2,4-Dichlorophenoloxyacetic Acid

By Jeanette Jacobs

2,4-dichlorophenoxyacetic acid (2,4-D)is a highly selective broadleaf herbicide.⁹ Herbicides are a group of compounds that can control unwanted plants. A selective herbicide is one that controls weeds in a crop without damaging that crop. In 1945, 2,4-D was introduced as one of the first selective herbicides. At that time many cash crops were grass-like plants. This herbicide was unique because it effectively killed broadleaf plants typical of many "weeds" but not grass-like plants.⁹This was a significant breakthrough.

2,4-D, a member of the phenoxy family of herbicides, rapidly became the most widely used herbicide in the world.After 50 years of use, 2,4-D is still the third most widely used herbicide in the United States and Canada, and the most widely used worldwide.¹⁵ Its major uses in agriculture are on wheat and small grains, sorghum, corn, rice, sugar cane, low-till soybeans, rangeland, and pasture. It is also used on rights-of-way, roadsides, non-crop areas, forestry, lawn and turf care, and on aquatic weeds.⁹

The most common use for 2,4-D, is post-emergent weed control in agricultural crops.2,4-D comes in the formulation of granular, amine and ester liquids and aerosol spray (foam).2,4-D has complex mechanisms of action against weeds, resembling those of auxins (growth hormones).Once absorbed 2,4-D is translocated within the plant and accumulates at the growing points of roots and shoots where it inhibits growth.⁸

2,4-D/a-ketoglutarate (a-KG) dioxygenase (TfdA) is an Fe(II) and a-ketoglutarate-dependent enzyme that catalyzes the first step in degradation of the herbicide 2,4-D.This enzyme couples the oxidative decarboxylation of a-KG to the hydroxylation of a side chain carbon atom.The resultant hemiacetal spontaneously decomposes to form 2,4-dichlorophenol (2,4-DCP), succinate, glyoxalate and carbon dioxide.⁴

Figure 1: 2,4-D reaction with a-ketoglutarate and O₂ , catalyzed by the enzyme TfdA and the cofactor Fe²⁺which produces 2,4-DCP, glyoxylate, succinate and carbon dioxide.

The enzyme TfdA possesses multiple essential histidine residues, whereas catalytically essential cysteine and lysine groups do not appear to be present.⁴

Figure 2.Model for the active site of TfdA.

The three residues proposed to ligate the ferrous ion of TfdA are shown along with a possible coordination mode for the substrates, a-KG and 2,4-D.Histidine implicated in catalysis and/or binding of 2,4-D, is also illustrated.

Mechanistically, TfdA resembles numerous other a-KG-dependent dioxygenases from plants, animals, fungi, and bacteria that catalyze similar hydroxylation reactions at unactivated carbon centers.The genes encoding the enzymes involved in these processes in Alcaligenes eutrophus JMP 134 have been localized to the pJP4 plamid, cloned and sequenced.⁴

This plasmid was transformed into Escherichiacoli JM109 and the recombinant cells were shown to synthesize high levels of a peptide with relative molecular mass 32000 g/mol.Despite the abundance of TfdA, the 2,4-D-degrading activity in cell extracts was very low compared to the rate of degradation in whole cells of A. eutrophus JMP134.The presence or absence of reducing agents had no effect on activity.The presence of a protease inhibitor during early stages of TfdA purification enhances the stability of the enzyme by preventing conversion of the subunit to an inactive TfdA fragment of apparent molecular mass 27000 g/mol.Conversions are found to occur after cell disruption rather than during the cell cultivation period, and shows homodimeric structure.The enzyme exhibits maximum activity at pH 6.5-7: however, it is stable over a pH range of 6.5-11.³

Ferrous ion is absolutely required for activity of TfdA and cannot be replaced by other divalent cations tested such as, Co(II), Cu(II), Li(II), Mg(II), Mn(II), Ni(II), or Zn(II).Ascorbic acid stimulates dioxygenase activity and reduces the rate of enzyme inactivation by a metal ion-mediate processⁿ.Ferrous ion alone is unable to sustain enzyme catalysis over long time periods, which was shown by a time-dependent decreases in enzyme activity.The enzyme inactivation results from a metal ion-mediated event by the retention of activity when the enzyme was stored in the absence of metal ions and in the presence of EDTA, which exhibits the greatest affinity and highest catalytic efficiency for 2,4-D.³

