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POZNAN UNIVERSITY OF TECHNOLOGY
FACULTY OF CHEMICAL TECHNOLOGY
INSTITUTE OF CHEMISTRY AND TECHNICAL ELECTROCHEMISTRY
Ph.D. THESIS
A STUDY OF POTENTIAL TOXIC EFFECTS OF DESIGNED IONIC LIQUIDS ON BACTERIUM PSEUDOMONAS PUTIDA AND EMBRYOS
OF THE ZEBRAFISH (DANIO RERIO) AS MODEL ORGANISMS
presented by
ALEKSANDRA PIOTROWSKA, M.Sc., Eng.
born on 22.10.1987 in Gora
to obtain the academic grade Doctor of Philosophy
Specialty: Chemistry
Supervisors 1. Dr. BOGDAN WYRWAS (PUT, Poznan) 2. Dr. HERMANN J. HEIPIEPER (UFZ, Leipzig)
Poznan 2017
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This PhD thesis is based on the following scientific publications
I. Aleksandra Piotrowska, Anna Syguda, Bogdan Wyrwas, Łukasz Chrzanowski, Hermann J. Heipieper, Toxicity evaluation of selected ammonium-based ionic liquid forms with MCPP and dicamba moieties on Pseudomonas putida. Chemosphere.
2017;167:114-119 [IF 2016 = 4.208; Journal ISSN: 0045-6535]
II. Aleksandra Piotrowska, Anna Syguda, Łukasz Chrzanowski, Hermann J. Heipieper, Toxicity of synthetic herbicides containing 2,4-D and MCPA moieties towards Pseudomonas putida mt-2 and its response at the level of membrane fatty acid composition. Chemosphere. 2016;144:107-112 [IF 2016 = 4.208; Journal ISSN: 0045- 6535]
III. Aleksandra Piotrowska, Anna Syguda, Bogdan Wyrwas, Łukasz Chrzanowski, Till Luckenbach, Hermann J. Heipieper, Adaptation of zebrafish embryos to toxic concentrations of ammonium-based ionic liquids and 2,4-dichlorophenol on the level of their phospholipid fatty acid composition. PLoS ONE; Journal ISSN: 1932-6203;
submitted 06.07.2017.
The above publications have been published or submitted respectively to international, peer- reviewed journals. Aleksandra Piotrowska has carried out the experimental work, data preparation and has been responsible for conception and writing of manuscripts. Anna Syguda, Bogdan Wyrwas, Łukasz Chrzanowski, Till Luckenbach and Hermann J. Heipieper have taken part in feedback or discussion and manuscripts editing.
Authors publishing in Chemosphere journal have wide rights to use their works for inclusion in a thesis without needing to seek permission.
https://www.elsevier.com/__data/assets/pdf_file/0007/55654/AuthorUserRights.pdf Last accessed on 23.09.2017
If the paper was submitted for publication by PLOS, the CC BY license applied to this work. Under this Open Access license, anyone can reuse article in whole or part for any purpose, for free.
http://journals.plos.org/plosone/s/licenses-and-copyright Last accessed on 23.09.2017
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Funding
Aleksandra Piotrowska had been financially supported within the project ‘Engineer of the Future. Improving the didactic potential of the Poznan University of Technology’ - POKL.04.03.00- 00-259/12, implemented within the Human Capital Operational Programme, co-financed by the European Union within the European Social Fund (20.03.2015-20.09.2015).
This research has been founded by the National Science Centre in Poland conferred on the basis of the decision DEC-2011/03/B/NZ9/00731.
This work has been a part of the research topic ‘Chemicals in the Environment’ (CITE) within the research program of the UFZ - Helmholtz Centre for Environmental Research (POF II- U45).
5 TABLE OF CONTENTS
ABBREVIATIONS ... 8
1. INTRODUCTION ... 10
1.1. PREFACE ... 10
1.2. GOALS AND OUTLINE OF THE THESIS ... 11
2. OVERVIEW ... 14
2.1. Ionic liquids ... 14
2.1.1. Structure and biological activity of HILs ... 14
2.1.2. The case of ionic liquids toxicity ... 16
2.1.3. Mechanism of ionic liquids toxicity on the level of cellular membrane ... 17
2.2. Assessment of the toxic effect of ionic liquids ... 21
2.2.1. Principles of toxicity testing ... 21
2.2.2. Biomarkers as useful tools in toxicity testing ... 23
2.2.3. Investigation of toxic effects towards Pseudomonas putida ... 26
2.2.4. Investigation of toxic effects towards zebrafish embryos ... 28
3. MATERIALS AND METHODS ... 34
3.1. Chemical reagents ... 34
3.1.1. 2,4-dichlorophenol ... 34
3.1.2. Herbicides ... 34
3.1.3. Esterquats with 2,4-D and MCPA moieties... 34
3.1.4. Dialkyldimethylammonium salts ... 36
3.2. Experiments on Pseudomonas putida ... 38
3.2.1. Solutions and media... 38
3.2.2. Sterilisation method ... 39
3.2.3. Growth experiment with Pseudomonas putida ... 39
3.2.4. Determination of growth inhibition ... 40
3.2.5. Lipid extraction and derivatisation to FAME ... 41
3.2.6. Determination of the fatty acid composition and the trans/cis ratio ... 41
3.2.7. Statistical analysis ... 42
3.3. Experiments on zebrafish embryos ... 43
3.3.1. Solutions and media... 43
3.3.2. Maintenance of adult zebrafish ... 43
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3.3.3. Fish embryo acute toxicity test ... 43
3.3.4. Exposure experiment ... 44
3.3.5. Lipid extraction ... 44
3.3.6. Lipid separation and derivatisation to FAME ... 45
3.3.7. Determination of the fatty acid composition and the UI ... 45
3.3.8. Statistical analysis ... 46
3.3.9. Ethics statement ... 47
4. RESULTS ... 48
4.1. Experiments on Pseudomonas putida ... 48
4.1.1. Esterquats with 2,4-D and MCPA moieties... 48
4.1.2. Dialkyldimethylammonium salts ... 53
4.1.3. Statistical analysis ... 56
4.2. Experiments on zebrafish (Danio rerio) embryos ... 57
4.2.1. Acute toxicity studies ... 57
4.2.2. Toxic effects on the lipid fractions ... 58
4.2.4. Changes in the phospholipid fatty acid pattern after exposure... 64
4.2.5. Effect of toxic exposure on UI values ... 65
4.2.6. Influence of different chemical structures on UI values ... 66
4.2.7. Statistical analysis ... 67
5. DISCUSSION ... 68
5.1. Experiments on Pseudomonas putida ... 68
5.1.1. Toxicity experiments ... 68
5.1.2. Compounds effects on cis-trans isomerisation of fatty acids in P. putida ... 70
5.2. Experiments on zebrafish (Danio rerio) embryos ... 70
