Photodegradation of 2-Substituted Benzimidazole Fungicides by Ultraviolet Radiation
by
Dong Chen
A Thesis Submitted to the Faculty of The College of Science in Partial Fulfillment of the Requirements for the Degree of Master of Science
Florida Atlantic University Boca Raton, Florida August 1994 PHOTODEGRADATION OF 2-SUBSTITUTED BENZIMIDAZOLE FUNGICIDES
BY ULTRAVIOLET RADIATION
BY
DONG CHEN
This thesis was prepared under the direction of the candidate's thesis advisor, Dr.Cyril Parkanyi, Department of Chemistry, and has been approved by the members of the supervisory committee. It was submitted to the faculty of the College of Science and was accepted in partial fulfillment of the requirements for the degree of Master of Science in Chemistry.
SUPERVISORY COMMITTEE
Thesis Advisor
Chairman, Department of Chemistry
D an, College of Sc1ence
I I Date
II Acknowledgments
I wish to express my sincere thanks to my thesis advisor, Dr. Cyril Parkanyi,
Professor and Chairman of the Department of Chemistry, for his guidance, suggestions
and enthusiastic support. His patience and keen interest throughout this study are greatly
appreciated.
I would also like to acknowledge the valuable suggestions of my committe
members Dr. Russell G. Kerr, Assistant Professor, and Dr. Earl W Baker, Professor,
Department of Chemistry.
Special thanks are due to my dear friend and colleague, Ms. Guoping Deng, for
her constant support and help with NMR data. I am most grateful to Dr. J William Louda
for the mass spectra, without his help this work would not have been completed.
My deepest and sincerest thanks go to my parents, Mrs. & Mr. Chen, for their
love, encouragement and support. Also, I thank all my friends who have helped me with this project.
Finally, I thank DuPont (benomyl), Merck Sharp & Dohme (thiabendazole) and
Bayer (fuberidazole) for supplying the samples of fungicides for this research.
Ill Abstract
Author: Dong Chen
Title: Photodegradation of 2-Substituted Benzimidazole Fungicides by
Ultraviolet Radiation
Advisor: Dr. Cyril Parkanyi
Institution: Florida Atlantic University
Degree: Master of Science in Chemjstry
Year: 1994
The photodegradation of benzimidazole-based fungicides: benomyl ( { 1-
[ (butylamine) carbonyl]-1 H-benzimidazol-2-yl} carbamic acid methyl ester), thiabendazole
(2-( 4-thiazolyl)-1 H-benzimidazole) and fuberidazole (2-(2-furyl)-1 H-benzimidazole) by ultraviolet radiation has been studied. Benomyl in an organic solvent (chloroform) undergoes a rapid degradation even without any UV radiation; UV radiation and air agitation will increase the rate of the reaction. The degradation product was found to be methyl 2-benzimidazolecarbamate (MBC) in all cases. The degradation of thiabendazole and fuberidazole in methanol was also studied under different conditions but degradation was observed only under UV radiation and in the presence of oxygen, 7-8
Photodegradation products were found in the mixture after irradiation of thiabendazole but only 2 of them were separated from the rruxture and identified. Benzimidazole-2- carboxamide and dimethyl oxalate were two of the final photodegradation products of thiabendazole in methanol. It is clear that the thiazole ring is the most vulnerable part of the molecule and suffers ring cleavage. No photodegradation products of fuberidazole were identified so far.
IV TO MY DEAR PARENTS Contents
Chapter 1. Introduction
1. 1 Light and Energy ...... 1
1.2 Laws ofPhotochemistry .. . 4
1.3 Ultraviolet Spectroscopy ...... 5
14 The Excited States ...... 7
1.5 Fate ofthe Excited Species ...... 9
1.6 Light Sources in Photochemistry ...... 11
1. 7 Fungicides ...... 13
1.7.1 Fate ofFungicides in Soil ...... 13
1. 7.2 Benzimidazoles as Fungicides ...... 15
Chapter 2. Literature Review
2. 1 Introduction ...... 18
2.2 Photochemistry ofHeterocyclic Fungicides ...... 19
2.2.1 Carboxamides ...... 19
2.2.2 Pyrimidines and Pyridines ...... 20
2.2.3 Thiophanates ...... 21
v 2.2.4 Benzimidazoles ...... 22
Chapter 3. Experimental
3 . I Materials ...... 28
3.2 Instrumentation ...... 28
3.2.1 Photolysis ...... 28
3.2.2 Monitoring of the Photodegradation ...... 36
3. 3 Identification and Isolation ...... 36
3.3 .1 TLC ...... 36
3.3.2 Column Chromatography ...... 36
3.4 Analytical Methods ...... 36
3 .4. 1 Infrared Spectroscopy ...... 36
3.4.2 Nulear Magnetic Resonance Spectroscopy ... . . 36
3.4.3 Mass Spectrometry ...... 36
3. 5 Procedure ...... 3 7
3. 5. 1 Photodegradation ...... 3 7
3. 5. 2 Detection and Isolation ...... 3 7
3. 5. 3 Characterization ...... 37
Chapter 4. Results and Discussion
4.1 Study of the UV Spectra ...... 38
4.2 Photodegradation ...... 43
4.2. 1 Degradation ofBenomyl in Chloroform ...... 43
VI 4.2.2 Degradation ofThiabendazole and Fuberidazole in Methanol ... 43
4.3 Detection, Isolation and Analysis ...... 50
4.3.1 Benomyl ...... 50
4.3 . 2 Thiabendazole ...... 55
4.3.3 Fuberidazole ...... 67
Chapter 5. Conclusions ...... 69
References ...... 71
VII List of Figures
Chapter 1. Introduction
1.1 Electromagnetic Spectrum ...... 2
1.2 Potential Energies of a Molecule ...... 6
1. 3 Scheme of Electronic Levels ...... 8
1.4 The Several Routes to Loss of Electronic Excitation ...... 10
1.5 Structural Formulas ofBenzimidazole-Based Fungicides ...... 16
Chapter 2. Literature Review
2. 1 Structural Formulas of Carboxin and Oxycarboxin ...... 19
2.2 Degradation ofCarboxin and Oxycarboxin ...... 20
2.3 Structural Formulas ofDimethirimol and Pyroxychlor ...... 20
2.4 Photodegradation of Thiophanates ...... 21
2.5 Structural Formulas ofBenzimidazole and its Photoproducts A and B ...... 23
2.6 Degradation ofBenomyl...... 24
2. 7 Degradation of Thiabendazole ...... 24
2.8 Degradation ofFuberidazole ...... 25
2.9 Degradation ofBenzimidazole-2-amine ...... 26
Vlll 2. 10 Degradation of 2-Chlorobenzimidazole ...... 27
Chapter 3. Experimental
3.1 IR Spectrum ofBenomyl .... 29
3.2 IR Spectrum ofThiabendazole ...... 30
3.3 IR Spectrum ofFuberidazole ...... 31
3.4 NMR Spectrum ofBenomyl ...... 32
3.5 NMR Spectrum ofThiabendazole ...... 33
3.6 NMR Spectrum ofFuberidazole ...... 34
3.7 Mass Spectrum ofThiabendazole ...... 35
Chapter 4. Results and Discussion
4.1 UV Spectrum ofBenomyl in Chloroform ...... 39
4.2 UV Spectrum ofThiabendazole in Methanol...... 40
4.3 UV Spectrum ofFuberidazole in Methanol ...... 41
4.4 UV Spectrum ofBenzimidazole in Methanol ...... 42
4.5 UV Spectrum ofReaction 1 ofBenomyl in Chloroform ...... 44
4.6 UV Spectrum ofReaction 2 ofBenomyl in Chloroform ...... 45
4.7 UV Spectrum ofReaction 3 o(Benomyl in Chloroform ...... 46
4.8 UV Spectra of Reaction 4 ofBenomyl in Chloroform ...... 47
4.9 UV Spectra ofReaction of Thiabendazole in Methanol ...... 48
4.10 UV Spectra ofReaction ofFuberidazole in Methanol ...... 49
4.11 IR Spectrum ofBenomyl Degraded Product ...... 52
IX 4.12 NMR Spectrum ofBenomyl Degraded Product ...... 53
4. 13 Degradation Pathway of Benomyl ...... 54
4.14 Thin-Layer Plate Resulting from Two-Dimensional Chromatography of
Thiabendazole Photolysis Reaction Product ...... 55
4. 15 IR Spectrum efFraction 2 ofThiabendazole Degradation ...... 57
4.16 NMR Spectrum efFraction 2 ofThiabendazole Degradation ...... 58
4.17 Mass Spectrum efFraction 2 ofThiabendazo1e Degradation ...... 59
4.18 UV Spectrum efFraction 2 ofThiabendazole Degradation ...... 61
4.19 IR Spectrum ofUnknown of Thiabendazole Degradation ...... 63
4.20 NMR Spectrum ofUnknown ofThiabendazole Degradation ...... 64
4.21 Mass Spectrum of Unknown of Thiabendazole Degradation ...... 65
4.22 Photodegradation Pathway ofThiabendazole ...... 68
X List of Tables
Chapter 1. Introduction
1.1 Typical Bond Dissociation Energies ...... 3
1.2 Properties ofthe n~1t* and 7t~7t* States ...... 9
1.3 Properties ofBenzimidazole-Based Fungicides ...... 17
Chapter 4. Results and Discussion
4.1 "-max Values ofBenzimidazole and Benzimidazole-Based Fungicides ...... 3 8
4.2 Rf Values for Benomyl Products ...... 51
4.3 NMR Data of Benomyl Degradation Product ...... 51
4.4 IR Data ofBenomyl Degradation Product ...... 54
4.5 NMR Data ofFraction 2 of Thiabendazole Degradation ...... 56
4.6 IR Data ofFraction 2 of Thiabendazole Degradation ...... 62
4.7 NMR Data ofUnknown of Thiabendazole Degradation ...... 62
4.8 IR Data ofUnknown of Thiabendazole Degradation ...... 67
XI Chapter 1
Introduction
Photochemistry is concerned with the chemical changes brought about by the absorption of light. It is normally initiated by absorption of light resulting in electronically excited molecules. The excitation of the molecules can take place by direct absorption of radiation or by transfer of energy from molecules called sensitizers which can easily undergo excitation by absorbing light radiation.