Nonhalogenated phenoxyacetate possesses a larger K_m value than the halogenated substrates.Similarly, the K_m value for 2-phenoxypropionate is substantially larger that that of 2-(2,4-dichlorophenoxy)propionate.The additional methyl group in the side chain of these two compounds greatly decreases the k_cat values for hydroxylation, due to the change from a secondary to a tertiary carbon atom. Although a-KG is the preferred cosubstrate for the enzyme, TfdA can use a range of other a-ketoacids with lower efficiencies.³Addition of an extra methylene groups between the a-ketoacid group and the free carboxyl group, as in a-ketoadipate can lead to small changes in the kinetic constants.In contrast, removal of one of the methylene groups, as in oxalacetate, led to an ineffective substrate.In the absence of 2,4-D, there was no decomposition of a-KG.³

Chemical modification studies were used to provide evidence consistent with the absence of essential thiol or arginine residues and the presence of multiple essential histidine residues in the enzyme.Studies were used on iodoacetamide, N-ethylmaleimide, and butanedione which failed to affect TfdA activity, but the addition of diethylpyrocarbonate (DEP), a histidine-selective reagent, led to rapid pseudo-first-order loss of activity.The ability of 2,4-D, a-KG, Fe(II) plus ascorbate and combinations of these substances to protect the enzyme against DEP inactivation was examined.What was found was that none of the individual compounds are able to protect the enzyme against inactivation by DEP, but the combinations of 2,4-D plus Fe(II) or a-KG was very effective in protecting the enzyme from inactivation by DEP.This indicates that there are requirements for positive charges at the binding sites of 2,4-D and a-KG, which are essential histidine residues that are present at the binding sites for each of these substrates.One or more additional histidine residues may be buried in the protein at the Fe(II) binding site.Binding of 2,4-D and a-KG protects the histidine residues at the substrate binding sites and protects the Fe(II) ligands by steric constraints.³

Figure 3.Model of the TfdA active site.

Pesticides and herbicides used in agriculture are the classic example of anthropogenic chemicals that enter the Earth’s environment in large amounts via non-point sources.These compounds can adversely affect non-target organisms and may be detrimental to human health if people are exposed by direct contact or to residues of the molecules in soil, water or agricultural products.⁹

Occupational exposure to 2,4-D has produced serious eye and skin irritation.Other symptoms of 2,4-D poisoning include nausea, weakness and fatigue and in some cases neurotoxic effects including inflammation of nerve endings.Some medical reports from practitioners who have treated victims of acute exposure to 2,4-D mention severe and sometimes long lasting or even permanent symptoms.This include as well as those listed above, diarrhea, temporary loss of vision, respiratory tract irritation, confusion, numbness and tingling, bleeding and chemical hypersensitivity.⁶

2,4-D enters the body through inhalation and the skin during occupational exposure. Its mechanism of action is related to uncoupled oxidative phosphorylation and decreased oxygen consumption in tissues, as well as to disturbances in carbohydrate and other metabolic processes.⁹

Symptoms vary with the different commercial products because of the specific amounts and types of additives such as surfactants and solvents. Only poor occupational practices make possible massive dermal and inhalation overexposure with signs and symptoms of acute or chronic intoxication.⁹

2,4-D was a major component (about 50%) of the product Agent Orange used extensively throughout Vietnam.However most of the problems associated with the use of Agent Orange were associated with a contaminant (dioxin) in the 2,4,5-T component of the defoliant.The association of 2,4-D with Agent Orange has prompted a vast amount of study on the herbicide.2,4-D is a Restricted Use Pesticide (RUP) in the United States.Restricted Use Pesticides may be purchased and used only by certified applicators.⁷

In general the molecular mechanisms of pesticide action are poorly understood.However, the lipophilicity of most of them makes lipid-rich membranes a possible target of their interaction with living organisms.The fluidity of membranes is considered one of the most sensitive parameters to their exposure to pesticides.An unequivocal relation between changes in membrane fluidity and the pesticide toxicity has not been established so far.However, some effects directly related to toxicity could primarily be due to change in membrane fluidity, e.g., permeability alteration for electrolytes and nonelectrolytes inhibition of acetylcholinesterase or changes in lipid composition.In spite of the implications that at least part of the toxic effects of pesticides could be due to the perturbation of the lipid phase of the membranes, very little work has been done on the possible changes in lipid fluidity due to their interactions.⁷

