## 4.2.6. Effects of herbicides

Author: Nico M. van Straalen

Reviewers: Cornelia Kienle, Henk Schat

Learning objectives

You should be able to

- Explain the different ways in which herbicides are applied in modern agriculture
- Enumerate the eight major modes of action of herbicides
- Provide some examples of side-effects of herbicides

Keywords: Amino acid inhibitor, growth regulator, photosynthesis inhibitor, pre-emergence application, selectivity

Introduction

Herbicides are pesticides (see section on Crop protection products) that aim to kill unwanted weeds in agricultural systems, and weeds growing on infrastructure such as pavement and train tracks. Herbicides are also applied to the crop itself, e.g. as a pre-harvest treatment in crops like potato and oilseed rape, to prevent growth of pathogens on older plants, or to ease mechanical harvest. In a similar fashion, herbicides are used to destroy grass of pastures in preparation of their conversion to cropland. These applications are designated “desiccation”. Finally herbicides are used to kill broad-leaved weeds in pure grass-fields (e.g. golf courts).

Herbicides represent the largest volume of pesticides applied to date (about 60%), partly because mechanical and hand-executed weed control has declined considerably. The tendency to limit soil tillage (as a strategy to maintain a diverse and healthy soil life) has also stimulated the use of chemical herbicides.

Herbicides are obviously designed to kill plants and therefore act upon biochemical targets that are specific to plants. As the crop itself is also a plant, selectivity is a very important issue in herbicide application. This is achieved in several ways.

- Application of herbicides before emergence of the crop (pre-emergence application). This will keep the field free of weeds before germination, while the closed canopy of the crop prevents the later growth of weeds. This strategy is often applied in fast-growing crops that make a high canopy with a lot of shading on ground level, such as maize. Examples of herbicides used in pre-emergence application are glyphosate and metolachlor. Also the selectivity of seedling growth inhibitors such as EPTC is due to the fact that these compounds are applied as part of a soil preparation and act on germinating plants before the crop emerges.
- Broad-leaved plants are more susceptible to herbicides that rely on contact with the leaves, because they intercept more of a herbicide spray than small-leaved plants such as grass. This type of selectivity allows some herbicides to be used in grassland and cereal crops, to control broad-leaved weeds; the herbicide itself is not intercepted by the crop. Examples are the chlorophenoxy-acetic acids such as MCPA and 2,4-D.
- In some cases the crop plant is naturally tolerant to a herbicide due to specific metabolic pathways. The selectivity of ACCase inhibitors such as diclofop-methyl, fenoxaprop-ethyl, and fluazifop-butyl is mostly due to this mechanism. These compounds inhibit acetyl-CoA carboxylases, a group of enzymes essential to fatty acid synthesis. However, in wheat the herbicidal compounds are quickly hydrolysed to non-toxic metabolites, while weeds are not capable of such detoxification. This allows such herbicides to be used in wheat fields. Another type of physiological selectivity is due to differential translocation, that is, some plants quickly transport the herbicide throughout the plant, enabling it to exert toxicity in the leaves, while others keep the substance in the roots and so remain less susceptible.
- Several crops have been genetically modified (gm) to become resistant to herbicides; one of the best-known modifications is the insertion of an altered version of the enzyme EPSP synthase. This enzyme is part of the shikimate pathway and is specifically inhibited by glyphosate (Figure 1). The modified version of the enzyme renders the plant insensitive to glyphosate, allowing herbicide use without damage to the crop. Various plant species have been modified in this way, although their culture is limited to countries that allow gm-crops (USA and many other countries, but not European countries).

Classification by mode of action

The diversity of chemical compounds that have been synthesized to attack specific biochemical targets in plants is enormous. In an attempt to classify herbicides by mode of action a system of 22 different categories is often used (Sherwani et al. 2015). Here we present a simplified classification specifying only eight categories (Plant & Soil Sciences eLibrary 2019, Table 1).

