NCN), Poland on the basis
of the decision number UMO-2013/09/B/ST5/03541.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in the online version,
at...
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