1.1 Light and Energy
Light is electromagnetic radiation. Although it may be considered to exist in both particle-like and wave-like forms, while the wave-like properties successfully describe the effects associated with light propagation, reflection, refraction, interference, diffraction and polarization, its energy is measured most commonly in terms of either wavelength (/....) or frequency (v). The common units of measure of /.... are the angstrom (A) and the millimicron or nanometer (nm), whereas v is measured in cycles per second (Hz) or represented as wavenumbers, in a modified form .
On the other hand, when disscussing the photochemical reaction, it is convenient to regard electromagnetic radiation as a particle or packet of energy as described by the quantum theory. A photochemical reaction is produced by the absorption of electromagnetic radiation by a molecule. According to Planck's quantum theory, radiant energy occurs in discrete parcels or quanta. The energy E of this quantum is given by
Planck's equation.
E = hv =he/A...... (1)
where h ---Planck's constant ( 6.65 x 1o-27 erg. sec)
c ---velocity oflight ( 2.998 x1ol0 em/sec)
A --- wavelength of the radiation in em
Electromagnetic radiation extends over a wide range of wavelength and the following
Figure 1. 1 shows the regions of electromagnetic spectrum and the.respective energies.
0.01 keel/ M I I
r ray x roy Microwave Radio I •• I I I X 0 .0 1 1 Unit .a nm 11-m mm em m
Figure 1.1 Electromagnetic Spectrum
Although the regton of electromagnetic radiation open to practical laboratory measurement is very broad, it is important to note that the absorption of light of a wavelength shorter than 750 nm (visible and UV) supplies an amount of energy which is sufficient to effect electronic transitions.
The energy available to bring about direct photochemical transformations amounts to about 143 kcallmole at 200 nm, 95 kcal/mole at 300 nm and 68 kcallmole at 420 nm .
For comparison, the energy required to break the carbon carbon bond in ethane is about
2 88 kcallmole, and a carbon-hydrogen bond in the same molecule requires about 98 kcaVmol. Other typical bond energies for free radical (homolytic) bond leavage are shown in Table 1.1. Bond strengths may vary, depending on the type of molecule, physical state, and reaction mechanism. It is apparent that UV light possesses sufficient energy to cause many kinds of chemical transfonnations.
Table 1.1 Typical Bond Dissociation Energies
Bond E (kcaVmole) A (nm)
HO-H 119 239
CnH'i-H 112 254
CH10-H 104 274
CH1CO-NH2 99 288
C2H'i-H 98 291
CH~CO-OCH~ 97 294
C6Hs-Cl 97 294
(CH3)2N-H 95 300
H-CH20H 94 303
CH1-0H 01 313
C{)H~Br 82 347
CoH~CH2-COOH 68 419
C6HsS-CH3 60 475
CH1-HgCH1 58 491
3 The energy continues to fall off as the wavelength increases. In the comparatively simple types of compounds represented by the large majority of fungicides, light of wavelength greater than about 450 nm (blue-violet region of the visible spectrum) representing energies of less than 65 kcaVmole would not be expected to bring about chemical changes under most circumstances even if the compound were extremely efficient at absorbing energy in this region. This comparatively low energy is generally sufficient only to increase the amplitude of vibration, rotation, or tumbling of the molecules. On the other hand, energies corresponding to wavelengths below 200 nm are too large and will lead to dissociation of the molecules.
Air and quartz are transparent in this region (200 nm-450 nm) and thus quartz optics can be used for the respective measurements.
1.2 Laws of Photochemistry
There are two fundamental laws of photochemistry:
1: First law (Grotthus, 1817, Draper, 1843):
The light absorbed by a molecule is the only light which can produce
photochemical change in the molecule.
2: Second law (Stark-Einstein, 1913):
If a species absorbs radiation, then one particle is excited for each quantum
of radiation absorbed.
According to this law a molecule absorbs one quantum of light and from the resulting excited molecules all the primary processes arise. The primary processes may be defined as a photochemical reaction, or as the processes involved in the formation of an
4 excited state due to direct absorption of a quantum of light. Secondary processes involve reactions of molecular fragments and dissipation of energy as the molecule returns to the ground state.
1.3 Ultraviolet Spectroscopy
The absorption of a photon by a molecule results in a change in its electronic level.
The absorption of energy brings .about the excitation of electrons from orbitals in the ground state to orbitals in the higher energy state. The excitation depends on the amount of energy absorbed and the nature of the molecule.
The ground state of a molecule is its nonnal state and corresponds to the minimum energy. Each molecule has only one ground state.
The absorption of energy by a molecule results in three types of transitions: ( 1) electronic transitions; (2) vibrational transitions; (3) rotational transitions.
Like electronic transitions, the rotations and vibrations of a molecule are quantized and
Eelec > Evib > Erot For each electronic level of a molecule, there are a number of vibrational sub- levels, and for each vibrational level there are a number of rotational sub-levels.
The electronically excited state of a molecule is a state resulting from an electronic transition. Figure 1.2 is a schematic representation of potential energies of a molecule.
5 Excited state \
\ kotational Vtbr:1tional ~levels levels
Ground state
Figure 1.2 Potential Energies of a Molecule
The amount of energy absorbed is quantized and is given by the same relation as
equation (1).
.::\£ = hv =he/A...... (2)
where .::\£ is the energy absorbed during the electronic transition of a molecule from
ground state to an excited state. The amount of energy absorbed depends upon the energy
difference between the two states. Any excess energy absorbed may result in the
dissociation or ionization of the molecule or it may be re-emitted as heat or light. The
release of energy as light results in fluorescence or phosphorescence.
Since absorption of energy is quantized the ultraviolet spectrum of a molecule
would be expected to consist of a single discrete line. Actually the ultraviolet spectrum
appears as a broad band because of the various vibrational and rotational transitions associated with each electronic transition.
An absorption spectrum is characterized by the wavelength of maximum absorption and by the intensity of this absorption, which is generally reported as the molar
6 extinction coefficient e. The absorption wavelength is a measure of the electronic energy level attained by the molecule, whereas the intensity of the absorption band is a measure of the probability of an electronic transition.
The intensity of absorption may be expressed as transmittance T (T = UI 0 ) where I and I0 are the transmitted and incident light intensities, respectively. The fraction of light transmitted through an absorbing system is very frequently found to be represented by the relation called the Beer-Lambert law.
T = UI 0 = 1o-ecb ...... (3) Also : log Iofl = ecb = A A ------absorbance c ------concentration of the solute ( mol/liter) b ------path length through the sample (em )
E ------molar absorptivity It can also be given as A = abc, where c is defined as grams per liter and a is the absorptivity. The intensity of an absorption band in the ultraviolet spectrum is usually expressed as the molar absorptivity at maximum absorption Emax or log Emax· Absorption with E max value greater than 1Q4 is a high intensity absorption whereas Emax lower than 103 corresponds to a low intensity absorption. Transitions of low probability correspond to forbidden transitions.