5.2.1. Acute toxicity ... 70
5.2.2. The effect of toxic exposure on lipid composition of zebrafish embryos ... 71
6. GENERAL SUMMARY ... 73
7. APPENDIX ... 75
8. LIST OF FIGURS ... 86
9. LIST OF TABLES ... 90
10. REFERENCES ... 92
ACKNOWLEDGMENTS ...100
ABSTRACT ...101
STRESZCZENIE ...103
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CURRICULUM VITAE ...105 SCIENTIFIC ACTIVITY ...106
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ABBREVIATIONS
1 M = 1 mol/L
1 mM = 1 mmol/L
2,4-D 2,4-dichlorophenoxyacetic acid 2,4-DCP 2,4-dichlorophenol
2,4-DNP 2,4-dinitrophenol ANOVA analysis of variance
DDT dichlorodiphenyltrichloroethane
DI water deionised water
dicamba 3,6-dichloro-2-methoxybenzoic acid
EC50 concentration of the compound which causes 50% of a growth inhibition measured with Pseudomonas putida mt-2 cells
FAMEs fatty acid methyl esters
FET fish embryo test
Fig figure
HILs herbicidal ionic liquids hpf hours post fertilisation
GC gas chromatography
GC-MS gas chromatography-mass spectrometry
ILs ionic liquids
LB lysogeny broth
LC10,30,50 concentration of the compound which causes death of 10%, 30% 50% of a group of zebrafish (Danio rerio) embryos
9 log P logoctanol/water partitioning coefficient MCPA (4-chloro-2-methylphenoxy)acetic acid
MCPP (R/S)-2-(4-chloro-2-methylphenoxy)propionic acid
n number of carbon atoms in alkyl chains
O.D.560 optical density at wavelength 560 nm
OECD Organisation for Economic Cooperation and Development
p probability value
PAHs polycyclic aromatic hydrocarbons
pKa logarithm of the acid dissociation constant
PLs phospholipids
PLFAs phospholipid-derived fatty acids PUFAs polyunsaturated fatty acids
rpm revolutions per minute
STD standard deviation
trans/cis 50% concentrations of compounds causing a half-maximum increase in the trans/cis ratio of unsaturated fatty acid in Pseudomonas putida mt-2 cells
UI unsaturation index
QSAR quantitative structure-activity relationship
w/v weight per volume
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1. INTRODUCTION 1.1. PREFACE
In agriculture, large scale applications of chemicals affect terrestrial as well as aquatic ecosystems. Therefore attempts are made to create more effective herbicides. Herbicidal ionic liquids exhibit higher biological activity and superior efficiency in comparison to typically employed herbicides. However, knowledge of their potential toxic effects is still limited.
Studying toxicity of newly designed compounds allows to classify them, eventually making a decision about their future application. Furthermore, understanding toxic effects and modes of toxicity can lead to development of assays that can be used to demonstrate and measure impact of chemicals on living organisms. That is why integration of chemical analysis with carefully chosen biological mechanism of the living organism to adapt to environmental contamination can reveal the impact of a toxic chemical on terrestrial and aquatic ecosystems.
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1.2. GOALS AND OUTLINE OF THE THESIS
The intention of this work is to provide information about toxic impacts of herbicidal ammonium-based ionic liquids and their precursors in terms of their possible use as an alternative to common herbicides (2,4-D, MCPA, MCPP, dicamba). This work contains studies on bacterium Pseudomonas putida and embryos of the zebrafish (Danio rerio) as model organisms for assessment of potential toxic effects, including adaptive mechanisms on the level of the cellular membrane. The aim of the thesis is to contribute to a better understanding of toxicity of ionic liquids and interpretation of the mode of their toxic action as well as the organism adaptation to its exposure. Therefore, experimental methods and chemical analyses have been applied to obtain information on toxic effects of chosen compounds.
This work focuses on:
toxicity of chosen compounds towards Pseudomonas putida mt-2 and it adaptation response, expressed in changes in fatty acid composition
adaptation response of zebrafish embryos to toxic concentrations of ammonium-based ionic liquid precursors and 2,4-dichlorophenol, expressed in changes in phospholipid fatty acid composition
Toxic effects of herbicidal ionic liquids (HILs) and their precursors on growth inhibition of P. putida have been investigated. The potential toxicity of herbicidal esterquats containing 2,4-D and MCPA moieties ([2,4-DDAEC6][Br], [2,4- DDAEC10][Br], [MCPADAEC6][Br] [MCPADAEC10][Br]) and dialkyldi- methylammonium salts, with herbicidal anions ([dicamba]-, [MCPP]-) and cations with various alkyl chain lengths ([C6,C6,C1,C1N][dicamba], [C8,C8,C1,C1N][dicamba], [C10,C10,C1,C1N][dicamba], [C6,C6,C1,C1N][MCPP], [C8,C8,C1,C1N][MCPP], [C10,C10,C1,C1N][MCPP]), has been estimated. For herbicidal esterquats the results have been compared to those obtained for 2,4-D and MCPA. For dialkyldi-
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methylammonium salts, the results have been compared to those obtained for MCPP, dicamba and ammonium-based ionic liquid precursors ([C6,C6,C1,C1N][Br], [C8,C8,C1,C1N][Br], [C10,C10,C1,C1N][Br]). Subsequently, cis-trans isomerisation of unsaturated fatty acids in P. putida cells has been applied as proxy for cellular stress adaptation to toxic compounds, and the trans/cis ratio has been determined.
Experiments on P. putida are carried out in order to determine the toxicity of ammonium-based ionic liquids and their precursors on the bacterium occurring in the environment potentially affected by herbicides. Furthermore, the intention of the experiments on P. putida is to study the relationship between ammonium- based ILs structure (considering the length of the alkyl chains and the corresponding anionic species) and their toxicity. This part of the work aims to provide knowledge about design ILs structure and their behaviour as environmental toxicants.