The negative effects of herbicides/pesticides on the environment caused by residues remaining in the soil can be eliminateded by applying a short decontamination step after harvest of the crops that could diminish resulting leaching into the groundwater.Microorganisms with specific genetic information for the degradation of the supplied herbicides can be inoculated in bulk soil or in the rhizophere of resistant plants with dense roots to increase the degradation rate of the herbicide.⁹

Bioremediation of soils containing resistant chemicals by inoculation with specialized strains has been shown to fail quite often due to several reasons.A major reason is often the poor survival of the inoculated strain due to competition with the indigenous well-established populations.To over come this problem, indigenous bacteria isolated from the same soil could be used either as such or after being equipped in the laboratory with the necessary degradative genes.Another alternative is to stimulate in situ dissemination of the degradative genes present in the applied strain to other soil bacteria that could result in several genera with the capability of degrading the novel herbicide.⁸

The genes coding the 2,4-D degradation are often located on plasmids, which can be transferred between wide ranges of bacteria.Recently bioagumentation of a soil by distribution of a 2,4-D degradative plasmid was shown to be feasible in some soils but not in all.⁸

Despite the abundance of information on the biochemistry and genetics related to 2,4-D biodegradation, information on the feasibility of continuously running systems for the treatment of concentrated liquid wastes is limited.A concentrated liquid waste stream containing 10 mg/l L of 2,4-D was tested and treatment of municipal wasterwater by an activated sludge unit was used, and the results were about 25^o_o removal.Further testing on other wastewater by other activated slude systems was found to have ranges of 16 ^o_o to 50^o_o removal of 2,4-D⁵.

2,4-D can enter the environment through discharges and spills arising from a variety of different manufacturers, transporters and through direct application as a weed control agent.It is removed from the environment by biodegradation through several possible pathways with the formation of 2,4-dichlorophenol as an intermediate.2,4-D is removed from the atmosphere by photo-oxidation and rainfall with a half-life of less than 1 day.The half-life of 2,4-D in soil is reported to range from 4-7 days in most soil types up to 6 weeks in acidic soils.2,4-D is rapidly biodegraded in water although some may be degraded by photolysis near the surface.Half-lives in water range from 1 to several weeks under aerobic conditions and can exceed 120 days under anaerobic condition.2,4-D is not expected to accumulate in bottom sediments and mud.Except from some algae it does not bioaccumulate in aquatic or terrestrial organisms because of its rapid degradation.²

Conventional methods employed to remove small concentrations of organic pollutants in ground water or industrial discharge, such as air stripping or adsorption by activated carbon, are not good alternatives for contaminants having low volatility or poor adsorption properties. Additionally, these methods only transfer the pollutant from one phase to the other leaving the problem only partially solved. On the contrary, effective oxidative treatments lead to the complete mineralization of a great variety of organic substances. Some of these detoxification methods employ strong oxidants such as hydrogen peroxide or ozone combined with one activation step initiated by UV radiation.¹

Several studies in the past have proposed different reaction mechanism for the photolysis of hydrogen peroxide. It is widely accepted that the main interactions between hydrogen peroxide with UV radiation and free radicals are well represented by reactions in Table 1, (1)-(6).Reaction (7) and (8) in Table 1correspond to the decomposition of any of organic compounds existing in the system by reaction with the generated free radical.¹

Table 1

Reaction mechanism

Initiation:H₂O₂¾^jP ®2OH^*(1)

Propagation:H₂O₂ + OH^*¾^K₂®HO₂^* + H₂O(2)

H₂O₂ + HO₂^*¾^K₃® HO₂^* + H₂O + O₂(3)

Termination:2OH^*¾^K₄® H₂O₂(4)

2OH^*¾^K₅®H₂O₂ + O₂(5)

OH^*+HO₂^*¾^K₆®H₂O + O₂(6)

Decomposition:RH + OH^*¾^K₇® products(7)

RH + OH₂* ¾^K₈®products(8)