Table 1. Classification of herbicides by mode of action

No. | Class (mode of action) | Examples of chemical groups | Example of active ingredient
1 | Amino acid synthesis inhibitors | Sulfonylureas, imidazolones, triazolopyrimidines, epsp synthase inhibitors | Glyphosate
2 | Seedling growth inhibitors | Carbamothiates, acetamides, dinitroanilines | EPTC
3 | Growth regulators (interfere with plant hormones) | Phenoxy-acetic acids, benzoic acid, carboxylic acids, picolinic acids | 2,4-D
4 | Inhibitors of photosynthesis | Triazines, uracils, phenylureas, benzothiadiazoles, nitriles, pyridazines | Atrazine
5 | Lipid synthesis inhibitors | Aryloxyphenoxypropionates, cyclohexanediones | Sethoxydim
6 | Cell membrane disrupters | Diphenylethers, aryl triazolinones, phenylphthalamides, bipyridilium | Paraquat
7 | Inhibitors of protective pigments | Isoxazolidones, isoxazoles, pyridazinones | Mesotrione
8 | Unknown | Chemical compounds with proven herbicide efficacy but unknown mode of action | Ethofumesate

To illustrate the diversity of herbicidal mode of action, two examples of well-investigated mechanisms are highlighted here.

Plants synthesize aromatic amino acids using the shikimate pathway. Also bacteria and fungi avail of this pathway, but it is not present in animals. They must obtain aromatic amino acids through their diet. The first step in this pathway is the conversion of shikimate-3-phosphate and phosphoenolpyruvate (PEP) to 5-enolpyruvylshikimate-3-phosphate (EPSP), by the enzyme EPSP synthase (Figure 1). EPSP is subsequently dephosphorylated and forms the substrate for the synthesis of aromatic amino acids such as phenylalanine, tyrosine and tryptophan.

Glyphosate bears a structural resemblance to PEP and competes with PEP as a substrate for EPSP synthase. However, in contrast to PEP it binds firmly to the active site of the enzyme and blocks its activity. The ensuing metabolic deficiency quickly leads to loss of growth potential of the plant.

Figure 1. The first step in the shikimate pathway used by plants to synthesize aromatic amino acids. The enzyme EPSP synthase is inhibited by glyphosate due to competitive interaction with PEP. Redrawn by Steven Droge.

Another very well investigated mode of herbicidal action is photosynthesis inhibition by atrazine and other symmetrical triazines. In contrast to glyphosate, atrazine can only act in aboveground plants with active photosynthesis. Sunny weather stimulates the action of such herbicides. The action of atrazine is due to binding to the D1 quinone protein of the electron transport complex of photosystem II sitting in the inner membrane of the chloroplast (see Figure 2). Photosystem II (PSII) is a complex of macromolecules with light harvesting and antenna units, chlorophyll P680, and reaction centers that capture light energy and use it to split water, produce oxygen and transfer electrons to photosystem I, which uses them to eventually produce reduction equivalents. The D1 quinone has a “herbicide binding pocket” and binding of atrazine to this site blocks the function of PSII. A single amino acid in the binding pocket is critical for this; alterations in this amino acid provide a relatively easy possibility for the plant to become resistant to triazines.

Figure 2. Schematic representation of the light-induced electron transport phenomena across the inner membrane of the chloroplast, underlying photosynthesis in Cyanobacteria and plants. Quinone D1 of photosystem II has a binding pocket for triazine herbicides and binding of a herbicide blocks electron transport. Redrawn from Giardi and Pace (2005) by Evelin Karsten-Meessen.

Side-effects

Most herbicides are polar compounds with good water solubility, which is a crucial property for them to be taken up by plants. This implies that herbicides, especially the more persistent ones, tend to leach to groundwater and surface water and are sometimes also found in drinking water resources. Given the large volumes applied in agriculture, concern has arisen that such compounds, despite them being designed to affect only plants, might harm other, so called “non-target” organisms.

