BIODEGRADATION KINETICS OF THE HERBICIDE ROUNDUP BY SOME SOIL MICROORGANISMS

ABSTRACT

Bacterial biodegradation of xenobiotics has been seen as one of the biological means of bioremediation of polluted site. This study was designed to isolate bacteria from soil and to evaluate their ability to biodegrade glyphosate which is a water soluble, non-selective herbicide used to kill weeds, especially annual broadleaf weeds and grasses known to compete with commercial crops grown around the globe. The bacteria that were able to grow in the presence of glyphosate were isolated using culture techniques and were identified as Bacillus sp, and Pseudomonas sp.  All the isolates recorded the highest growth rate in the presence of glyphosate at the concentration of 7.2 mg/ml and least growth rate at concentration of 200 mg/ml.  The growth rate decreased with increase in glyphosate  concentration.  The  Monod  constants,  half  saturation  constant  (ks)  andmaximum growth rate (µmax  for Bacillus sp were determined as 7.15 mg/ml and 0.59 h¯1, that of Pseudomonas sp, 6.15 mg/ml and 0.62 hˉ1 respectively. The Monod constants for the consortium, half saturation constant (ks) and maximum specific growth rate (µmax) of Bacillus and Pseudomonas spp were 3.65 mg/ml and 0.65 hˉ1  respectively. This study demonstrates that the organisms were more effective in degrading glyphosate when used as consortium than when they are used separately.

CHAPTER ONE

INTRODUCTION

The  need  to  feed  the  world’s  increasing  population  has  prompted  the  use  of agrochemicals to increase food production and ensure the continuation of the human race. Such agrochemicals include pesticides like 2, 4-diphenoxyacetic acid, (2, 4-D), several formulations of inorganic fertilizer and the subject of this study Roundup. The increased use of pesticides in agricultural soils causes the contamination of the soil with toxic chemicals. When pesticides are applied, the possibilities exist that these pesticides may exert certain effects on non-target organisms, including soil microorganisms (Simon- Sylvestre and Fournier, 1979; Wardle and Parkinson, 1990). The microbial biomass plays an important role in the soil ecosystem where they play a crucial role in nutrient cycling and decomposition (De-Lorenzo et al., 2001). During the past  four decades, a large number of herbicides have been introduced as pre and post-emergent weed killers in many countries of the world. In Nigeria, herbicides have since effectively been used to control  weeds  in  agricultural  systems  (Adenikinju  and  Folarin,  1976).  As  farmers continue to realize the usefulness of herbicides, larger quantities are applied to the soil. However, the fate of these compounds in the soils is becoming increasingly important since they could be leached; in which case groundwater is contaminated or becomes immobile, and may persists on the top soil (Ayansinaet al., 2003). These herbicides could then accumulate to toxic levels in the soil and become harmful to microorganisms, plant, wild life and man (Amakiri, 1982).

Contamination of soil from pesticide mixing, loading, storage and rinsing at agricultural chemical dealership is a concern due to potential contamination of surface water and groundwater (Moormannet al., 1998). There is an increasing concern that herbicides not only affect the target organisms (weeds) but also the microbial communities present in soils,  and  these  non-target  effects  may  reduce  the  performance  of  important  soil functions. These important soil functions include organic matter degradation, nitrogen cycle and methane oxidation (Hutsch, 2001). Roundup is the clear herbicide of choice for most illiterate farmers; it is used either alone or in combination with other herbicide

preparations like 2, 4-Diphenoxyacetic acid apparently to achieve additive or synergistic action. It is mostly used in the rice farm to control post emergence weed. These farmers indulge  in  the  use  of  Roundup  and  other  herbicides  to  clear  their  farms  prior  to cultivation without cognizance to the obvious ecotoxicological impacts of such practices. The extensive use of Roundup and other herbicides by these farmers is attributable to the aggressive marketing strategies of the representative of the manufacturers in Nigeria who are  able  to  demonstrate to  these  farmers the  wonders their  products could  achieve, promising them less toiling on their farms with much better results. This is preached without  commensurate  caveat  on  the  possible  toxicity  of  these  chemicals,  and highlighting the possible danger of these chemicals to man and his environment