The scope of research on Pseudomonas putida includes the following stages:
1. growth experiments with Pseudomonas putida mt-2 in a mineral medium with toxicants
2. determination of growth inhibition, given as EC50 values, by monitoring the turbidity of cell suspensions
3. lipid extraction and derivatisation to fatty acid methyl esters 4. fatty acid analysis
5. determination of trans/cis ratio based on relative amounts of monounsaturated fatty acids
For ammonium-based ionic liquid precursors ([C6,C6,C1,C1N][Br], [C8,C8,C1,C1N][Br]) and 2,4-dichlorophenol, the acute toxicity on zebrafish (Danio rerio) embryos has been estimated. Subsequently, the changes in phospholipid fatty acid composition of zebrafish embryos, as an adaptation mechanism triggered by different lethal concentrations (LC10, LC30, LC50) of selected [C8,C8,C1,C1N][Br], have been investigated. Furthermore, the changes in the unsaturation index (UI) of phospholipid fatty acids (PLFAs), as the sum parameter of membrane fluidity in eukaryotic cells, have been presented. In addition, an influence of compounds hydrophobicity and different chemical structures on the UI values has been
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analysed. Therefore, 2,4-DCP with comparable hydrophobicity to [C8,C8,C1,C1N][Br]
has been chosen to observe whether the ammonium-based ionic liquid precursor can lead to similar changes in the UI as 2,4-DCP. As the second compound to compare, the less hydrophobic ammonium-based ionic liquid precursor with shorter alkyl chains ([C6,C6,C1,C1N][Br]) has been selected. Differences in the UI values for the LC50 between the compounds (2,4-DCP, [C6,C6,C1,C1N][Br]) and [C8,C8,C1,C1N][Br] have been investigated. The intention of the experiments on zebrafish embryos is to present the UI as a sum parameter for the adaptive mechanism to ionic liquid exposure. This part of work aims to provide innovative studies on zebrafish embryos and present the changes in the PLFAs as a possible biomarker of toxic effect for aquatic contaminations.
This scope of research on zebrafish (Danio rerio) embryos includes the following stages:
1. Fish embryo acute toxicity (FET) tests
2. exposure experiments up to 72 h of fertilised zebrafish eggs to solutions containing interpolated lethal concentration from concentration-response curves
3. lipid extraction, separation and derivatisation to fatty acid methyl esters 4. fatty acid analysis
5. determination of the phospholipid fatty acid unsaturation index (UI) based on relative amounts of fatty acids in phospholipid fraction
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2. OVERVIEW 2.1. Ionic liquids
2.1.1. Structure and biological activity of HILs
Ionic liquids (ILs) are ionic compounds characterised by a melting point below 100 oC. They contain organic cations like imidazolium (Fig 1 A), pyridinium (Fig 1 B), pyrrolidinium (Fig 1 C), ammonium (Fig 1 D), phosphonium (Fig 1 E) and inorganic (e.g. [BF4]-, [PF6]-, [ZnCl3]-, [NO3]-, [CF3SO3]-) and organic (e.g. salicylate, lactate) anions. Halogenates of ionic liquid cations are called ionic liquid precursors. Their chemical and physical properties have been modulated by selection of proper cation and anion.
N+ R
N N+
R R
N+
R R
R N+
R R
R
R P+
R R
R
A B C
D E
1
2 3
4 1
2
1 2
1
2 3
4
Fig 1. Organic cations used to synthesised ionic liquids. Selected cations species: imidazolium (A), pyridinium (B), pyrrolidinium (C), ammonium (D), phosphonium (E).
Their achievable properties of chemical design have become an impulse to obtain ILs containing herbicide moieties (Petkovic et al. 2010). Derivatives of phenoxy acid such as 2,4-D ((2,4-dichlorophenoxy)acetic acid), MCPA ((4-chloro-2- methylphenoxy)acetic acid) and MCPP ((R/S)-2-(4-chloro-2-methylphenoxy)- propionic acid)) next to dicamba (3,6-dichloro-2-methoxybenzoic acid) are common herbicides used in agriculture to provide unimpeded grow of plants (Cojocaru et al. 2013). However, their negative impact on the environment,
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associated with their high volatility and water solubility, has created the need to design better alternatives.
Herbicidal ionic liquids (HILs), are defined as ionic compounds with melting temperature below 100 oC, where one of the ions possesses herbicidal activity.
They belong to the third and the youngest generation of ionic liquids with defined biological activity (Pernak et al. 2011). In literature, HILs containing herbicidal anions: 2,4-D (Pernak et al. 2012a, Praczyk et al. 2012), MCPP (Pernak et al.
2012b) and dicamba (Cojocaru et al. 2013) have been presented. HILs with additional surfactant properties have been synthesised and firstly described by (Pernak et al. 2011, 2012b). In their structure, a hydrophilic anion possessing herbicidal activity from MCPA and 2,4-D moieties had been merged with a hydrophobic quaternary ammonium cation, with long alkyl chains. An ammonium- based ionic liquid with dicamba moiety ([C10,C10,C1,C1N][dicamba]) has presented by (Cojocaru et al. 2013) and the ammonium-based ionic liquid with herbicidal activity of MCPP has currently been synthesised ([C10,C10,C1,C1N][MCPP]) (Fig 2).
H21C10
N+ CH3 C
H3 C10H21 O
O CH3 O
Cl CH3
Fig 2. Herbicidal ionic liquids [C10,C10,C1,C1N][MCPP]; ammonium-based ionic liquid with MCPP moiety.
The addition of a quaternary ammonium cation in an ionic liquid with a herbicidal active anion, provides higher absorption of herbicide allowing lower application dose on a field, than herbicides without additional surfactant properties (Cojocaru et al. 2013). HILs with long alkyl chains exhibit surface activity and lower liquid- contact angle of the surface of the leaf, which for foliar applied herbicides plays a key role in the penetration of the active substance in plant (Praczyk et al. 2012).
Additionally, their higher biological activity and consequential reduction of mobility in soil and groundwater provide ionic liquids an advantageous edge over phenoxy herbicides (Pernak et al. 2011).
