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
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.
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:
- 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
- Toxicity of accumulated intermediates of the shikimic acid pathway (Fisher 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. Fig. 2: Inhibition of shikimic acid pathway by glyphosate.
1.4.3 Glyphosate Metabolism
Several researchers have studied glyphosate metabolism especially microbial degradation of glyphosate in the field. One of the organisms mostly studied is Pseudomonas sp PG 2982 which is shown to degrade glyphosate to glycine, a one carbon unit, and phosphate via the formation of sarcosine intermediate (Kishore and Jacob, 1987). In this study, Kishore and Jacob concluded that metabolism of glyphosate requires an energy source (in form of gluconate) and is generally inhibited by inorganic phosphate (Pi). Conversion of glyphosate to aminomethylphosphonic acid (AMPA) is achieved by Flavobacteriumsp (Balthazar and Hallas, 1986), as well as by mixed soil cultures of bacteria (Rueppelet al., 1977; Nomura and Hilton, 1977), but metabolism of glyphosate in Pseudomonas sp PG 2982 does not involve the formation of AMPA. In the same vein, conversion to AMPA occurs in the presence of inorganic phosphate (Pi) whereas inorganic phosphate inhibits the conversion of glyphosate to glycine via the sarcosine intermediate (Pipkeet al., 1987). Jacob et al., (1985) took the study further and reported that the glycine which is the breakdown product of glyphosate by Pseudomonas spPG 2982 which utilizes glyphosate as its sole source of phosphorus is utilized as follows; 20% of this glycine is used in the synthesis of purine, 35% is incorporated as seryl residues with an additional 35% incorporated as glycyl residues. The phosphonomethyl carbon of glyphosate is ultimately incorporated into a number of sites, including the C-2 and C-8 positions of the purine rings of nucleic acids, methyl groups of methionine and thymidine and the methylene group of serine. Using both single and double cross-polarization solid-state Nuclear Magnetic Resonance, (NMR), Jacob et al., (1985), were able to establish for the first time an indication of the involvement of tetrahydrofolate, a co-enzyme which facilitates single-carbon transfers and the complete determination of the metabolism of glyphosate in a pure culture. Shinarbarger and Braymer, (1986), using radiorespirometry techniques and in agreement with already cited studies, revealed that approximately 50-59% of the C-3 carbon of glyphosate was oxidized to CO2, 45-47% of the assimilated label is distributed to proteins and that the amino acids serine and methionine are highly labelled. The results indicated that the C-3 of glyphosate was at some point metabolisedto C-1 compound whose ultimate fate could be both oxidation to CO2 and distribution to amino acids and nucleic acid bases that receive C-1 group from C-1 donating co-enzyme, tetrahydrofolate. Furthermore, examination of crude extracts prepared from PG 2982 cells revealed the presence of sarcosine oxidizing enzyme oxidizes sarcosine to glycine and formaldehyde. These results further indicate that the first step in glyphosate degradation by PG 2982 is the cleavage of the carbon-phosphorus bond, resulting in the release of sarcosine and a phosphate group. The phosphate group is utilized as a source of phosphorus, and the sarcosine is degraded to glycine and formaldehyde. In addition to the above, Pseudomonas sp. Strain LBr. was found by Jacob et al., (1988), to have the ability of degrading high levels of glyphosate, primarily by converting it to AMPA followed by release into the growth medium. Only a small amount of AMPA, which is needed to supply phosphorus for growth, of about 0.5-0.7mM, could be metabolized by the micro-organism. About 5% of the glyphosate was degraded by a separate pathway-involving breakdown of glyphosate to glycine, a pathway first observed in Pseudomonas sp PG 2982 (Shinabarger and Braymer, 1986). Thus, Pseudomonas sp strain LBr appears to possess the two distinct routes of glyphosate detoxification.