1.4 The Excited States
oJc oJc (1t~1t ) and (n~1t ) Absorption in the wavelength region of photochemical interest leads to electronic excitation of the absorber. Although it would be a mistake to suppose that the only form
7 of excitation that can lead to a photochemical change is electronic, it is generally true that electronically excited states are involved in photochemical processes.
There are two types of electronic transitions which are most important in organic photochemistry: 1t ~ 1t • transitions which are involved in the excitation of C=C double bonds, and n ~ 1t • transitions, which are typical in the photochemistry of the carbonyl group and other groups with heteroatoms.
Five types of molecular orbitals are of importance in organic photochemistry: the bonding 1t orbitals, the antlbonding 1t • orbitals, the nonbonding n orbitals, the bonding cr orbitals, and the antibonding cr • orbitals. Single bonds between two atoms involve cr orbitals: the a-electrons in these orbitals are strongly bonding and are highly localized.
Double bonds involve cr orbitals and 1t orbitals at the same time. The 7t-electrons are delocalized and are readily excited. Finally, certain heteroatoms such as oxygen or nitrogen contain unshared electrons in n orbitals, which are localized on the heteroatoms.
These nonbonding n orbitals obviously have no corresponding aPtibonding orbital.
a* i 1t • E H n Lt 1t l ;r a ..
Figure 1.3 Scheme of Electronic Levels
8 The transitions may be arranged in the following sequence of increasing energies:
n ~ 1t • < n ~ cr • < 1t ~1t •< cr ~ cr •
Table 1.2 Properties of then ~ 1t" and 1t ~ 1t" States
n~7t • 1t~1t •
Absorption maximum, Em~x 10 -103 103 - 105
Vibrational structure Sharp in nonpolar solvents, Blurred: no solvent
blurred in polar solvents effect
Shift of absorption band in Blue shift Red or no shift
polar solvent
1.5 Fate of the Excited Species
Photochemical process involving the absorption of light can be divided into the act of absorption, which falls within the domain of spectroscopy, and the subsequent fate of the formally electronically excited species.
Figure 1.4 represents, in a simplified form, the vanous paths by which an electronically excited species may lose its energy.
1. Energy transfer, represented by paths (IV) and (V) in the diagram, leads to excited species, which can then participate in any ofthe general processes.
2. Chemical change can come about either as a result of dissociation of the absorbing molecule into reactive fragments (process I), or as a result of direct reaction of the
9 electronically excited spectes (process II). A special case of dissociation ts that of
ionization, shown as path (VITI).
Physical quenching Luminescence AB AB+hv AB• +e Ionization (vii) + M
AB' Intramolecular,.______------.A+ 8 energy transfer Dissociation (radiation less transition)
AE + 8 or ABE Direct reaction AB+CD: AB·~ + E·- or AB·- + E·• BA Intermolecular Charge transfer Isomerization energy transfer
Figure 1.4 The Several Routes to Loss of Electronic Excitation.
(The use of the symbols *, + and ± is only intended to illustrate the presence of electronic
excitation and not necessarily differences in states. One or both of the products in
processes (i)-(iii) may be excited.)
3. Electronically excited species may also undergo spontaneous isomerization, as indicated by path (ill).
10 4. Radiation loss of excitation energy (path VI) g1ve nse to the phenomenon of
luminescence. The terms fluorescence or phosphorescence are used to describe particular
aspects of this general phenomenon (fluorescence from an excited singlet state,
phosphorscence from an excited triplet state).
5. Path (VII) indicated in Figure 1.4 is physical quenching. In this process an atom or
molecule M can relieve AB • of its excess energy. Physical quenching differs only formally
from the intermolecular energy transfer in that M, which must initially take up some
excitation energy, does not make its increased energy felt in terms of its chemical
electronic excitation of AB • . It is frequently converted to translational or vibrational
energy ofM.
1.6 Light Sources in Photochemistry
Photochemical process may lead to a chemical change. The nature of the products,
and the rates of their formation may be determined by standard chemical techniques. We
are here more concerned with those parts of the experimental techniques that involve light.
In the case of photochemical studies a source emits a few intense frequencies in the
desired range and the intensity of the source (number of photons emitted per second)
should be stable with time. Moreover, the source itself should have a long life, be inexpensive, and easy to replace or repair. By far the most important consideration in choosing the source for photolysis is the absorption spectrum of the compound to be photolyzed. Photochemical reaction can take place only if the compound absorbs at the frequencies generated by the source. Another consideration is the rate of photolysis. Since the rate of most photochemical reaction is directly proportional to source intensity, a
11 source intensity must be available to bring about a desirable conversion of reactants to products in a convenient time interval.
Many types of ultraviolet light sources have become commercially available in recent years. These generally may be considered under three categories: (a) incandescent;
(b) arc; (c) fluorescent. Incandescent lamps are familiar to everyone in the form of the household light bulb. They employ a hot tungsten filament in a bulb filled with inert gas.
The arcs are an inefficient sources of ultraviolet radiation, but by the use of appropriate filters, they may be utilized as very weak sources of long-wavelength ultraviolet ("black") light.
The most common arc lamps are those in which an electric discharge is generated in a gaseous element such as neon, xenon, or mercury vapor. Open carbon arcs such as those used in emission spectroscopy have not received extensive application in photochemical experiments.
The mercury-vapor arcs, operating under both high and low pressure, have become the standard UV source for most photochemical research. They usually consist of an evacuated quartz envelope containing a small pool of metallic mercury and appropriate electrodes. They are manufactured in a wide variety of forms including spiral, tubular, and bayonet types. Those lamps are rich sources of ultraviolet light emitted over a broad wavelength range and usually consisting of a series of sharp spectral lines. A common variation of the low-pressure mercury arc is the well-known fluorescent lamp.
12 1. 7 Fungicides
l. 7.1 Fate of Fungicides in Soil
Fungicides control a variety of fungal diseases that would otherwise cause serious losses in agricultural production. Most of them are applied to surfaces to prevent attacks by fungi . The appropriate method of applying a fungicide to a surface depends upon several factors such as: chemical and physical properties of the compounds which restrict the type of formulations available and the type of treatments that are possible; relative effectiveness of the fungicide against the target organism; the nature and ecology of target pathogens; tolerance of the host to the fungicide and the cost of fungicide. Their fate in the soil is one of the most important aspects concerning their widespread use because it determines the amount of fungus control obtained, selectivity, persistence, effect on soil organisms, soil and water contamination and crop rotation. Fungicides must survive in the surface environment to kill the fungus. The term persistence is defined as residence time of a chemical species in a specially defined compartment of the environment.
Studies on persistence of fungicides have been reviewed by Alexander 1 and
Helling et al2 . As compared to insecticides and herbicides, very little work has been done on the persistence of fungicides in soil. Domsch and Brown3 indicated two possibilities of testing fungicide persistence in soil, i.e., determination of remaining activity of the fungicide or determination of the remaining viability of pathogenic fungicide.
Concentration of the fungicide, fungicide structure characteristics, edaphic and climatic conditions, soil moisture content, soil temperature and soil type are the factors controlling persistence. There is a tendency for greater persistence of toxicants in soils containing relatively more organic matter and clay than in light-textured sandy soils.
13 All organic compounds added to the soil must ultimately decompose or be altered
to become a part of the soil complex. The nature and speed of the alteration or loss is
determined by the intricate interaction of the chemical and physical properties of the
fungicides with the chemical, physical, edaphic and biological properties of the soil4
/ The effect of the residue of the fungicides on man and environment seems to be
indirect rather than direct. It has been proven that plants can absorb and translocate
residual fungicides from the contaminated soils. This leads to a link between the residual
fungicides and man's food chain. Hence it is necessary to study the fate of the fungicides in
the soil.
The various processes responsible for the decontamination of the fungicides in the
soil are sorption, diffusion, volatilization, leaching and runoff. Biological processes such as
root uptake and metabolism and chemical process such as oxidation, reduction and
hydrolysis play an important role.
Physical processes like leaching, vaporization, or adsorption in themselves have
little effect on the chemical, although decomposition may occur during these processes.
The three basic processes leading to decomposition of a chemical are chemical reactions,
biochemical or biological reactions, and photochemical reactions. Microbial degradation
accounts for much of the loss of the chemical from soil. Microbial degradation of
fungicides involves detoxification of the compound and its degradation by some soil
microorganisms. Microbial inactivation takes place in the following stages: initial uptake
of toxicant, metabolic breakdown and finally possible utilization of suitable fragments for
energy purposes.