1.1Pesticide toxicology

Pesticide is the umbrella term for chemicals or biological used to control pests. The Environmental Protection Agency (EPA) defines a pesticide as any substance or mixture of substances/chemicals intended to prevent, destroy, repel or mitigate any pest (US- EPA, 2006).  A pesticide need not always kill a pestː it could sterilize, or repel pests. Pests can be insects, mice and other animals, unwanted plants (weeds), fungi or microorganisms like bacteria and viruses.  Pesticides can be classified in various ways such as, by their target, chemical nature, physical state and mode of action (Ware, 2000). Classification based on the target is perhaps the most widely known as the following examples indicate; Pesticides used to manage insects are called insecticides; and those used to manage rodents are called rodenticide; those used to manage fungi are called fungicides (Ware and Whitacre, 2004). Pesticides also include plant growth regulators, defoliants, or desiccants otherwise known as herbicide, the presence of a xenobiotic in the environment always represents a risk for living organisms. However, to talk about impregnation there is a need to detect the toxin in the organism, and the concept of intoxication is related to specific organ alterations and clinical symptoms. Moreover, the relationship between the toxic levels within the organism and the toxic response is rather complex and has a difficult forecast since it depends on several factors, namely toxicokinetic  and  genetic  factors.  One  of  the  methods  to  quantify  the  exposure  to

xenobiotics and its potential impact on living organisms, including the human being, is the monitoring by the use of biomarkers.

1.2   Biomarkers in Ecotoxicology

One of the greatest challenges to humanity today is the endangerment of human health due to indiscriminate use of pesticides. To estimate the biological danger thereof, knowledge of their harmful effects is necessary. In revealing the risks of such substances, every living being and life function can be considered a potential biomarker or bioindicator. Biomarkers are ideal complement to the traditional analytical techniques employed in evaluating toxicity of pollutants or chemicals in the environment. Microorganisms can be used as indicator organisms (or biosensors) for toxicity tests or in risk assessment. The use of microorganisms present in a polluted environment is an approach that provides a link between exposure and effect because chemicals are known to elicit measurable and characteristic biological responses in exposed (microbial) cells. Those tests performed with bacteria are considered to be the most reproducible, sensitive, simple, economical and rapid (Matthews, 1980).   Risk assessment has relied on models that use toxicity data and physical properties of chemicals, and this approach has been effective at the ecosystem level.  The term “biological markers” (or biomarkers) can be taken to mean cellular, biochemical or molecular alterations which are measurable in biological media such as the human tissue, cells or fluids as a result of exposure to environmental chemicals.  Three  types  of  events  involved  are  exposure,  effect,  and susceptibility. In a broad sense, biological markers are measurements in any biological specimens (such as the blood plasma, bacterial cells) that will elucidate the relationship between exposure and effect such that adverse effects could be prevented (NRC, 1992).

A crucial aspect of Ecotoxicology is the measurement of the effects of toxic substances on organisms in ecosystems and on ecosystems as a whole. This has traditionally been done by determining levels (or bioaccumulation) of toxic substances in organisms and relating these levels to detrimental effects on the organisms (biomarkers). Biomarkers can be used to

Identify  causal  associations  and  to   make   better  quantitative  estimates  of  those associations at relevant levels of exposure. They may also make it possible to identify

susceptible groups or individuals who are at risk of exposure to certain types of environmental and  occupational agents.  A better  approach is  the  use  of biomarkers consisting of observations and measurements of alterations in biological components, structures, processes, or behaviours attributable to exposure to xenobiotic substances. Animals, microorganisms or plants can be used as biomarkers to evaluate the effect of chemical hazards to humans. Biomarkers, or biological markers, can also be chemicals or metabolites that can be measured in body fluid, such as urine, blood, saliva, and other body fluids. Metabolites are chemicals that were transformed by the body from original chemical or chemical constituents of the pesticide. The biological events detected can represent  variation  in  the  number,  structure, or  function of cellular  or  biochemical components. Recent advances in molecular and cellular biology allow for measurement of biologic events or substances that may provide markers of exposure, effect, or susceptibility in humans. Certain tests, such as DNA adduct formation, are used for measuring biologically effective dose, whereas others are considered to measure early effects, such as chromosomal aberrations. Biomarkers are predictive assays rather than diagnostic.