1.4.4 Adsorption of Glyphosate
Glyphosate is water-soluble, but it has an extremely high ability to bind to soil particles.Adsorption of glyphosate increases with increasing clay content, cation exchange capacity, and decreasing soil pH and phosphorous content (Sprankleet al., 1975a, b; Hance 1976; Nomura and Hilton 1977; Rueppelet al., 1977; Glass, 1987). Glyphosate is adsorbed to soil particles rapidly during the first hour following application and slowly thereafter (Sprankleetal., 1975b). Strong adsorption to soil particles slows microbial degradation, allowing glyphosate to persist in soils and aquatic environments. Because glyphosate rapidly binds to soils, it has little or no herbicidal activity (“killing power”) once it touches soil (Sprankleet al., 1975a; Hance 1976; Nomura and Hilton 1977). Glyphosate can also be inactivated by adsorption if mixed with muddy water. Adsorption prevents glyphosate from being mobile in the environment except when the soil particles themselves are washed away (Sprankleet al., 1975b; Rueppelet al., 1977; Roy et al., 1989a). Comes et al., (1976) found that glyphosate sprayed directly into a dry irrigation canal was not detectable in the first irrigation waters flowing through the canal several months later, although glyphosate residues remained in the canal soils. In most cases, glyphosate is quickly adsorbed to suspended and bottom sediments (Fenget al., 1990).
1.4.5 Chemical Decomposition of Glyphosate
Glyphosate is not readily hydrolyzed or oxidized in the field (Rueppeet al., 1977; Anton etal., 1993; Zaranyika and Nyandoro 1993).
1.5 Behaviors of Glyphosate in the Environment
Glyphosate binds readily with soil particles, which limits its movement in the environment. It is degraded through microbial metabolism with an average half-life of two months in soils and two to ten weeks in water. In plants, glyphosate is slowly metabolized.Glyphosate is highly water soluble, but unlike most water-soluble herbicides, glyphosate has a very high adsorption capacity. Once glyphosate contacts soil it is rapidly bound to soil particles rendering it essentially immobile (Roy et al., 1989a; Feng and Thompson 1990). Unbound glyphosate molecules are degraded at a steady and relatively rapid rate by soil microbes (Nomura and Hilton 1977; Rueppelet al., 1977). Bound glyphosate molecules also are biologically degraded at a steady, but slower rate. The half-life of glyphosate in soil averages two months but can range from weeks to years (Nomura and Hilton 1977; Rueppelet al., 1977; Newton et al., 1984; Roy et al., 1989a; Feng and Thompson 1990; Anton et al. 1993). Although the strong adsorption of glyphosate allows residues to persist for over a year, these residues are largely immobile and do not leach significantly. Feng and Thompson, (1990) found that >90% of glyphosate residues were present in the top 15 cm of soil and were present as low as 35 cm down the soil column in only one of 32 samples. Adsorption to soil particles prevents glyphosate from being taken-up by the roots of plants.
Because glyphosate binds strongly to soils, it is unlikely to enter waters through surface or subsurface runoff except when the soil itself is washed away by runoff, and even then, it remains bound to soil particles and unavailable to plants (Rueppelet al., 1977, Malik et al., 1989). Most glyphosate found in waters likely results from runoff from vegetation surfaces, spray drift, and intentional or unintentional direct overspray. In most cases, glyphosate will dissipate rapidly from natural water bodies through adsorption to organic substances and inorganic clays, degradation, and dilution (Folmaret al., 1979; Fenget al., 1990; Zaranyika and Nyandoro 1993; Paveglioet al., 1996). Residues adsorbed to suspended particles are precipitated into bottom sediments where they can persist until degraded microbially with a half-life that ranges from 12 days to 10 weeks (Goldsborough and Brown 1993). At least one study found that >50% of the glyphosate added directly to the waters of an irrigation canal were still present 14.4 km downstream (Comes et al., 1976),
Glyphosate is metabolized by some, but not all plants (Carlisle and Trevors, 1988). It is harmless to most plants once in the soil because it is quickly adsorbed to soil particles, and even when free, it is not readily absorbed by plant roots (Hance, 1976). The half-life of glyphosate on foliage has been estimated at 10.4 to 26.6 days (Newton et al., 1984). Roy etal., (1989) found 14% and 9% of applied glyphosate accumulated in the berries of treated blueberry and raspberry bushes, respectively. These residues dissipated from the fruit with a half-life of <20 days for blueberries and <13 days for raspberries (Roy et al., 1989).