14 Certain microorganisms which are effective in metabolizing foreign substances can be used for soil enhancement. ~egradation by light is likely to be a minor factor ~ n area with frequent rains and little intense sunshine. In sunny arid places it may be a major factor in the loss or modification of fungicides applied to the soil. The rate of breakdown and the potential of the products to contaminate the environment influence the usefulness of the parent compound. Solvent, its concentration, and aeration are important factors affecting rates of photolysis. Rates of photochemical reactions are seldom influenced by temperature. For environmental studies of photolysis, water is the predominant solvent.
Organic solvents are frequently limited by their ability to transmit light or by the fact that they themselves may participate in the chemical reactions. In the presence of high concentrations, many complex polymeric substances may be formed. In dilute aqueous solutions first-order rate laws apply to photodecomposition rates by photolysis. The course and products of reaction are also influenced by the presence or absence of oxygen.
Photooxidation reactions predominate in the presence of oxygen rather than nitrogen 5
I. 7.2 Benzimidazoles as Fungicides
Benzimidazoles are fungitoxic compounds, with a substituted 2-carbon atom of the heterocyclic ring of the benzimidazole molecule. The structural formulas of the benzimidazole molecule and benzimidazole-based fungicides are shown in Figure.l .5.
Two types of fungicidal derivatives of benzimidazole are known. The first type of derivatives includes thiabendazole (TBZ) and fuberidazole. In both these compounds, the heterocyclic rings are joined by a carbon-carbon bond. In the second type, methylbenzimidazol-2-yl carbamic acid (MBC) is the effective moiety. Benomyl (methyl
N-(1-butylcarbamoyl)-2-benzimidazolecarbamate) is the first compound ofthis group. The butylcarbamoyl substituent on one of the ring-nitrogen atoms is very readily hydrolyzed to give MBC and other products. This stable breakdown product is partly or even wholly
15 responsible for fungitoxicity. A transient toxicant, butyl isocyanate (BIC), is also produced
during the conversion ofbenomyl to MBC, but has no practical significance.
0 N H II >-~-C-OM~ H CX N ~~un I N>-0 ~0/ C=O (XN N I fuheridazolc HN-C4 H, thiabendazole hcnomyl
Figure 1.5 Structural Formulas of Benzimidazole-Based Fungicides
In 1960, for the first time, Klopping reported systemic fungicidal activity of various 2-sub~tituted benzimidazoles. This report led to the development and subsequent introduction of thiabendazole in 1964. Other toxicants available are carbendazim, cypendazole and micarbinzid. Fuberidazole is another important fungicide in this series.
They are general inhibitors of DNA synthesis (benomyl and carbendazim) and respiration (thiabendazole and fuberidazole). Benzimidazoles are inactive against phycomycetes, but active toward several ascomycetes and certain basidiomycetes, showing its broad spectrum of activity.
The benzimidazole-based fungicides to be discussed later are benomyl, thiabendazole and fuberidazole. They are white crystalline solids in their pure forms. Their properties are shown in Table 1.3 .
16 Table 1.3 Properties of Benzimidazole-Based Fungicides
Common name M.W m.p. (OC)
Benomyl 290.32 decomposed by heat
Thiabendazole 201 .26 304-305
F uberidazole 184.2 294.4
17 Chapter 2
Literature Review
2.1 Introduction
Systemic fungicides are subject to a number of natural processes which will alter
their chemical structure. Generally we may distinguish between two main processes by
which the structure of systemic fungicides can be altered: firstly, purely chemical break
down processes, and secondly, the metabolic systemic precesses in plants, animals and . . m1cro-orgarusms.
Among chemical, non-enzyrruc reactions, spontaneous hydrolysis and oxidation
may be mentioned, e.g. in the formation of carboxin sulfoxide in soil. Irradiation of
fungicides with sunlight, either in the presence or absence of biological material, may lead
to photochemical reactions.
By contrast, most reactions occurring in the metabolic system of organisms are enzyme-catalyzed. When the substrate specificity of an enzyme is wide enough, it may accept a foreign organic molecule as a substrate. The biochemical conversions which systemic fungicides undergo in this way can be roughly grouped as oxidation, reduction, hydrolysis and conjugate formation.
In view of the toxicological and environmental implications of the use of crop protection agents, it is important to have thorough knowledge of the chemical biochemical conversion of systemic fungicides. Therefore, in most countries these data are
18 required by the registration authorities, and agrochemical manufacturers are requested to submit these.
The following sections deal with the photochemical alteration of most systemic fungicides used in practice.
2.1 Photochemistry of Heterocyclic Fungicides
2.2.1 Carbo:s:amides
0 Me ( Xc-N-Q~ ~S~ II H _ 0 0 °
a b Figure 2.1 Structural Fonnulas of Carboxin and O:s:ycarboxin
Carboxin ( 5, 6-dibydro-2-methyl-1, 4-oxathin-3 -caboxanilide) was discovered and developed by Uniroyal International Division of Uniroyal, Inc., Bethany, CT, under the tradename vitavax. The structural formula of carboxin is shown in Figure 2.1a. It is a colorless solid having two crystal structures. Oxycarboxin (5,6-dihydro-2-methyl-1,4- oxathiin-3-carboxanilide-4,4-dioxide) was introduced in 1966 by Uniroyal Chemical, U.S under the trademark Plantvax. It is produced by the oxidation of carboxin with hydrogen peroxide and it forms a colorless crystalline, nonvolatile solid.
Lyr et al.6 reported photochemical sulfoxide formation occurred in the presence of flavin compounds and by the action of ultraviolet light 7.
19 0 Me CsX]-~-c o" ~o o . carboxin carboxin sulfoxide oxycarboxin
Figure 2.2 Degradation of Carboxin and Oxycarboxin
2.2.2 Pyrimidines and Pyridines
Pyrimidines, developed by ICI, UK, are hydroxyaminopyrimidines with alkyl
substituted in the nucleus. Dimethinnol was introduced in 1968 by the ICI plant protection
division. It is a white crystalline solid with on odor and is stable to heat in alkaline and acid
solution.
CtD N OCH3
Figure 2.3 Stnlctural Formulas of Dimethirimol and Pyroxychlor
Pyroxychlor, based on pyridine, was the first chemical demonstrated to be capable of controlling root-fungal infection. It was discovered by Johnson and Tomita, and developed
20 by Dow Chemical Co., U.S. It has low water solubility (II J.lg/ml), but high solubility in organic solvents. No photochemical alteration has ever been reported.
Ethirimol is broken down in soil by sunlight, the rate of degradation being dependent on the type of the soils. Cleavage of the pyrimidine ring was indicated by
I4co2 evolution from appropriately labeled parent compound, but no other degradation products were identified.
2.2.3 Thiophanates
Buchenauer et al. 9 observed photochemical formation of carbendazim from thiophanate-methyl on the surface of cotton leaves, and Matta and Gentile 10 reported that tissue fragments of different plants accelerated this conversion. Studies with potato tuber cell-free extracts revealed that intermediary o-quinones formed by polyphenoloxidase activity may catalyze this conversion 11 .
s 0 0 0 H II H II .H II H II ~N-C-N-C-OR--~ N-C-N-C-OR ~N-C-N-C-OR CXN-C-N-C-OR H II H II H II H II s 0 0 0 thiophanates ~ 4.4'-o-phenylene-bis-allophanates
R = CH 3 or C 2 H 5 ~ N H ~ >-N-C-OR CXN H alk ylbenzimidazole carbamates
Figure 2.4 Photodegradation of Thiophanates
21 Besides carbendazim, small amounts of dimethyl-4,4'-o-phenylene-bis-allophanate were
formed on leaf-treated plants and evidence was obtained for the photochemical nature of this conversion 12.
2.2.4 Benzimidazoles
In contrast to other heterocyclic systems, the photochemistry of benzimidazole has received only brief attention 13, 14 We have now found that exposure of a 1% ethanolic
solution ofbenzimidazole to UV light (253 .7 nm) for 48 hours resulted in the formation of two major products: A (16% yield) and B (25% yield) which were separated by column chromatography (alumina). The recovery of unchanged benzimidazole (50%) from the mixture accounted for 92% of starting material.
Compound A (m.p 255-257°C) had a molecular formula C14H10N4 and was identified as a dehydrodimer of benzimidazole, 2,4'-bisbenzimidazole. Compound B (m.p
350-352°C) also had a molecular formula C14H10N4 and was similarly confirmed to be another dehydrodimer of benzimidazole, 2,5'-bis-benzimidazole. The formation of compounds A and B may be explained by assumption of the intermediate 2-benzimidazolyl radical generated either directly by the action of UV radiation or produced by rearrangement of a pre-formed 1-benzimidazolyl radical. The existence of a 2- benzimidazolyl radical has already been suggested in both esr 15 and a chemical study 16.