16 2.1.2. The case of ionic liquids toxicity
Ionic liquids are promoted as ‘green solvents’ in publications and on green and sustainable chemistry conferences. They are known for fulfilling at least three principles (marked below) of the twelve principles of green chemistry developed by (Anastas and Warner 1998):
1. prevent waste 2. atom economy
3. less hazardous synthesis 4. design benign chemicals 5. benign solvents & auxiliaries 6. design for energy efficiency 7. use of renewable feedstocks 8. reduce derivatives
9. catalysis
10. design for degradation
11. real-time analysis for chemistry
12. inherently benign chemistry for accident prevention
Ionic liquids can berepeatedly used and show higher efficiency and selectivity in low temperature as compared to volatile organic compounds. They are known as effective solvents and catalysts. Furthermore, with the growing interests in the use of ILs, it can be expected that they could fulfil other criteria in future. However, the argument about their relatively non-toxic character has been constantlychallenged (Renke et al. 2007). Depending on chemical structures and model systems for toxicity testing, ionic liquids have presented moderate to high toxicity in comparison to conventional organic compounds such as benzene, methanol or propan-2-ol (Bubalo et al. 2014, Hermandez-Fernandez et al. 2015). In Literature, studies testing the toxicity of ILstowards bacteria, algae, Daphnia magna (water flea), worms, embryos (frog), fish (loach, zebrafish, goldfish) and mammals (mice, rats) have be found. ILs toxicity on different systems of biological organisation, considering aquatic and terrestrial compartments, has been successively discussed by (Pham et al. 2010).
So far, the largest database on ILs aquatic toxicity has been collected for Daphnia
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magna (Ranke et al. 2007). However, toxic effect of ILs on humans and large mammals have not been thoroughly studied (Hackenbach et al. 2016, Ranke et al.
2007).
According to a meta-analysis from 2016, literature on ILs toxicity for all cation species represented 0.55% ± 0.27% of total ILs publishing activity during the years 2000-2014 (Heckenbach et al. 2016). Compared with other industrial chemicals, with similar number of publications where the amount of publishing activity is over four times the amount of total body of ILs literature (12% for phthalates vs. 2.9% for ILs), there is an obvious lack of attention concerning ionic liquids’ environmental impact (Hackenbach et al. 2016) (Fig 3).
Fig 3. Percentage of toxicity-associated literature presented by (Heckenbach et al. 2016) using the SicFinder database for chosen chemical groups: linear alkyl sulfonates (LASs), alkylphenol etoxylates (AEs), polybrominated diphenyl ethers (PBDEs), phthalates, bisphenols, perfluorinated compunds (PFCs) and ionic liquids (ILs). The percentage of literate related to toxicity is shown in blue. Source: Open Access articles; www.sciencedirect.com.
2.1.3. Mechanism of ionic liquids toxicity on the level of cellular membrane 2.1.3.1. Cell membrane function and mechanisms of membrane toxicity
Cytoplasmic membranes play a crucial role in regulation of the intracellular environment, signal transduction and energy-transducing processes (Sikkema et al. 1995). Membranes of prokaryotic (bacteria) as well as eukaryotic cells (animals) are formed by a bimolecular layer of phospholipids forming matrix
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(Fluid-Mosaic Membrane Mode originally proposed by Singer and Nicolson 1972) with integral and peripheral proteins and glycoproteins (Fig 4).
Fig 4. A schematic illustration of membrane model, as presented by (Escribá et al. 2008). Different phospholipids, in various colours, forming domains around integral proteins and glycoproteins as well as being asymmetrically distributed across the membrane. Illustration and commentary adapted from (Nicolson 2014). Source: Open Access articles; www.sciencedirect.com.
Cell envelopes are different between organisms and even between strains (subtypes of a bacterium) (Rose 1989). For bacteria, two major groups have been be recognised on the basis of their cell envelope composition: Gram-positive and Gram-negative bacteria. The Gram-positive bacteria have only one membrane (cytoplasmic membrane) which is surrounded by a cell wall. The Gram-negative bacteria have a cytoplasmic membrane and an outer membrane that consists of phospholipids and lipoproteins. Between the two membranes there is a thin layer of peptidoglycans (Nikaido and Vaara 1987).
Phospholipids (PLs) are present in all living forms being the major constituent of membranes that protect cells from external environment and segregate organelles. PLs are composed of two hydrophobic tails, coming from diacylglycerol, and a hydrophilic head group containing a phosphate (Fig 5). This amphipathic structure assembles into bilayers, which compartmentalise the cell and harbour an assortment of proteins and glycans that play critical roles in cell structure, function and metabolism (Zalba and Hagen 2017).
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Fig 5. Example of a molecular structure of a phospholipid, phosphatidylcholine, detected in zebrafish (Danio rerio) embryos by (Pirro et al. 2016). Image adapted from (van Hoogevest 2017).
Source: Open Access articles; www.sciencedirect.com.
The membrane bilayer contains also lipid-soluble compounds such as cholesterol (lipid molecule biosynthesised by animal cells), hopanoids (pentacyclic components of bacteria), and carotenoids (produced by plants and algae), which are responsible for maintaining membrane fluidity. However, the phospholipid fraction is most important in the partitioning process of toxic compounds into the membrane. The area occupied by phospholipid molecules is determined by the large volume of their acyl chains (formula RCO- where R represents an alkyl group) and the accumulation of a compound into this area could be the reason of its toxic properties (Sikkema et al. 1994, 1995).
A cytoplasmic membrane exhibits a high permeability for hydrophobic (lipophilic) compounds (e.g. hydrocarbons) which can easily penetrate the lipid bilayer, leading to non-specific reversible disturbance and increase in fluidity (van Wezel and Opperhuizen 1995). Hydrophobicity (or lipophilicity), as a physicochemical feature of compounds, depends on various physical and chemical characteristics, e.g. molecular surface and polarity (Leo et al. 1976). Higher hydrophobicity, expressed in terms of the log P value (logoctanol/water partitioning coefficient), increases the toxic effect of a compound indicating its partitioning from aqueous medium to membranes (Bubalo et al. 2014, de Bont 1998, Heipieper et al. 1994, 1995; Hermandez-Fernandez et al. 2015, Sikkena et al. 1995).