1.6 Persistence and Movement of glyphosate in the Soil
Glyphosate’s persistence in soil varies widely, so giving a simple answer to the question “How long does glyphosate persist in soil?” is not possible. Half-lives (the time required for half of the amount of glyphosate applied to break down or move away) as low as 3 days (in Texas) and as long as 141 days (in Iowa) have been measured by glyphosate’s manufacturers. Initial degradation (breakdown) is faster than the subsequent degradation of what remains, (Torstensson and Stark, 1979). Long persistence has been measured in the following studies: 55 days on an Oregon Coast Range forestry site (Newtonet al., 1984). 249 days on Finnish agricultural soils (Newton et al., 1984); between 259 and 296 days on eight Finnish forestry sites (Torstensson and Stark, 1979); 335 days on an Ontario (Canada) forestry site (Feng and Thompson, 1990); 360 days on 3 British Columbia forestry sites (Roy et al., 1989); and, from 1 to 3 years on eleven Swedish forestry sites (Torstenssonet al., 1989). EPA’s Ecological Effect’s Branch wrote, “In summary, this herbicide is extremely persistent under typical application conditions.
Glyphosate is thought to be “tightly complexed bound by most soils and therefore “in most soils, glyphosate is essentially immobile. This means that the glyphosate will be unlikely to contaminate water or soil away from the application site. However, this binding to soil is “reversible.” For example, one study found that glyphosate bound readily to four different soils. However, desorption, when glyphosate unbinds from soil particles, also occurred readily. In one soil, 80 percent of the added glyphosate desorbed in a two hour period. The study concluded that this herbicide could be extensively mobile in the soil (Feng and Thompson, 1990).
1.6.1 Glyphosate and Water Contamination
When glyphosate binds readily to soil particles, it does not have the chemical characteristics of a pesticide that is likely to leach into water. However, glyphosate can move into surface water when the soil particles to which it is bound are washed into streams or rivers. How often this happens is not known, because routine monitoring for glyphosate in water is infrequent. Glyphosate has been found in both ground and surface water. Examples include farm ponds in Ontario, Canada, contaminated by runoff from an agricultural treatment and a spill(Frank et al., 1990). The runoff from a watersheds treated with Roundup during production of no-till corn and fescuecontaminated surface water in the Netherlands ‘(Edward et al., 1980); seven U.S. wells (one in Texas, six in Virginia contaminated with glyphosate; contaminated forest streams in Oregon and Washington (Rashinet al., 1993). contaminated streams near Puget Sound, Washington and contaminated wells under electrical substations treated with glyphosate (Smith et al., 1996). Glyphosate’s persistence in water is shorter than its persistence in soils. Two Canadian studies found glyphosate persisted 12 to 60 days in pond water (Goldsborough et al., 1989). Glyphosate persists longer in pond sediments (mud at the bottom of a pond). For example, the half-life in pond sediments in a Missouri study was 120 days; persistence was over a year in pond sediments in Michigan and Oregon (Goldsboroughet al., 1989).
1.7 Glyphosate Toxicity
Given the marketing of glyphosate herbicides as benign, it is striking that laboratory studies have found adverse effects in all standard categories of laboratory toxicology testing. These include medium-term toxicity (salivary gland lesions), long-term toxicity (inflamed stomach linings), genetic damage (in human blood cells), effects on reproduction (reduced sperm counts in rats; increased frequency of abnormal sperm in rabbits), and carcinogenicity (increased frequency of liver tumors in male rats and thyroid cancer in female rats). Glyphosate treatment has reduced populations of beneficial insects, birds, and small mammals by destroying vegetation on which they depend for food and shelter.In laboratory tests, glyphosate increased plants’ susceptibility to disease and reduced the growth of nitrogen-fixing bacteria.Commercial glyphosate herbicides are more acutely toxic than pure glyphosate. The amount of Roundup (containing glyphosate and the surfactant POEA) required to kill rats is about 1/3 the amount of glyphosate alone.’ Roundup is also more acutely toxic than POEA (Martinez et al., 1991).