The preferred substrate for this radical in the present case must be the benzimidazole.
Hydrogen abstraction from the solvent, a common photochemical reaction of other heterocyclic systems, is ruled out by the absence of reduced products or benzimidazole-
22 solvent adducts. The absence of 2,2'-bisbenzimidazole, a major product which could be
formed by coupling of two 2-benzimidazolyl radicals, is another noteworthy result.
CX>H OC>-Q cr~H, V-N> NVNH H A B
Figure 2.5 Structunl Formulas of Benzimidazole and Photoproducts A and B
During the past few years, considerable effort has been devoted to the
development of effective and economically useful systemic fungicides. One compound in
particular, benomyl ("Benlate" DuPont) has received considerable attention because of its
broad spectrum fungicidal properties. It has been found that this fungicide is relatively
unstable ~d decomposes to a highly insoluble compound also having fungicidal
properties. Studies of the decomposition product were therefore undertak:en.17, 18, 19,20
It is reported to be unstable in aqueous media and breaks down rapidly to methyl 2- benzimidazolecarbamate (MBC)21 , 22.
Baude et at23, however, demonstrated by using 14C-labeled benomyl that benomyl is rather stable in water" and on plates after application. Chiba and Doombos20 reported a rapid degradation of benomyl in organic solvents and its degradation product was confirmed to be MBC.
23 N H ~ ')-N-C-OMt N H 9 N (Jc >-N-c-o\1~ er I N C=O H I carhcnda1im HN-C4 H9 ocnomyl
Figure 2.6 Degradation of Benomyl
Thiabendazole is an important agricultural fungicide and is used under conditions
where it is likely to suffer photodecomposition. Crank and Mursyidi24 indicated that when
exposed to light, thiabendazole was slowly photolyzed in ethanol to give benzimidazole-2-
carboxamide and ethyl benzimidazole-2-carboxylate. It is clear that the thiazole ring is the
most vulnerable part of the molecule and suffers ring cleavage as shown in Figure 2.7. The
ester is an artifact from the solvent.
~ ;--s ~j. (X>- Figure 2. 7 Degradation of Thiabendazole 24 The use of fuberidazole as a fungicide has increased considerably during the last two decades. However, only limited information is available in the literature regarding decomposition studies of fuberidazole. Mahran and co-workers are the only ones who studied the behavior of fuberidazole under UV radiation. A 1% methanolic solution of fuberidazole in the presence of methylene blue was irradiated in a pyrex vessel (A. > 3 13 run) with a Hg high-pressure lamp (Philips HPK 125) resulting in disappearance of fuberidazole after 18 hours.The degradation products were identified as methyl y (benzimidazole )-y-hydroxybutyric acid and trace of benzimidazole. ~~~ ~~>--~ -CH ,-CH 2-COOH ~0/ - ~N 6H - f u beridazole y-(benzimidazol-2-yl )-y-hydroxybutyric acid + benzimidazole Figure 2.8 Degradation of Fuberidazole Photolysis of various 2-substituted benzimidazoles was also studied by Crank and co-workers24. Photolysis of benzimidazole-2-amine in aqueous hydrochloric acid gave a mixture of products. Two colorless crystalline products were isolated and were identified as (2,5'-bibenzimidazole)-2'-amine and (2,4'-bibenzimidazole)-2'-amine. Figure 2.9 on the next page shows the degradation pathway ofbenzimidazole-2-amine. 25 Ri:i/ R- alkyl -/ ~ 0 II (X~) RC~) + r"'Y NH, H, ~N H ~~ ~NH : ~(tt):H ~~ NH1 • I }-NH: :::::::..... N H CX>-Q N~y .NH NH1 Figure 2.9 Degradation of Benzimidazole-2-Amine The photolysis of 2-chlorobenzimidazole was carried out in ethanol and gave a mixture separated by column chromatography to yield two products: l-(benzimidazole-2' yl)benzimidazole-2(3H)-one and benzimidazole-2(3H)-one. These products are quite different from the products from other benzimidazoles and point to a different process for the photolysis of 2-chlorobenzimidazole. It seems reasonable to suggest that the initial excited species expels a chlorine atom and then reacts with water, present in the solvent, to give benzimidazole-2(3H)-one. The dimeric compound (5) appears to result from a further 26 reaction of the reactive species (4a) or (4b) with (6). It is possible that the reactions are ionic rather than radical. Photosubstitution of hydroxyl for halogen is known for various aromatic compounds such as 2, 4-dichlorophenoxyacetic acid or pentachlorophenol25. ----~ (X)-a +a· - N [CX?·] H rL cx >H ·l (I) (4a) (4b) IH,O 0 H (r)-.ANH (4a\ or (4bl (X">=o - (:(}-oH N N H H ·o~5) ~-/) (6) Figure 2.10 Degradation of 2-Chlorobenzimidazole Benzimidazole-2(3H)-one is itself inert to photochemical change, as proved by its exposure to ultraviolet light for 168 h without any sign of reaction. 27 CHAPTER3 EXPERIMENTAL 3.1 Materials The fungicide benomyl (Benlate) was provided by E. I. DuPont de Nemours & Co., Inc., Wilffiington, DE. Thiabendazole (Mertect) was provided by Merck Sharp & Dohme, Rahway, NY. Fuberidazole (Voronit) was provided by Bayer AG, Leverkusen, Germany. The purity of these compounds was verified by their infrared and nmr spectra. The infrared spectra of these compounds are given in Figures 3. 1 to 3. 3 and the nmr spectra in Figures 3.4 to 3.6. All solvents used were ofspectro-grade quality. 3.2 Instrumentation 3.2.1 Photolysis The irradiation of the fungicide solutions were carried out in a Rayonet photoreactor which was equipped with two types of lamps: ( 1) Mercury vapor lamps emitting far UV light primarily in the 240 to 260 nm range with peak emission at 253 .7 nm; (2) Fluorescent lamps with emission between 320 and 450 nm with peak emission near 360 nm. 28 II 0 II ,.. f'. Cl N 0 0 • II II II Ill II.. • II II 0 Cl II ! II ,II .. II • II 0 0 .. 0.. .. II 0 II II 0 0 -~ II - .. ~c. I.. CQ ~ 0 -~ 0 0 •L s .. Ill II .a• = , E ~ :I = 0"' c: CQ II '- .. •> II u•II = , ox• s 0 Ill 0 Ill II I.. 0 0 f'. Ill = II Ill .. 1'..,1 ::r:: -~ I II .. c. u II 0 ~ II , , u f'. II II == 0 'B II• «S 0 - 0 E 0 -t"i II 'I: , ~ Ill" CJ I.. ~= -::cI z ~ 0 0 ,II 0 ~------L--~------~r------r------r-----~g 0 0 0 0 II Ill • • I-L8C:81i"'+'+'8C:U8 • 29 100 c:,,th1ebend ....__ 8 T r •n • ao8a.8•e 87.88 =C-H (unsaturated rin_gJ w • 0 n c • 1311.1578 151i1.150 8015 . 1501 7 411.-48lil 73.401 C6H4- -4000 31500 3000 eeoo eooo 11500 1000 1500 wevenulllbere Figure 3.2 IR Spectrum of Thiabendazole (KBr pellet) T r •n •Ill C=C-H (unsaturated ring} 1 t w t .... 183-4.1520 88.015 •n c • C6H4- eo t-41&.15ee 7 738 . 783 72 . -40 .4000 31500 3000 aeoo 2000 11500 1000 eoo Wavanulllbara Figure 3.3 IR Spectrum of Fuberidazole (KBr pellet) 0 ...•- I ' ...z- ' a u- I 0 -•0 I 00 ..... ,., -e Q =~ u•- "o = '-0 0 - Q "0 I I - - 0 2 e r:/J ~ J.= :E ~ CJ -~ 0 c. r:/J ~ z.. :; u 0 z z I ~ - uao u ~ I I ~ z-a •-z J. 0(1= -~--~ ~ o' d - ID 32 1--~s=~~--~~~\J + ~~ '+- :::r::z ~z Go~ -4 \1 33 A b b+c+f DMSO-d6 h- J lo.,J." w ~ r ------. ·-- I I I .. 7.0 8 .0 ppm Figure 3.6 NMR Spectrum of Fuberidazole (Assignments are in agreement with a private communication from Bayer AG) M+ == 201 m/e (M-CHN)+ == 174 H N, ijs (M-C2SNH)+ == 130 ,)---'-N) N N a (C6H 11 CNH2)+ == 111 thiabendazole (M-C7H6N2)+ == 83 . , .'