Surfactants (surface-active compounds) are also able to affect cellular membranes (Helenius and Simons 1975). Lipophilic compounds as disinfectants,
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detergents and antiseptics, including quaternary ammonium compounds, accumulate in the lipid bilayer, leading to lipid-protein as well as lipid-lipid interactions. They cause permeabilisation of cells (Miozzari et al. 1978) and release of membrane-incorporated proteins (Helenius and Simons 1975). Toxic effects of surfactants have mostly been determined by the critical micelle concentration (CMC; concentration of surfactant above which micelles form) and the partition coefficient of surfactant (how much surfactant prefers to be in the oil phase than in water phase). These parameters depend on the type of surfactant, medium, temperature, pH, ionic strength, and ionic composition (Kalmanzon et al.
1992). In studies presented by (Kalmazon et al. 1992) the effectiveness of detergents had been tested, and anionic sodium dodecyl sulfate (SDS) and non- ionic Triton X-100 had been compared. SDS caused permeabilisation of membranes at concentrations below the CMC, whereas for Triton X-100 the same effectiveness had been observed above its critical micelle concentration (Kalmazon et al. 1992).
Other chemical stressors, like 2,4-dinitrophenol (2,4-DNP) and 2,4- dichlorophenol (2,4-DCP), are expected to interfere with lipid metabolism. These compounds are protonophores, shown to disrupt the proton-motive-force (PMF) across the membrane. As the PMF is known to be an energy supply for the translocation of membrane proteins, protonophores therefore inhibit the translocation phenomenon (Bimal et al. 2009) and increase the membrane fluidity by interacting with membrane components (Nunes et al. 2008).
2.1.3.2. Effects of functional side chains on the toxicity of ionic liquids
The proposed model of ILs toxic mechanism is based on cellular membrane disruption, similar for detergents, pesticides and antibiotics (Bubalo et al. 2014).
However, the toxicity pathway for ionic liquids has not yet been entirely understood. From literature it can be concluded that ILs toxicity is mostly affected by (Heckenbach et al. 2016):
21 1. cation size or branching 2. cation species
3. length or branching of cation alkyl chains 4. hydrophobicity
5. anion species
6. surfactant behaviour
The long alkyl chains occurring in cations of ionic liquids are responsible for the lipophilic character of these compounds, and thus enhance the possibility of membrane disruption. This is known as the side chain effect and describes a tendency of increasing toxicity with the elongation of alkyl chains (Ventura et al.
2012). In several studies, it has been demonstrated that imidazolium-based ILs with longer alkyl side chains exhibit higher toxicity (Xuan et al. 2013, Hermandez- Fernandez et al. 2015). However, results published by (Ventura et al. 2012) have showed that guanidinium-based ILs with longer alkyl chain do not demonstrate higher toxicity than their derivatives with shorter alkyl side chain. Authors have claimed that at a defined number of carbon atoms in alkyl chain, toxicity is not increasing any further. This phenomenon is called cut-off effect (Xuan et al. 2013, Ventura et al. 2012) and it depends on the size of the molecules, low solubility, insufficient bioavailability as well as their influence on slower uptake by cell membranes (Ventura et al. 2012, 2014).
2.2. Assessment of the toxic effect of ionic liquids
2.2.1. Principles of toxicity testing
Toxicity testing is the evaluation and interpolation of a harmful effect of a toxicant by testing it on a living organism. The foundation principle of toxicity tests requires an understanding that the response of an organism to an exposure depends upon the toxic dose or the toxic concentration. Toxicity tests have been designed to describe a dose-response (concentration-response) relationship as a dose-response (concentration-response) curve, when the measured effect is plotted graphically against the dose (concentration).
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Short-term tests aim to determine the LC50 or EC50 values (Hoffman et al.
1995).
LC50 is the concentration of a compound which causes death of 50% of a group of test organisms.
EC50 is the concentration of a compound which gives the half-maximal response, other than death (e.g. growth inhibition).
Toxicity of a chemical has be observed in terms of in vitro studies, using cells or cell lines, as well as in vivo exposure on an experimental organism (Parasuraman 2011). The model organism is non-human species that is extensively studied and has been chosen to proof toxicity of a chemical, assuming that data may be applicable to other organisms; especially those more complex than the model organism (Leonelli and Ankeny 2013). Model organisms have been found among prokaryotes, fungi, plants and animals. However, in past decades, the term ‘model organism’ has been usually applied to those species, that are small in size, have short generation times or facilitate laboratory research (Hedges 2002).
In the first step research processes, mostly non-mammalian model organisms are used in order to deliver fast answers to a research problem (Kaletta and Hengartner 2006).
In recent years, data on toxic effects of xenobiotics have been collected and in combination with the physicochemical characteristics have been used to rationalise results and predict the fate of pollutants in an environment.
Quantitative structure-activity relationships (QSARs) of baseline toxicity (also called narcosis) haven been helpful to estimate the minimum toxicity. Based on an existing fish embryo toxicity database, the baseline toxicity QSAR for fish embryo (LC50), using chemicals that act according to the non-specific mode of action, have been established. The octanol-water partition coefficient (log P) has been commonly applied to discriminate between non-polar and polar narcotic (Klüver et al. 2016). In studies presented by (Couling et al. 2006) attempts have been made to generate predictive equations using the QSAR modelling based on ILs toxicity towards Vibrio fischeri (bioluminescent bacterium) and Daphnia magna (water flea). However, the modelling does not provide further information on toxic effects
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and their underlying mechanisms (Sikkema et al. 1995). Furthermore, it has to be taken into consideration that unspecific modes of action can occur, especially for small aquatic organisms. That is why independent studies on applicability for various organisms are required in order to predict toxic effect of untested chemicals (Renke et al. 2007).
In case of soluble in water ionic liquids, distinct fates of cation and anion in the environment suggest to evaluate their toxicity separately. Ionic liquid anions in a form of alkali salts and halogenates cations form a recommended strategy in attempts to explain the whole effect of ionic liquid on living organism (Renke et al.
2007). As a starting point for toxicity testing, the bacterium as a model organism to study potential antibacterial activity is recommended, mostly because of the short generation time (Pham et al. 2010).
2.2.2. Biomarkers as useful tools in toxicity testing 2.2.2.1. Biomarkers for measuring toxic effects
When a toxicant enters living organisms, it causes two kinds of changes. On the one hand, the organism undergoes automatic changes that serve as attempts to protect itself against harmful effects of a chemical, and the other hand, the toxicant causes malign changes for the organism. They latter may be specific or non-specific for a particular type of chemical. Furthermore, similar responses to any toxicant could be specific to only certain groups of organisms or could be observed in all of them (Walker et al. 2006). Biomarkers (short for biological markers) defined as any biological responses to a chemical, demonstrating a shift from normal status, can confirm whether or not an organism was exposed to toxicant (Douben 1998, Okoro et al. 2011). The term ’biomarker’ or ‘biomarker response’ usually refers to cellular, biochemical, molecular, or physiological changes that have been measured in cells, body fluids, tissues, or organs within an organism (Torres et al. 2008).