1.7.1 Acute Toxicity of glyphosate
Both glyphosate and the commercial products that contain glyphosate are acutely toxic to animals in general, but glyphosate alone is less toxic than the common glyphosate product, Roundup, and other glyphosate products have intermediate toxicity. Part of these differences can be explained by the toxicity of the surfactant (detergent-like ingredient) in Roundup, The amount of Roundup (containing glyphosate and the surfactant POEA) required to kill rats is about 1/3 the amount of glyphosate alone’ (Mitchel et al., 1987).
220.127.116.11Acute Toxicity of Glyphosate to Laboratory Animals
Inhalation of roundup by rats caused signs of toxicity in all test groups even at the lowest concentration tested. These signs include reduced activity and body weight loss, lungs were red or blood congested. The dose required to cause lung damage and mortality following pulmonary administration of two roundup and POEA was only 1/10 of the dose causing oral damage (Martinez et al., 1990). When dogs were given intravenous injection of Glyphosate, POEA or Roundup, Glyphosate increased the ability of the heart muscle to contract. POEA reduced the output of the heart and the pressure in the arteries. Roundup caused cardiac depression (Martinez et al., 1990).
18.104.22.168 Acute Toxicity of Glyphosate to Humans
The acute toxicity of glyphosate products to humans was first publicized by physicians in Japan who studied suicide attempts, (Lordiet al., 1993), nine cases were fatal. Symptoms included intestinal pain, vomiting, excess fluid in the lungs, pneumonia, clouding of consciousness, and destruction of red blood cells (Sawada et al.,1988) They calculated that the fatal cases ingested on average about 200 milliliters (3/4 of a cup). They believed that POEA was the cause of Roundup’s toxicity (Sawada et al., 1988). More reviews of poisoning incidents have found similar symptoms, as well as lung dysfunction, (Tominack 1991; Talbot, 1991), erosion of the gastrointestinal tract, (Tominack 1991; Talbot, 1991), abnormal electrocardiograms (Talbot 1991), low blood pressure (Tominack 1991; Talbot 1991), kidney damage and damage to the larynx (Hung et al., 1997). Smaller amounts of Roundup cause adverse effects, usually skin or eye irritation as well as symptoms like painful eyes, blurred vision, blisters, skin rash, rapid heartbeat, heart palpitations, elevated blood pressure, chest pain, itchy skin, headache, tingling skin , recurrent eczema and so on (Giiordano-Labadie et al., 1996).
1.7.2Subchronic Toxicity of Glyphosate
In subchronic (medium term) studies of rats and mice done by the National Toxicology Program (NTP), microscopic salivary gland lesions were found in all doses tested in rats (200 – 3400 mg/kg per day) and in all but the lowest dose tested in mice (1,000-12,000 mg/kg per day). A follow-up study by NTP found that the mechanism by which glyphosate caused these lesions involved the hormone adrenalin (Nordstrom, 1998). The NTP study also found increases in two liver enzymes at all but the two lowest doses tested. Other effects found in at least two doses in this study were reduced weight gain in rats and mice; diarrhea in rats; and changes in kidney and liver weights in male rats and mice (Nordstrom, 1998).
Another subchronic laboratory test found that blood levels of potassium and phosphorus in rats increased at all doses tested (60-1600 mg/kg/day) U.S. EPA (1993). In a 7-day study of calves, 790mg/kg per day of Roundup caused pneumonia, and death of 1/3 of the animals tested. At lower doses, decreased food intake and diarrhea were observed.
1.7.3 Chronic Toxicity of Glyphosate
Glyphosate is also toxic in long-term studies. At all but the lowest dose tested, excessive cell division in the urinary bladder occurred in male mice and inflammation of the stomach lining occurred in both sexes of rats (Nordstrom, 1998).