- w . , Vt 111 . 7 ~ 291. u 3 12U~ Ia Ia -: c I1J ·a 21alalala-: c . :l .: 69 89 .a 1 ra rarata • / , 1 1 1 t 9 B I[ : ,/ J/ I I ~ r ~ .L Ia~-~-~-~-~~-~-~--~-~-~,-~- ~-,-~-~-~~-~-~ L,.------~~. 3 J 1 12 12 1 2 ~a 1 4 ra 1 s ~ 1 3 a :~ ta ta t-'1ass/Chat"'g e ------~------ Figure 3.7 Mass Spectrum of Thiabendazole 3.2.2 Monitoring of the Photodegradation The photodegradation of the fungicides was monitored using a Varian Cary 3 UV visible spectrophotometer. 3.3 Identification and Isolation 3.3.1 TLC Detection of photoproducts was accomplished by TLC with commercial Whatman silica gel 60A plates and silica gel film F254· The detection of the starting materials and the photoproducts were done by uv-light. 3.3.2 Column Chromatography Isolation of photoproducts was accomplished by chromatography on silica gel (kiesel gel 60, particle size 0.2-0.5 mm, E. Merck, Darmstadt) packed in a column. 3.4 Analytical Methods 3.4.1 Infrared Spectroscopy Mattson Model 4020 Fourier Transform Infrared Spectrometer was used for analysis and identification of the starting materials and the photoproducts in K.Br pellets. 3.4.2 Nuclear Magnetic Resonance Spectroscopy Macintosh IT-Based Real Time NMR Station with a QE 300 General Electric NMR Spectrometer (300 MHz) was used to measure the lH-NMR spectra of the fungicides and their photoproducts. 3.4.3 Mass Spectrometry Hewlett-Packard model 5988A quadropole mass spectrometer was used to measure the mass spectra of thiabendazole and their photoproducts. The spectrometer was calibrated with PFTBA (perfluorotributylarnine), using peaks at m/e 69, 219, 502. 36 Analysis was DIP-El-MS. 70 eV electron impact (EI), ion source 22ooc, probe 20-22ooc at 1OOC/min . 3.5 Procedure 3.5.1 Photodegradation First, the ultraviolet spectra of the solutions of the fungicides were recorded in variety solvents such as methanol, cyclohexane, chloroform and water-methanol (v/v, 1:1 ). The photodegradation of thiabendazole and fuberidazole was studied in methanol and benomyl was studied in chloroform. The photodegradation was carried out as follows: samples of fungicide solutions ( 1. 5 mglml) were exposed to ultraviolet radiation in the photoreactor under continuous aeration at ambient temperature. The experiments were performed in a quartz flask which contained a stopper fitted with a glass tube which the air was bubbled through. The air used for aeration was provided by a laboratory pump and was cleaned by passing through a flask containing deionized water. Reference experiments were performed with nitrogen from tank bubbled through the solution exposed to the UV radiation and blank experiments were carried out by without exposing the solutions to the UV radiation. 3.5.2 Detection and Isolation After the reactions were completed, the photodegraded fungicide solutions were evaporated on a rotary evaporator to almost dryness. The mixture of photoproducts was analyzed by TLC to determine the number of photoproducts formed. Then the solutions were evaporated to dryness under the presence of silica gel on the rotary evaporator. The components were separated by column chromatography. 3.5.3 Characterization The separated photoproducts were characterized by their IR and NMR spectra. 37 CHAPTER4 RESULTS AND DISCUSSION . 4.1 Study of the UV spectra The UV spectrum of benomyl, thiabendazole and fuberidazole are shown in Figures 4. I to 4.3 . Their Amax values and that of benzimidazole are shown in Table 4. 1. The first bands are n~1t * transitions, caused by electronic transition from nitrogen, oxygen or sulfur lone pair non-bonding orbital to an empty ring 7t-orbital. The second bands can be ascribed to a transition from the occupied 7t-orbital of the ring having the highest energy(HOMO) to the empty 7t-orbital oflowest energy(LUMO) 1t~1t* transition. On comparing the uv spectra of benomyl (Figure 4.1), thiabendazole (Figure 4.2) and fuberidazole (Figure 4.3) with benzimidazole (Figure 4.4), we observed that there is a bathochromic shift of the n~1t * band which is due to the electron-releasing substituents present in these compounds. Table 4.1 Amax Values of Benzimidazole and Benzimidazole-Based Fungicides 1st band ( nm.lllo..s.. El 2nd band _f_nml Benzimidazole 274.3 (3.49), 279.0 (3.49) 242.0 Benomyl 293 .3 (4.95_1, 285 .5_(4 .86_1 220.8 Thiabendazole 298.6 (4.39) 235.0 Fuberidazole 319.7 (4.42), 305 .0 (4.49) 248.6 38 1.8888 8.8888 . . • • . }\: • • . . • 8.6888 w I.D 8.4888 B.21188i . . . . • • • • 8.8888 288.88 228.88 248.88 268.88 288.88 388.88 . . ADS . 8.8888 -> 1.8888 "" . 288.88 -> 388.88 Figure 4.1 UV Spectrum of Benomyl in Chloroform (6.2 PPM) ""0 8.8888;------,------~------~------.------.,------. 288.88 248.88 288.88 328.88 ADS 8.8888 -> 8.5888 "" 288.88 -> 328.88 Figure 4.2 UV Spectrum of Thiabendazole in Methanol ( 14 .2 PPM) 8.6888 8.4888 -+>- 8.2888 8.8888 - 288.88 248.88 288.88 328.88 ABS 8.8888 -) . 8.68~ "" : 288.88 -> 338.88 Figure 4.3 UV Spectrum of Fuberidazole in Methanol (17.8 PPM) / _/ ~ N (J Ol 0 ~00 240 mp 280 320 Figure 4.4 UV Spectrum of Benzimidazole in Methanol13 4.2 Photodegradation Reactions were carried out under different conditions. They were monitored by a Varian Cary 3 UV-Vis Spectrophotometer. 4.2.1 Degradation of benomyl in chloroform Four reactions were performed under different conditions: 1. In a Pyrex glass flask without any uv radiation. 2. In a Pyrex glass flask with uv radiation. 3. In a quartz flask with uv radiation. 4. In a quartz flask with uv radiation and air agitation. The uv spectra of reactions of benomyl are shown in Figure 4.5 to Figure 4.9. After 6 hours, reaction 1 was about 38% degraded, reaction 2 was 60% degraded. reaction 3 was 77% degraded and reaction 4 was 90% degraded. These reactions prov~ that benomyl is extremely unstable. It undergoes degradation rapidly even without U\ radiation. The reaction rate is reaction 4> reaction 3> reaction 2> reaction 1. It is no· possible to separate the photoreaction from the dark reaction or even to distinguist between the two reactions. 4.2.2 Degradation of thiabendazole and fuberidazole in methanol Four reactions were carried out under different conditions: 1. Blank reaction: in the absence of ultraviolet radiation 2. In the presence ofultraviolet radiation with air (from air pump) bubbling through the solution in a Pyrex glass flask. 43 8.28881 1-initial· • • 2-after 4 hr • • • ~1 8.1688 I 3-after 6 hr • 2 +>- ~ +>- 8.1288j _____---: ~ ./ ~ . 3~ • • • . \ ~ 8.8888~ • • • • . ' ~ ' \ • • • • ' . ""·"-' • • • ' ' • • . . 8.8888 I 278.88 288.88 298.88 388.tiU ABS . 8.8888 -) 8.2888 NM . 278.EJEt - ) 3EtEt.EtEJ Fieure 4.5 UV Spectra of Reaction I of Benomyl in Chloroform (initial: 1.8 ppm) 1-initial 2-after 1 hr 3-after 2 hr 4-after 3 hr 5-after 4 hr 6-after 5 hr 7-after 6 hr 1 • ~ VI r-- ~---- ~ • • • 8.8481 • • • • • • • • 8.8888 278.88 288.88 l98.88 388.88 ADS . 8.8888 -> 8.2888 NH : 278.88 -> 388.88 Figure 4.6 UV Spectra of Reaction 2 of Benomyl in Chloroform (initial: 2. 