1.3      Roundup

Roundup is the brand name of a systemic, broad-spectrum herbicide produced by the U.S. company, Monsanto, and contains the active ingredient glyphosate.

1.3.1  Glyphosate

Glyphosate, with IUPAC name, N-(phosphonomethyl) -glycine is a non-selective, broad spectrum, post emergent systemic herbicidewidely used to kill unwanted plants both in agriculture and in nonagricultural landscapes.  Its herbicidal activity is expressed through direct contact with the leaves and subsequent translocation throughout the plant (Piesova,

2005). Glyphosate was first synthesized by Monsanto in May 1970 and was tested in the greenhouse in July of that year. The molecule advanced through the greenhouse screens and field testing System rapidly and was first introduced as Roundup ® herbicide by Monsanto Company (Baird, et al., 1971).

Fig. 1: Structures of Glyphosate

In pure chemical terms, glyphosate is an organophosphate, however, it does not affect the nervous system in the same way as organophosphate insecticides, and it is exploited for its anticholinesterase effects (Marrs, 1993). Glyphosate represents about 60% of global non-selective herbicide sales (Aspelin, 1997). Most glyphosate containing herbicides are either made or used with a surfactant and chemicals that help glyphosate to penetrate plant cells. Formulated glyphosate (like Roundup®) is highly soluble in water and could be mobile in aquatic systems. Glyphosate is an acid molecule, but it is formulated as a salt for packaging and handling. Various salt formulations include isopropylamine, diammonium, monoammonium, or potassium. Some brands include more than one salt. Some companies report their product as acid equivalent (ae) of glyphosate acid; some report it as active ingredient (ai) of glyphosate plus the salt, and others report both. In order to compare performance of different formulations it is critical to know how the products  were  formulated.  Since  the  salt  does  not  contribute  to  weed  control. Glyphosates products are supplied most commonly in formulations of 120, 240, 360, 480 and 680g active ingredient per litre. The most common formulation in agriculture is 360g active ingredients either alone or with added cationic surfactants. Glyphosate is also usually    sold    with    surfactants    like    polyoxyethyleneamine    (POEA),    methyl

pyrrolidinoneamongst many other surfactants.   Mostly these formulations may also be spiked with other active ingredients such as simazine, and 2, 4- D.

1.3.2   Glyphosate Trade Names

Glyphosate is marketed by many agrochemical companies in different solution strengths under many trade names; which includes the following: Aquaneat, Aquamaster, Buccaneer, Clearout 41 plus, Genesis Extra 1, Glyfos induce, Glystar induce, Glyphomax induce, Razor pro, Rodeo. Roundup 1, Roundup pro concentrate, Roundup ultraMax, Roundup weatherMax, Touchdound I Q and so on.

1.3.3   Uses of Glyphosate

Glyphosate is believed to be the world’s most heavily used pesticide (Duke and Powles,

2008b), with over 600 thousand tonnes used annually. Glyphosate is effective in killing a wide variety of plants, including grasses, broadleaf, and woody plants.  It has a relatively small effect on some clover species, by volume; it  is one of the most widely used herbicides. It is commonly used for agriculture, horticulture, and silviculture purposes, as well as garden maintenance (including home use). It is also used for pre-harvest desiccation of cotton, cereals, peas, beans, and other crops; for root sucker control; and for weed control in aquatic areas. The sodium salt (Quotamaster) is used as a growth regulator on sugar cane to hasten ripening, enhance sugar content, and promote earlier harvesting and on peanuts. Glyphosate is also  used to destroy drug crops grown in Colombia  (Leahy,  2007).Weak  solutions  of  the  Roundup  formulation  are  used  to devitalise some plant materials before importation into Australia and New Zealand to reduce biosecurity risks by preventing propagation of the plant material. For example, the New Zealand biosecurity authority requires that about 50 mm of the stems of cut flowers and foliage be immersed in a 0.5% solution of Roundup for 20 minutes—this reputedly prevents propagation but allows about a week of shelf life (Leahy, 2007).