Biomarkers have been classified into three general types (Committee on Biological Markers of the National Research Council 1987, Peakall and Fairbrother 1998):
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1. Biomarker of exposure - proves the presence of a compound, its metabolites or other products, or the change in physical, biochemical parameters, which can be connected with exposure to the stressor. Biomarker of exposure has been be considered a substitute for the dose or the concentration in the dose-response model or in concentration-response model.
2. Biomarker of effect - evidence of a change in the status, or functional capacity of parameters which is connected with exposure to the stressor or other adverse response. Biomarker of effect has been considered a substitute for the dose or the concentration in the dose-response model or in concentration-response model.
3. Biomarker of susceptibility or sensitivity - evidence of a presence or a change in some parameters (physical, biochemical) that prove that system has a potential to be sensitive to the effect of certain stressor. Biomarker of susceptibility or sensitivity includes genetic factors and changes in receptors which change the susceptibility of an organism to chemical exposure.
Biomarkers offer a way in which the effect of environmental contamination can be monitored. They have an advantage over chemical analysis, demonstrating whether or not an organism is exposed to chemical contamination (Lam and Gray 2003). With biomarkers, it is possible to determine whether the physiology of an organism is significantly shifted from normal status. If a physiological parameter is measured and no significant difference is observed, then the organism is not considered to be exposed to potentially toxic chemicals; even when such a chemical was detected in the environment (Peakall and Fairbrother 1998).
Biomarkers can be a sensitive tool of chemical stress detection within organisms (Okoro et al. 2011). They can be also used for testing hypotheses about the mechanism of chemical impact at the chosen level of biological organisation (Brain and Cedergreen 2008). The application of biomarkers is not only limited to laboratory work but is also used in field studies. However, the development of biomarkers starts with experimentation to first identify potential responses and
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causal mechanisms. Each biomarker have to be validated by establishing a relationship between the exposure and the biological change of interest (Committee on Biological Markers of the National Research Council, 1987). Criteria for validity of biomarkers have presented by (Peakall and Fairbrother 1998):
1. clarity of interpretation - ability to define the biomarker
2. chemical specificity - knowledge of concentration-response or dose- response relation of the biomarker to a specific chemical
3. biological specificity - knowledge of organism classes for which the biomarker can be used
4. time of response
5. permanence of response
6. reliability - knowledge of environmental influences and variations in biological responses
7. methodological considerations e.g. reproducibility, cost, ease of the assay 8. relative sensitivity - the biomarker should be sensitive enough to be defined
before other effects set in (e.g. mortality) 9. validation in the field
Currently, the most commonly used biomarkers in environmental hazard assessment are acetylcholinesterase (AchE) inhibition, aminolaevulinic acid dehydratase (ALA-d) (sensitive biomarker of lead exposure in avian species and mammalians), porphyrins (biomarkers of polyhalogenated aromatic hydrocarbon- inducted changes) and eggshell thinning (DDT exposure) (Okoro et al. 2011, Peakall and Fairbrother 1998). The inhibition of acetylcholinesterase (AChE) has been used to study effects of ILs in an enzyme level. AChE plays an important role in nerve response and function of mammalians nervous system. Any inhibition of AChE leads to various effects in neuronal processes (Lionetto et al. 2013). The ILs toxicity data includes those of AChE from the enzyme system of mouse liver (Yu et al. 2009) and from electric eel (Electrophorus electricus) (Arning et al. 2008, Jastorff et al. 2005, Matzke et al. 2007, Ranke et al. 2007). The results of these studies have showed that cations moiety is the dominating factor of ILs toxicity and anions have a non-inhibiting effect.
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2.2.2.2. Biomarkers on the level of membrane fatty acid composition
General conditions for metabolic processes depend on the integrity of cellular membranes (Zhang et al. 2014). Therefore, any membrane disruption can be observed as a toxic effect on a model organism. As mentioned in section 2.1.3.
(‘Mechanism of ionic liquids toxicity on the level of cellular membrane’) a disturbance of phospholipid integrity by lipophilic compounds eventually could result in swelling of the bilayer. To resist changes caused by external stimuli cells need to adapt by modifying their membrane lipid composition (Sikkema et al.
1995). Therefore, adaptive responses to chemical stressors on the level of membrane composition could be expressed as a possible lipidomic biomarker.
Lipidomic studies allow a comprehensive overview of lipids in specific biological conditions. They often rely on chromatography (e.g. liquid and gas chromatography) coupled to mass spectrometry (MS). These approaches require sample preparation and lipid derivatisation. Alternatively, direct MS analysis can provide lipid profiling. Among the direct techniques, desorption electrospray ionisation mass spectrometry (DESI-MS) as an ambient ionisation MS has been developed (Pirro et al. 2016).
2.2.3. Investigation of toxic effects towards Pseudomonas putida
Environmental toxicity is an important factor revealing concentration of such compounds that have negative effects on soil bacteria including those responsible for biodegradation processes. Because of a considerable potential for ecosystem contamination through direct use on a field, it is crucial to value the possible negative impact of herbicides on soil microorganism (Ahtiainen et al.
2003, Ławniczak et al. 2016).
In the present work, the bacterium Pseudomonas putida as one of the best investigated model organisms for toxicity and adaptation mechanisms (Heipieper et al. 1995) has been chosen. Strains of Pseudomonas belong to heterotrophic microorganisms occurring globally. They are well-characterised, metabolically versatile soil species with highly adaptable abilities to survive and proliferate in various habitats (Ahtiainen et al. 2003, Heipieper et al. 1992 1994). Those Gram- negative bacteria are mostly found in soil and water habitats, where oxygen is present. P. putida has been reported as being able to degrade natural products as
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well as xenobiotics. Besides this, P. putida has been often used as a model organism for measuring toxicity of different agents including organic pollutants, heavy metals and antibiotics (Hachicho et al. 2014, Keweloh and Heipieper 1996). In toxicity tests P. putida is cultivated in a defined medium with different concentrations of a chemical. During the exposure the bacterial strain multiplies slower and cell growth is inhibited due the toxic effect (Ahtiainen et al. 2003).