1 ppm) 8.28881 1-initial· • • • 2-after 4 hr • • • .-1 8.1688 I 3-after 6 hr • • • • • 8.1288 • 0\""" • 8.8888 • • • 2 • . -3======------8.8488~ · • • • ' • • • ·------• I 8.8888 278.88 288.88 298.88 388.88 ADS . 8.8888 -> 8.2888 tttt . 278.88 -> 388.88 Figure 4.7 UV Spectra ofReacHQn 3 ofBenomyl in Chloroform (initial: 2.0 ppm) 1-lnltial 2-after 1 hr 3-after 2 hr 4-after 3 hr 5-after 4 hr 6-after 5 hr ?-after 6 hr . • • 8.2888~ • • • 8.1688 • .. ~ - -....) 8.1288 ~ ~ 8.8888 ------4 8.8488 . :s~ 6, 7 • 8.8888;------~------~------~------~------~------~ 278.88 288.88 298.88 388.88 ADS 8.8888 -> 8.288~ JiM 278.88 - > 388.88 Fismre 4.8 lJV Snedra nf RP.artion 4 of RPnomvl in Chloroform (initial : 1. 8 oom) 1-initial • 2-after 2 days 3-after 5 days 4-after 6 days 5-after 8 days 6-after 9 days 8.4888 • • • 1 8.3888 +:>. 00 8.2888 8.1888 8.8888 - 228.88 248.88 268.88 288.88 388.88 328.88 ABS 8.8888 -> 8.4888 tttt 228.88 -> 328.88 v: ...... f Q IIV "nPrtn of Reaction of Thiabendazole in Methanol (initial 14 .2 ppm) 1-initial 2-after 2 hr 3-after 4 hr 4-after 6 hr 5-after 18 hr 6-after 24 hr - 8.6888 • • • • 8.4888 • 1.0"""' • • • • ~ ~ 8.2888 ~~· 8.8888 238.88 258.88 278.88 298.88 318.88 338.88 ADS . 8.8888 - > 8.6888 NM . 238 .88 - > 338 .88 Figure 4.10 UV Spectrum of Reaction of Fuberidazole in Methanol (initial: 17 8 ppm) 3. In the presence of ultraviolet radiation with air bubbling through the solution in a quartz flask. 4. In the presence ofultraviolet radiation with nitrogen (from tank) bubbling through the solution in a quartz flask. The uv spectrum of reaction 3 of thiabendazole is shown in Figure 4.9 and of fuberidazole in Figure 4. 10. For both thiabendazole and fuberidazole, reactions l , 2 and 4 showed no changes in the concentration for 9 days in the case of thiabendazole and 24 hours in the case of fuberidazole. However, when they were irradiated with ultraviolet radiation at 253 .7 nm with oxygen bubbling through the solution, appreciable changes in the concentration of both were observed. After 9 continuous days, the degradation of thiabendazole is 80% complete and in 24 hours the reaction of fuberidazole is about 72%. This tells us that both thiabendazole and fuberidazole are fairly stable towards sunlight and undergo decomposition under UV radiation. Oxygen plays an important role in this photoreaction. 4.3 Detection, Isolation and Analysis 4.3.1 Benomyl After 6 hours, reaction mixtures of benomyl were analyzed on commercially prepared silica gel film containing fluorescent indicator. Petroleum ether and acetone (90:10) were used to develop the chromatogram, and ultraviolet light (253.7 nm) was used for detection. Table 4.2 shows Rfvalues of the products. The Rf values show us that the 0.5 spot is obviously benomyl itself and the 0.0 spot is its degradation product. After reaction, the original clear solution became cloudy, 50 and a white precipitate fonned. Then the white precipitate was separated from the solutio n by filtration and analyzed later. Table 4.2 Rf Values for Benomyl Products Reaction Rfl Rt? Reaction 1 00 0.5 Reaction 2 0.0 0.5 Reaction 3 0.0 0.5 Reaction 4 0.0 0.5 Solution after filtered out white precipitate 0.5 Freshly prepared solution 0.5 The white precipitate from reaction 1, 2, 3, 4 was analyzed separately. TheIR and NMR spectra prove that they are the same compound. Figure 4. 11 and Figure 4. 12 are the IR spectrum and NMR spectrum of this compound, respectively. It is a highly insoluble compound. The chemical structure of this compound was determined by its IR and NMR data. The IR data is shown in Table 4.4 and the NMR data in Table 4.3 below. Oxygen does not play any role in the degradation of benomyl. Table 4.3 NMR Data of Bnomyl Degradation Product Proton Chemical shift (ppm) Cr;!Lt- 7.1, 7.4 -OCH1 3.7 51 so~r------~------~ ,..T •n • eo "'1 t: t: Vl • 7~e.7ae ae.o~• N n c • ea.ao~ ~0 =C-H 31111.7011 ~0.708 N-H 117.11!lo a~.eso 80 OIH. 7711 21.833 (=0 117.181 ~000 3800 3000 IIBOO aooo tBOO 1000 BOO Wavanulllbera Figure 4.11 IR Spectrum of Benomyl Degradation Product (KBr pellet) I I I 1I ..;o I lI ;: l I ~ ! o E - <0 c. c. 53 Table 4.4 IR Data of Benomyl Degradation Product Functional group Wavenumber ( cm-11 N-H 3327.4 =C-H 3063 .1 -CH3-, (C=C) 2955-2671 C=O 1709, 1631 C=C 1593, 1483, 1452 C-0-C 1265, 1099 1, 2-substituted 732.9 Since the IR and NMR data for unknown compound matches well with those of MBC, it can be concluded that unknown compound is methyl 2-benzimidazolecarbamate and following decomposition pathway could be suggested for its formation26 ~z'c-NH-C-OCH, ~w 11 0 MBC Figure 4.13 Degradation Pathway ofBenomyl 54 4.3.2 Thiabendazole The photodegradation mixture of thiabendazole after 9 days of exposure under uv with oxygen was analyzed by two-dimensional TLC on 8 in2 silica gel plate (Whatman 80). A small amount of mixture was applied in one comer. The plate was developed in one direction with benzene-dioxane-ammonium hydroxide (10:80: 10, v/v) (l) and, after drying, in the second direction with 1-butanol-water-acetic acid (65:25 :10, v/v) (2). The plate was examined under ultraviolet light for uv absorbing compounds. It showed 7 spots of different intensities. Figure 4.14 ~hows the thin-layer plate chromatogram. 1 ------+) 2 Figure 4.14 Thin-layer Plate Resulting from Two-dimensional Chromatography of Thiabendazole Photolysis Reaction Product Concentration of the reaction mixture under reduced pressure at 40° affords a yellow oily residue. An aqueous solution of the residual oil was rotary evaporated at 40° and the distillate treated with ammonia. Vacuum evaporation of the ammoniacal solution left a white solid. 55 The oily residue after removmg the water was dissolved in the nurumum of methanol, followed by addition of acetone. This produced a yellowish precipitate. The filtrate was then evaporated to dryness in the presence of silica gel. The mixture was added to a silica gel column packed in light petroleum (b.p.40-6QO) and the column was eluted with toluene, methylene chloride, 10% methanol in diethyl ether and 25% methanol in diethyl ether and yielded fractions 1, 2, 3 and 4. They were evaporated to dryness on a rotary evaporator. Each of these fractions and the precipitate were analyzed by IR NMR MS (some fractions) and UV-Vis. Toluene-eluted fraction 1 gave a yellowish crystalline substance. The structure of this compound has not been identified so far. Methylene chloride-eluted fraction 2 was further purified by scraping out the spot from two dimensional developed TLC plate and extracting with methanol. The extract was centrifuged first then separated the solution from the silica gel and evaporated the solvent by air. The colorless crystalline compound was identified as iJenzimidazole-2-carboxamide for the following reasons: (a) The 1H-NMR (Figure 4.16) data of fraction 2 shown in Table 4.5. (b) TheIR (Figure 4.15) data of fraction 2 shown in Table 4.6. Table 4.5 NMR Data of Fraction 2 of Thiabendazole Degradation Proton Chemical shift (ppm) C4!4- 7.7 C6fLt- 8.0 NH (hydrogen bondin_B} 8.2 NH 8.7 56 ao~r-~;K~------.