Glyphosate  is  patented  as  a  synergist  for  mycoherbicides  (natural  fungi  used  for biological control of weeds), as it enhances the virulence of the fungi (Johal and Huber,

2009). In many cities, glyphosate is sprayed along the sidewalks and streets, as well as crevices in between pavement where weeds often grow.

1.4. Physicochemical Properties of Glyphosate

Pure glyphosate is a colourless, odourless, crystalline solid with a melting point of 1800C and decomposes at 1870C producing toxic fumes including nitrogen oxides and phosphorous oxides Pure glyphosate is slightly soluble in water (12g/litre at 250C), and is practically insoluble in  most  organic solvents. The alkali metal and amine salts are

readily soluble in water. Glyphosate formulations are stable over extended periods below

600C. It has a vapour pressure of less than 1 x 10-5 Pa at 250. Chemically, glyphosate is a weak acid comprising a glycine moiety and a phosphonomethyl moiety (Solomon and Thompson, 2003). Glyphosate closely resembles naturally occurring substances and does not possess chemical groups that will confer great reactivity, atmospheric mobility or biological persistence.  Its  physical  and  chemical  properties  indicate  that  it  will  not bioaccumulate,  nor  biomagnifies  through  the  food  chain  to  any  appreciable  extent (Giesyet al., 2000). Although it’s apparent great solubility would lead one to expect glyphosate to be mobile in water. It is readily ionized and, as the anion, will be strongly adsorbed to sediments and soils of pH greater than 3.5.  It thus has almost no mobility in soils and is rapidly removed from water to sediments and suspended particulate matter. When applied to soil, glyphosate shows low activity because the strong binding to soil organic matter makes the substance biologically unavailable to plants (Solomon and Thompson, 2003).

1.4.1   Method of Application of Glyphosate

In the application of glyphosate the precaution taken are to ensure that the vegetation is not wet or if rain is expected within 6 hours of application (and preferably not within 24 hours of application) this is because rain after an application can wash glyphosate off before it has a chance to enter the leaf. Rain also reduces the activity by dilution, so plant may not receive a lethal dose of the herbicide. Glyphosate products are formulated to be mixed with water to facilitate application (Hartzleet al., 2006). Water quality (soft or hard) affects glyphosate’s effectiveness. Hard water contains large amounts of dissolved

salts as calcium and magnesium, these salts have a positive charge and may associate with the negatively charged glyphosate molecule, displacing the isopropylamine or other salt used in the formulated product. Plants absorb less glyphosate bound with calcium or magnesium salts than the formulated salt of glyphosate, thus reducing glyphosate activity (Hartzle et al., 2006).

Glyphosate’s performance is affected by many factors, and applicators have little or no control over many of them. The primary cause of weed control failures is a delay in application that allows weeds to reach sizes that are difficult to kill consistently. Timely application and using the proper rate for the specific situation minimizes the effects of factors outside of the applicator’s control and reduces the likelihood of performance failures (Hartzle, et al., 2006).

1.4.2 Mode of Action

Glyphosate penetrates the plant leaf cuticle shortly after contact and begins a cell-by-cell migration  to  the  phloem,  from  which  it  is  transported  throughout  the  plants.  The herbicidal action usually occurs within 7 days and up to 30 days for woody plants (McLaren and Hart, 1995).Researchers have found that glyphosate kills plants by inhibiting  5-enolpyruvylshikimate-3-phosphate  synthase  (EPSPS).  EPSPS  is  a  key enzyme in the shikimate biosynthetic pathway that is necessary for the production of the aromatic amino acids, auxin, phytoalexins, folic acid, lignin, plastoquinones and many other secondary products (Williams et al 2000). Over 30% of the carbon fixed by plants passes through this pathway. ESPS catalyses the reaction of shikimate-3- phosphate (S3P) and phosphoenolpyruvate to form 5-enolpyruvyl-shikimate-3-phosphate (ESP) which is subsequently dephosphorylated to chorismate, an essential precursor for the synthesis of proteins  and  amino  acids  mentioned  above.  Inhibition  of  EPSPS  by  glyphosate deregulates the pathway, leading to even more carbon flowing through the pathway with accumulation of shikimate and shikimate-3-phosphate. Up to 16% of the plant’s dry matter can accumulate as shikimate. X-ray crystallographic studies of glyphosate and EPSPS show that glyphosate functions by occupying the binding site of the phosphoenolpyruvate, mimicking an intermediate state of the ternary enzyme substrates complex. There are two forms of EPSPS in nature, EPSPS I, which is found in plants,