Single-species bacterial tests have been widely accepted to assess potential toxicity of chemicals. They are known as generally more sensitive than multispecies toxicity tests, in which complex systems can lead to wrong interpretations and problems with repeatability need to be addressed (Ahtiainen et al. 2003, Landis 1997). The main advantages of using P. putida as a model organism are firstly a very short time of measurement, which takes approximately 5 h (Hage et al. 2000), and secondly the use of the stress response systems as an additional biomarker.
The genus Pseudomonas has shown wide range of stress responses to environmental changes (Heipieper et al. 2007). For example, Pseudomonas strain is able to grow in presence of toluene at concentrations up to 50%, however, it is not capable of metabolising toluene (Inoue and Horikoshi 1989, Inoue et al. 1991).
Abiotic stresses such as organic compounds affect bacteria, causing an increase of membrane fluidity. The stress response buffers the harmful effect by changing the confirmation of membrane fatty acids. The cis configuration of phospholipid fatty acids causes an increase in membrane fluidity by its bended steric structure (Fig 6 A). The trans configuration reduces the membrane fluidity due to its long and extended steric structure that allows to insert itself into membranes (Fig 6 B) (Macdonald et al. 1985). That is why, P. putida is able to adapt to toxicants by isomerisation of unsaturated fatty acids, which lowers its membrane fluidity (Heipieper et al. 1992, Udaondo et al. 2012).
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Fig 6. Different conformations of phospholipids and their effect on the structure of the lipid bilayer (according to Sikkena et al. 1995). (A) cis-unsaturated fatty acids; (B) trans-unsaturated fatty acids.
The trans-unsaturated fatty acids are synthesised by a direct isomerisation of cis- unsaturated fatty acids without a shift in the position of a double bond (de Bont 1998). Cis-trans isomerisation of unsaturated fatty acids has been proven to be independent of de novo fatty acid biosynthesis (synthesis of complex molecules from simple molecules) allowing bacteria to adapt to a presence of toxicant, even under environmental conditions not supporting their growth (Heipieper et al.
1994, 2007). Furthermore, cis-trans isomerisation of unsaturated fatty acids occurs rapidly taking less than 15 minutes to response to the exposure. A shift in the trans to cis ratio (trans/cis ratio) indicates the presence of an environmental stressor and depends on hydrophobicity and the concentrations of toxic chemicals (de Bont 1998, Heipieper and de Bont 1994). Therefore, cis-trans isomerisation of unsaturated membrane fatty acids in P. putida can be applied as a proxy for cellular stress adaptation to toxic compounds.
2.2.4. Investigation of toxic effects towards zebrafish embryos
2.2.4.1. Zebrafish embryos as a model organism for investigating aquatic pollutants Water solubility enables ionic liquids and their precursors with long alkyl chains to influence aquatic habitats. Negative effects could be expected in large- scale agriculture, where accidental water contamination can occur. The impact of farming on water quality is well known and has been thoroughly studied (Jaynes et al. 1999). It has been shown that in the USA 72% of water quality problems by river side are caused by agriculture activity, 37% of which are due to herbicide utilisation (USEPA 1992). Many field-scale studies have shown the correlation
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between herbicide usage (e.g. 2,4-D) and surface water contamination from runoff, heavy rain events and tile drainage (Baker and Laflen 1982, Buhler et al. 1993, Gaynor et al. 1995, Kladivko et al. 1991, White et al. 1976).
The first data on toxicity of ionic liquids to various aquatic organisms have been showed that the negative effects of ammonium-based ILs especially on aquatic habitats have to be taken into consideration (Pretti et al. 2006). In this work, embryos of the zebrafish (Danio rerio) have been chosen to study potential toxic effects of ammonium based-ionic liquid precursors and 2,4-DCP. Zebrafish is a fresh water fish, from the family of Cyprinidae (Fig 7). They are easy to obtain and inexpensive to maintain (Laale 1977).
Fig 7. Illustration of adult female and male zebrafish (Danio rerio). Illustration adapted from
‘Central Illustration. Phenotypic Screens in Zebrafish’ presented by (Kithcart et al. 2017). Source:
Open Access articles; www.sciencedirect.com.
Zebrafish embryos develop very quickly (Fig 8) and five days after fertilisation the yolk supply, used for growth, development and as energy source, is almost completely consumed and the zebrafish starts active external feeding (Kimmel et al. 1995). However, according to (Hachicho et al. 2015), in spite of fundamental changes during zebrafish development the amount of carbon, nitrogen, proteins and fatty acid fractions change relatively little over time.
Embryos of the zebrafish have become one of the most popular model system in (eco)toxicology (Belanger et al. 2013, Goldsmith 2004, Nagel 2002, Pelster 2008, Scholz et al. 2008). In contrast to experiments with adult fish, experiments with fish embryos are not considered as animal testing and zebrafish embryos is therefore regarded as an alternative to fish tests (Scholz et al. 2013).
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Fig 8. Consecutive embryonic stages of zebrafish (Danio rerio)embryos: 0.5 hpf, 24 hpf, 48 hpf and
72 hpf (according to Kimmel et al. 1995). Photos were taken at Department of Bioanalytical Ecotoxicology, Helmholtz Centre for Environmental Research-UFZ, Leipzig (Germany).
Zebrafish embryos provide also a comprehensive experimental platform for assessing water contaminations. The example of how zebrafish embryos can be affected by water pollution has been presented in studies on toxic urban runoff by (McIntyre et al. 2014). Urban stormwater contains a mixture of contaminants (e.g.
polycyclic aromatic hydrocarbons; PAHs) that can be toxic to aquatic life. Zebrafish embryos were exposed to a runoff (up to 96 h) and showed a number of sublethal and lethal developmental abnormalities, including reduced growth, pericardial edema, microphthalmia (small eyes) and reduced swim bladder inflation. The untreated urban runoff caused also heart failure, pericardial edema and looping defects (Fig 9) (McIntyre et al. 2014).
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Fig 9. Examples of developmental abnormalities after exposure of zebrafish (Danio rerio) embryos (up to 96 h) to urban stormwater containing PAHs, compared to control. Illustration adapter from
‘Graphical abstract’ presented by (McIntyre et al. 2014). Source: Open Access articles;
www.scienedirect.com.