In agricultural systems and their immediate surroundings, complete removal of weeds will reduce plant biodiversity, with secondary effects on plant-feeding insects and insectivorous birds. In the short term however herbicides will increase the amount of dead plant remains on the soil, which may benefit invertebrates that are less susceptible to the herbicidal effect, and find shelter in plant litter and feed on dead organic matter. Studies show that there is often a positive effect of herbicides on Collembola, mites and other surface-active arthropods (e.g. Fratello et al. 1985). Other secondary effects may occur when herbicides reach field-bordering ditches, where suppression of macrophytes and algae can affect populations of macro-invertebrates such as gammarids and snails.

Direct toxicity to non-target organisms is expected from broad-spectrum herbicides that kill plants due to a general mechanism of toxicity. This holds for paraquat, a bipyridilium herbicide (cf. Table 1) that acts as a contact agent and rapidly damages plant leaves by redox-cycling; enhanced by sunshine, it generates oxygen radicals that disrupt biological membranes. Paraquat is obviously toxic to all life and represents an acute hazard to humans. Consequently, its use as a herbicide is forbidden in the EU since 2007.

In other cases the situation is more complex. Glyphosate, the herbicide with by far the largest application volume worldwide is suspect of ecological side-effects and has even been labelled “a probable carcinogen” by the IUCR (Tarazona et al., 2017). However, glyphosate is an active ingredient contained in various herbicide formulations, e.g. Roundup Ready, Roundup 360 plus, etc. Evidence indicates that most of the toxicity attributed to glyphosate is actually due to adjuvants in the formulation, specifically polyethoxylated tallowamines (Mesnage et al., 2013).

Another case of an unexpected side-effect from a herbicide is due to atrazine. In 2002 a group of American ecologists (Hayes et al., 2002) reported that the incidence of developmental abnormalities in wild frogs was correlated with the volume of atrazine sold in the area where frogs were monitored, across a large number of sites in the U.S. Male Rana pipiens exposed to atrazine in concentrations higher than 0.1 µg/L during their larval stages showed an increased rate of feminization, i.e. the development of oocytes in the testis. This would be due to induction of aromatase, a cytochrome P450 activity responsible for the conversion of testosterone to estradiol.

Finally the development of resistance may also be considered an undesirable side-effect. There are currently (2018) 499 unique cases (255 species of plant, combined with 167 active ingredients) of herbicide resistance, indicating the agronomical seriousness of this issue. A full discussion of this topic falls, however, beyond the scope of this module.

Conclusions

Herbicides are currently an indispensable, high-volume component of modern agriculture. They represent a very large number of chemical groups and different modes of action, often plant-specific. While some of the older herbicides (paraquat, atrazine, glyphosate) have raised concern regarding their adverse effects on non-plant targets, the development of new chemicals and the discovery of new biochemical targets in plant-specific metabolic pathways remains an active field of research.

References

Fratello, B. et al. (1985) Effects of atrazine on soil microarthropods in experimental maize fields. Pedobiologia 28: 161-168.

Giardi, M.T., Pace, E. (2005) Photosynthetic proteins for technological applications. Trends in Biotechnology 23, 257-263.

Hayes, T., Haston, K., Tsui, M., Hoang, A., Haeffele, C., Vonk, A. (2002). Feminization of male frogs in the wild. Nature 419, 895-896.

Mesnage, R., Bernay, B., Séralini, G.-E. (2013). Ethoxylated adjuvants of glyphosate-based herbicides are active principles of human cell toxicity. Toxicology 313, 122-128.

Plant & Soil Sciences eLibrary (2019), https://passel.unl.edu.

Sherwani, S.I., Arif, I.A., Khan, H.A. (2015). Modes of action of different classes of herbicides. In: Price, J., Kelton, E., Suranaite, L. (Eds.). Herbicides. Physiological Action and Safety. Chapter 8, IntechOpen.

Tarazona, J.V., Court-Marques, D., Tiramani, M., Reich, H., Pfeil, R., Istace, F., Crivellente, F. (2017). Glyphosate toxicity and carcinogenicity: a review of the scientific basis of the European Union Assessment and its differences with IARC. Archives of Toxicology 91, 2723-2743.