^*In this table, RH represents 2,4-D (2,4-dichlorophenoxyacetic acid); DCP, (2,4-dichlorophenol); and

CHQ (chlorohydroquinone)

Figure 3 shows the main reaction paths proposed for the 2,4-D photodegradation, where the principal reaction intermediates are chlorohydroquinone.2.4-dichlorophenol and humic acids (this last poorly defined compound is usually natural components of river waters and hence considered non-toxic).¹

Figure 3. Main reaction paths for the 2,4-dichlorophenoxyacetic acid photodegradation. Keys: D (2,4-dichlorophenoxyacetic acid); DCP (2,4-dichlorophenol); CHQ (chlorohydroquinone)

Atmospheric contamination by 2,4-D may occur as a result of ability to vaporize and drift from application by spraying. Residues in the atmosphere are predominantly in the form of isopropyl and butyl esters. In large-scale studies in areas of intense 2,4-D use in Canada, about 40% of all air samples were found to contain between 0.01 and 0.1 µg of 2,4-D per m³. In a general program of air quality monitoring undertaken in citrus-growing regions in the USA, only 1 out of 880 samples analyzed was found to contain 2,4-D, at a level of 4 µg/m³.No quality standards exist for the amount of 2,4-D allowed in the air of homes.⁹

.2,4-D is an herbicide that has been heavily used in agriculture all over the world for some fifty years or more.There continues to be high levels of concern about long-term adverse effects of 2,4-D on human health and water pollution.Research chemists and biologists are constantly trying to reduce the environmental impact of 2,4-D and other herbicides. One factor is the amount of chemical required in an application. Other environmental characteristics that are considered include environmental persistence (measured in half-life degradability), soil mobility, volatility, and bioaccumulation. New methods of plant control include biotechnology of the TfdA enzyme with kinetic characteristics that will turn on and off the degradation of 2,4-D.This mechanism will allow less herbicide to be applied for weed control because 2,4-D will not degraded until needed.Other new methods are bioengineering strains of crop seeds that are more resistant to the herbicide used to control weeds.Increased food production, along with concern for the environment, will continue to be important issues.

References

1.Alfano, M., Brandi, R., and Cassano, A. (2001)Degradation Kinetics of 2,4-D in water employing hydrogen peroxide and UV radiation.Chemical Engineering Journal. 82, 209-218.

2.Balagué, C., Stürtz, N., Duffard, R., and Evangelista de Duffard, A. (2000)Effect of 2,4-dichlorophenoxyacetic acid herbicide on Escherichia coli growth, chemical composition, and cellular envelope.Environmental Toxicology 16, 43-53.

3.Hausinger, R and Fukumori, F. (1995)Characterization of the first enzyme in 2,4-dichlorophenoxyacetic acid metabolism.Environmental health Perspectives 103, 37-39

4.Hogan, D., Smith, S., Saari, E., McCracken, J., and Hausinger, R. (2000)Site-directed mutagenesis of 2,4-dichlorophenoxyacetic acid/a-ketoglutarate dioxygenase.The Journal of Biological Chemistry. 275:17, 12400-12409

5.Mangat, S., and Elefsiniotis, P. (1998)Biodegradation of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) in sequencing batch reactors. Water Reserve. 33:3, 861-867.

6.Prescott, A. G., and John, P. (1996)Annual Review Plant Physiology, Plant Molecular Biology 47, 245-271

7.Suwalsky, M., Benites, M., Villena, F., Aguilar, F., and Sotomayoer, C.(1996) Interaction of 2,4-dichlorophenoxyacetic acid (2,4-D) with cell and model membranes. Biochimica et Biophysica Acta 1285, 267-276

8.Top E., Maila, M., Clerinx, M., Goris, J., Vos, P., and Verstraete, W.(1998)Methane oxidation as a method to evaluate the removal of 2,4-dichlorophenoxyacetic acid (2,4-D) from soil by plasmid-mediated bioaugmentation. FEMS Microbiology Ecology. 28, 203-213.

9.Water, Sanitation and Health. (1998)2,4-dichlorophenoxyacetic acid (2,4-D) Guidelines for drinking-water quality, 2^nd ed.. World Health Organization. 2, 191-199

Copyright © 2001 Jeanette Jacobs and Koni Stone

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