------ T r n• • •1 t t • •• 0.7 •n a V'l Nl-!2_ (bendi!!g) 'isoa.a•s •11.0 -.1 • ••••. 8711 711. ·ua-- I I 77., ..I NH 1 -CONH- ~t• \ C=O lt444.774 74.8DO C=C-H , .... 7411 ...... r ' 4000 saoo aooo eaoo 8000 saoo 1000 aoo H•venulllb•r• Figure 4.15 IR Spectrum of Fraction 2 of Thiabendazole Degradation (KBr pellet) H-bond ~ t t b~N· ·-- H c.VN~c - ~H d He. II ~ 0 DMSO-d6 ~0., b, c, J ____.,.. V'o 00 ----,--~ 8 .0 I --- ppm Figure 4.16 NMR Spectrum of Fraction 2 of Thiabendazole Degradation ~N ~N~C - NH2 H II 0 I, I, VI 1.0 Ill ~oooc. - s 1 u l 1 B c 30000 -: ·' {Q n c 20000-: 69 9J :l ..0 [t lODO:}~u-r~:~LJ~J~~~~~l}dilikl~~J~~&.JM~~~U~U~~Jd~··-L~~-L . 5~ lfala 150 21tH?! 25~ t1a::: S: . Char' [t E ------ Figure 4.17 Mass Spectrum of Fraction 2 of Thiabendazole Degradation ~N ~N~C-NH2 H II 0 m/e 161 (W) . -CO m/e 145 m/e 118 ~+ ~NH m/e 9! 60 n.mmel I l t\ . }%b , I 8 . 6BBEL~l ~ .J. \ , ~- I r · ~ ~ I , ,I \ 1 I_ I _,. 0'1 8.40001 - - / -l I o --u~ Q'1 I U.~UU Uj . ) I ' ' . ' ' ~ l.f7 •.f I \ -- J t 4 " ·"" _.....,..----_,_..... t - - _,_ I - ~-.~-- - ~ I ~ --....,_,__ 0 .0000 I - ZBB.0B 300.00 400.00 Figure 4.18 UV Spectrum _of Fraction 2 of Thiabendazole Degradation (in methanol) Table 4.6 IR Data of Fraction 2 of Thiabendazole Degradation Wavenumber (cm-1) Functional group 3275,3195,3140 NH 1689.7 C=O 1618.4 NH2 (bending) 1535.4, 1491 , 1439 C=C-H (unsaturated ring) 1342.4 -CONH- 738.5 1, 2-Substituted (c) The mass spectrum (Figure 4. 17) of fraction 2 showed: m/e 161 (W), 145 (M-NH2)+, 118 (M-CONH)+, 91 (M-C2H2N20)+ (d) The UV-Vis spectrum (Figure 4.18) of fraction 2 showed a Amax at 347.5 nm. The aqueous solution distillate of the yellowish residual oil was treated with ammonia to give a colorless crystalline compound which proved to be oxamide by its IR NMR and MS spectrum. Figure 4.19, Figure 4.20 and Figure 4.21 are the IR NMR and MS spectrum of this compound. Table 4.7 contains NMR data on this compound and Table 4.8 theIR data on this compound. Table 4. 7 NMR data of unknown of thiabendazole degradation Proton Chemical shift (ppm) NHz 1.3 62 100~------r~ T r 113b.7113 n• m• 1 t t ~10~.281 117 . 778 n• c ~ I I I --et88 . 178 78.22~ \ 3~0.11~11 81.~32 ~ • C-N N-H (primary) 381 . 073 117 . 138 C==O 1111~.1173 ~~~ . ~~~~ aa- ~------r------~------~------,------~--.r------r------r~ -4000 3~00 3000 2~00 2000 1~00 1000 aoo W•v•numb•r• Figure 4.19 IR Spectrum of Unknown of Thiabendazole Degradation (KBr pellet) CONH2 I CONH2 CDCl3 11 20 \ 0\ ~ ,, ----r I 1.0 ppm 2.0 Figure 4.20 NMR Spectrum of Unknown of Thiabendazole Degradation CONH2 I CONH2 r, l . rJE:t-51 a.l 0\ u 1~ 88 Vl 8 ~JC t~ c l - m E . uE+'1 I ' - '"'0 c 4 . OE+41 :l .n a: 2 0C+4J I I I IJ•k, ,,, kl•"' .,. I .JL..U..-1 1 ' 4. I .....&.' ,. I I I A • 1 0 . DE 1-0 __.Lj JlLu l ....---. ' •I.,. I , "' ~~~ -- -T 50 100 150 200 ?S O t 1a. S: :;;; / C h a. r [1 E Figure 4.21 Mass Spectrum of Unknown of Thiabendazole Degradation 0 0 II II 0 0 C-C -N 11 II ...... H2C-CH2 N +4H ftY ·+ m/e 70 m/e 60 0 0 II II H2NC-CNH2 • nv'e 88 < 0 0 II II ~ HC-NH2 -H +C-NH2 mle 45 m/e 44 66 Table 4.8 IR Data of Unknown of Thiabendazole Degradation Wavenumber ( cm-1) Functional group 3396.8, 3198.2 N-H (primary) 1664.7 C=O 1356 C-N 636.5 N-H (out-of -plane bend) From the mass spectrum: m/e 88 (W), 70 (M-NH4)+, 60 (M-N2)+, 45 (M CONH)+, 44 (M-CONH2)+ Since dimethyl oxalate on treatment with ammonia yielded oxamide, it could be concluded that dimethyl oxalate was present originally. The oil remaining after removing the water and then adding acetone produced a yellowish precipitate proved to be the original compound thiabendazole by compareison of its IR spectrum with that ofthiabendazole. Further elution with methanol-diethyl ether ( 1 :9) and methanol-diethyl ether ( 1:4) yielded fractions so far unidentified. According to the present study, a pathway of the thiabendazole photodegradation could be suggested as shown in Figure 4.2227 4.3.3 Fuberidazole The photodegradation mixture of fuberidazole after 24 hours of exposure under uv with oxygen was analyzed on a silica gel film with toluene-ethyl acetate-ethanol (6:3: 1, v/v) as a developing solvent. Five spots appeared on the film . 67 The reaction mixture that acquired a brownish-yellow coloration was evaporated under reduced pressure in the presence of silica gel, then subjected to column chromatography (on silica gel). The column was eluted by toluene, chloroform, ether methanol (5 : 1, v/v), giving fractions 1, 2 and 3 from fuberidazole (so far unidentified). hY '"' [O:NI l I I Ot:-N 0:\:NH I jJ N .!.l._ C-N H2 s LJ" ~\=;J.j H II 0 • H • O:N "A" ([eN N)J. N;J ~ H H + • N "8" LJJs [Qsj ~s~ ,; l11 CH]OH 2 1 102 0 II 1o2 HCOOH HCHO • t-:JJ'~H - S s-7C'OCH [~sj] ?Jl H. • [C.OOH] :, J,o2 0 0 ~ II II COOH -so N~'NH I ~-=rf'~H I· I I COOH ltS C02CH 3 S v),'OCHJ - Or- 0 . "! Clt]OH COOCH3 2 NH] CONH2 I I COOCH3 - 2 CH30H CONH2 Figure 4.22 Photodegradation Pathway of Thiabendazole 27 68 Chapter 5 Conclusions The following conclusions were drawn from the results obtained from the experiments conducted. 1) Benomyl in chloroform undergoes rapid thermal degradation at room temperature in the dark. UV radiation and agitation will increase the rate of degradation. In 6 hours, the degradation in the dark was 38% complete, the reaction in the Pyrex glass flask under UV irradiation was 60% complete, the reaction in the quartz flask under UV irradiation was 77% complete and the reaction in the quartz flask under UV irradiation with air agitation was 90% complete. 2) In all degradation reactions of benomyl, whether with or without uv radiation, the final product is the same-methyl 2-benzirnidazolecarbamate (MBC). 3) Thiabendazole undergoes degradation in the presence of oxygen under UV irradiation when compared to its stability in the presence of nitrogen under UV irradiation and in the absence ofuv radiation. In 9 days, thiabendazole was about 80% degraded. 4) Thiabendazole gives rise to 7-8 photoproducts at the end of the photodegradation process. Benzirnidazole-2-carboxarnide and dimethyl oxalate were separated from the photodegraded mixture and identified. 69 5) Fuberidazole underwent no degradation in the absence of UV irradiation and in the presence of UV irradiation with nitrogen bubbling through the solution. It underwent a rapid degradation in the presence of UV irradiation and oxygen. In 24 hours, fuberidazole in methanol was about 78% degraded. The structures of the degradation products have not been identified so far . 70 References 1. Alexander, M., Soil Biology, Review of Research, UNESCO Publication, New York, NY, 209 (1969). 2. Hilling, C.S., Keaney, P.C., and Alexander, M., Behavior of Pesticides in Soils, Advances in Agronomy, Vol. 23, Brady, N. C., Ed., Academic Press, New York, NY, 147 (1971). 3. Brown, A.W.A. , Fungicides and the Soil Microflore, Ecology of Pesticides, J. Wiley, New York, NY (1978). 4. Sinha, A. P ., Singh, K. and Mukhopadhyay, A. N ., Soil Fungicides, Vol. I I II. 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