fungi, and most bacteria, and is sensitive to glyphosate, and EPSP II, which is found in glyphosate resistant bacteria and is not inhibited by glyphosate. It is the gene for an EPSPS II that has been used to genetically engineer resistance of glyphosate in crops. The shikimate pathway is most active in meristematic tissue. Hence, glyphosate has to translocate to the meristematic tissue to be effective. Glyphosate translocates in the plant from a source to sink direction. Up to 70% of absorbed glyphosate can translocate out of the treated leaves to the root and shoot apices. However, glyphosate translocation is self- limiting and only occurs for the first 48-72 h after application (Kremer and Means 2009). The reason for this self-limiting phenomenon is not clear, but is related to the site of action of the herbicide, since there is greater translocation in glyphosate resistant crops compared to  susceptible  plants.  Glyphosate’s ability  to  translocate  readily  in  plants results in it controlling not only annual, but also perennial weeds.  The extremely broad spectrum of activity of glyphosate is primarily due to the inability of most plant species to rapidly metabolize the herbicide to non-toxic forms. While certain species, such as soybeans, can cleave glyphosate into glyoxylate and aminomethylphophonate, the rate of degradation is not rapid enough for tolerance. The two metabolism genes that have been used to generate glyphosate resistant plants, glyphosate oxidase and glyphosate acetyltransferase, were derived from bacteria.   Given the mechanism of action of glyphosate and the difficulty in genetically engineering glyphosate resistant crops, it was speculated that selection of resistance in weeds would be a very rare event. However, there are now 11 species in which resistant biotypes have been selected. The two mechanisms of resistance are (1) alterations of the target site, EPSPS, and (2) decreased uptake or translocation of glyphosate to the meristematic tissues. The levels of resistance that have been selected to date are between 2 and 10 fold. Both mechanisms of resistance appear to be overcome by increasing the rate of glyphosate application.  Glyphosphate is absorbed through foliage. Because of this mode of action, it is only effective on actively growing plants; it is not effective in preventing seeds from germinating (Kremer and Means 2009).

Toxic effects of glyphosate may be attributed to the following:

i.          the inability of the organism to synthesize aromatic amino acids; an energy drain on    the    organism    resulting    from    adenosine    triphosphate    (ATP)    and

phosphoenolpyruvate (PEP) spent in the accumulation of shikimate-3-deoxy-D- arabinoheptulose-7-phosphate (DAHP) and hydroxybenzoic acids; and

ii.        Toxicity  of  accumulated  intermediates  of  the  shikimic  acid  pathway (Fisher etal.1986).

(Smart et al., 1985) and (Huynh, 1988) in their various studies adduced evidence that glyphosate uniquely binds to and inhibits EPSP.   According to Lamb et al., (1998) glyphosate also  inhibits plant  cytochrome P-450, an enzyme that  is  involved in the detoxification of some herbicides. Biochemical symptoms of toxicity in plant include decreases in concentrations of the aromatic acids, tryptophan, phenyalanine, and tyrosine, as well as decreased rates of synthesis of proteins, indole acetic acid (cessation of growth, followed by chlorosis and the necrosis of plant tissues. Due to the fact that many animals do not possess the shikimate pathway, they depend on plants and other sources for obtaining the aromatic amino acids mentioned above. Hence, glyphosate is relatively non-toxic        to        animals        but        is        an        effective        herbicide        in plants.

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