2.2.4.2. Adaptive mechanism of zebrafish embryos on the level of membrane lipid composition
Different stressors are affecting living systems on organism as well as on cellular level. At the cellular level, the effect of a stressor is regarded as any kind of disturbance to normal development. To resist or eliminate the damaging chemical, the organism evolves cellular stress response, by changing its membrane viscosity (Kroes et al. 1972, Padmini 2010).
Zebrafish embryos contain polyunsaturated fatty acids (PUFAs) (Pirro et al.
2016) which are responsible for adjusting the physicochemical properties of the cellular membrane and regulating its fluidity (Fig 10) in a process called homeoviscous adaptation (Hazel 1984). The general trend in adaptation to stressors is the increase in the relative amounts of phospholipid saturated fatty acids and the corresponding decrease in unsaturated fatty acids; in order to enhance the lipid membrane rigidity (Kaszycki et al. 2013, Sinensky 1974, Spector and Yorek 1985). Therefore for the zebrafish embryos, the adaptive response to a chemical stressor has been expected to be expressed in changes in the phospholipid fatty acid composition and in the unsaturation index (UI).
The unsaturation index (UI) is the sum parameter of membrane fluidity in eukaryotic cells which presents the average number of double bonds in the fatty acid lipid fraction (Couture and Hulbert 1995, Kaszycki et al. 2013).
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Fig 10. Visualisation of different composition of lipid bilayers. Presence of unsaturated phospholipids results in higher state of fluidity. The double bounds in lipids results in bends in the fatty chains causing steric hindrance between them. Image and commentary adapted from Zalba and ten Hagen 2017. Source: Open Access articles; www.sciencedirect.com.
Changes in the UI values for exposed zebrafish embryos to a toxic compound have been studied by (Hachicho et al. 2015), who examined the effect of 2,4-DNP for one toxic concentration (LC10). 2,4-DNP has caused a significant decrease in the UI of the phospholipid fatty acids (PLFAs) after the exposure of fertilized zebrafish eggs for 24 h, 30 h and 48 h (Table 1).
Table 1. Mean values (± STD) for unsaturation index (UI) of the PLFA fraction in untreated zebrafish embryos and embryos treated with 2,4-DNP at 24, 30 and 48 hpf, presented by (Hachicho et al. 2015). Statistically significantly different values from Student t-test (p < 0.05) are marked with (*)
PLFA UI
Untreated 2,4-DNP
24 hpf 1.91 ± 0.02 1.48 ± 0.02*
30 hpf 1.85 ± 0.01 1.44 ± 0.03*
48 hpf 1.89 ± 0.04 1.76 ± 0.05*
In addition, (Hachicho et al. 2015) examined to which degree the amount of proteins and different lipid fractions (phospholipids, glycolipids, neutral lipids) in zebrafish embryos is influenced by exposure to a chemical. According to (Hachicho
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et al. 2015) upon toxic exposure major changes have been observed only in the amount of glycolipids but not for proteins and other lipid fractions.
So far, the adaptive mechanism of zebrafish embryos, expressed in the changes in PLFA composition, triggered by toxic exposure, has not been presented as possible lipidomic biomarker of toxic effect. Furthermore, the influence of different compounds structures and properties on the PLFA’s UI values for zebrafish embryos, exposed to toxic chemicals, has not yet been studied.
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3. MATERIALS AND METHODS 3.1. Chemical reagents
3.1.1. 2,4-dichlorophenol
The compound used in the exposure experiments on zebrafish (Danio rerio) embryos, 2,4-DCP (99%) have been purchased from (Sigma Aldrich, Munich, Germany)
3.1.2. Herbicides
The herbicides used in the growth experiments with Pseudomonas putida, 2,4-D (97%), MCPA (≥95%), dicamba (97%) and MCPP (98%) have been purchased from Sigma-Aldrich (Munich, Germany) (Table 2). The herbicides have been examined in acid form, as according to ‘Human health and ecological risk assessment final report - 2,4-D’: exposures to 2,4-D in the use of both salt and ester formulations will be essentially identical in terms of acid equivalents (USDA 2003).
Chemical formula of the herbicides is presented in Table 2.
Table 2. Chemical structures of herbicides
Herbicide Chemical structure Herbicide Chemical structure
2,4-D
O O
Cl OH Cl
dicamba
Cl
Cl
OH O CH3 O
MCPA
O O
CH3 OH Cl
MCPP
O O
CH3 OH
Cl CH3
3.1.3. Esterquats with 2,4-D and MCPA moieties
The herbicidal esterquats have been designed and synthesised in the Department of Chemical Technology in Poznan University of Technology (Poland)
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and their synthesis has not been a part of the PhD work. Synthesis details have been presented in the supplementary material (Piotrowska et al. 2016). The esterquats with 2,4-D and MCPA moieties are compounds with ‘limited solubility’
in water, which applies to 0.1 g of compound that dissolved in 3 mL of water at 25 °C (according to the protocols described in Vogel et al. 1989). The chemical and structural formula, melting points and purity for the esterquats with 2,4-D and MCPA moietiesare provided in Table 3.
Table 3. Chemical and structural formula, melting points and purity of esterquats with 2,4-D and MCPA moieties; n.d.: no data
Chemical formula/Name Structural formula Melting point [oC]
Purity [%]
[2,4-DDAEC6][Br]
(2,4-dichlorophenoxy)-2-
acetoxyethylhexyldimethylammonium bromide
O
O O
Cl Cl
N+ CH3 C H3 H13C6
Br-
120.3 – 122.8 n.d.
[2,4-DDAEC10][Br]
(2,4-dichlorophenoxy)-2-
acetoxyethyldecyldimethylammonium bromide
O
O O
Cl Cl
N+ CH3 C H3 H21C10
Br-
127.8 – 128.8 97.0
[MCPADAEC6][Br]
(4-chloro-2-methylphenoxy)-2- acetoxyethylhexyldimethylammonium bromide
O
O O
CH3 Cl
N+ CH3 C H3 H13C6
Br-
120.9 – 123.3 n.d.
[MCPADAEC10][Br]
(4-chloro-2-methylphenoxy)-2- acetoxyethyldecyldimethlammonium bromide
O
O O
CH3 Cl
N+ CH3 C H3 H21C10
Br-
126.8 – 127.3 97.5