22.214.171.124 Glyphosate and Carcinogenicity
Toxicological data on cancer is inadequate and inconsistent. Glyphosate was originally classified by the US EPA as a ‘Group C’, ‘possible human carcinogen’, on the basis of increased incidence of renal tumours in mice. However, after “independent review of the slides, the classification was changed to D on the basis of a lack of statistical significance and uncertainty as to a treatment-related effect”. D classification means “not classifiable as to human carcinogenicity” but Hardell and Eriksson (1999) referred to a study, reported by Stauffer Chemical Company, which found increased incidence of hepatocellular carcinoma, leukaemia, and lymphoma in mice (Pavkov and Turnier 1986). The US EPA’s classification does not take into account recent epidemiological evidence. Increasingly this is suggesting that glyphosate might be causing non-Hodgkin’s lymphoma, and possibly other haematological cancers.
In addition a Swedish study of hairy cell leukemia (HCE), a form of the cancer non-Hodgkin’s lymphoma, found that people who were occupationally exposed to glyphosate herbicides had a threefold higher risk of HCE. A similar study of people with non-Hodgkin’s lymphoma found exposure to glyphosate herbicides was associated with an increase in risk of about the same size (Nordstrom, 1998).Additionally there is increasing toxicological evidence that glyphosate, Roundup, and the metabolite AMPA all have the potential to cause cancer through mechanisms such as genotoxicity, oxidative stress, and interference with hormonal functions.
126.96.36.199Genotoxicity and Mutagenicity of Glyphosate
A pesticide is genotoxic if it causes damage to a gene that could result in cell death, or result in change in the structure or function of the gene. The damage can be mutagenic (heritable) or non-mutagenic. Mutagenic means causing a change in the genetic structure, usually through base-pair substitution (change in amino acid sequence), deletion, or addition of gene fragments, or some other mechanism. Mechanisms involved include causing damage to the chromosome such as loss, breaks or rearrangements of chromosomal segments. It also includes “sister chromatid exchanges”, interchanges and re-attachments of strands in the chromosome during DNA replication, and induction (increase) in the frequency of micronuclei (small fragments formed when chromosomes break). One of the main health implications of genotoxicity is cancer.U.S.EPA (2006)
Although glyphosate’s manufacturer describes “a large battery of assays” showing that glyphosate does not cause genetic damage, However, there are many other studies that did not come from Monsanto which demonstrate glyphosate, Roundup, and/or the metabolite AMPA to be genotoxic. Even some industry papers show this: Hardell& Eriksson (1999) cite a number of papers from the Stauffer Chemical Company (Majeska and Matheson, 1982a, b and 1985a, b) showing gene mutations and chromosomal aberrations in mouse lymphoma cells. Most compelling are the studies that show genotoxicity in human cells: Glyphosate caused DNA damage in human liver cells at concentrations of 3 to 7.5 mM, but not in human lymphocytes at 0.2 to 6 mM (Mañaset al., 2009a). Roundup caused dose-dependent DNA damage in human liver cells, with 50% DNA strand breaks at 5 mg/kg, described by the authors as “residual levels corresponding to 120 nM of glyphosate” (Gasnieret al., 2009). Glyphosate was genotoxic in normal human cells at concentrations of 4 to 6.5 mM and in human cancer cells (fibrosarcoma) at 4.75 to 5.75 mM (Monroyet al., 2005). Glyphosate caused a dose-dependent increase in chromosomal aberrations and an increase in sister chromatid exchange in human lymphocytes (Lioiet al., 1998a). Glyphosate and Roundup caused dose-dependent increases in sister chromatid exchange in human lymphocytes; Roundup had a greater effect (Bolognesiet al., 1997). Roundup at high concentrations caused an increase in sister chromatid exchange in human lymphocytes (Vigfusson and Vyse 1980). In the first data to be published on the potential genotoxicity of the metabolite AMPA, Mañaset al (2009b) have shown that it is clearly genotoxic, causing DNA damage in human liver cells at concentrations of 2.5 to 7.5 mM. It also caused chromosomal damage in human lymphocytes at 1.8 mM. A variety of tests on animals, bacteria, and plant cells have further demonstrated the genotoxic ability of glyphosate, Roundup and AMPA: Glyphosate caused the induction of micronuclei at high doses, possibly through oxidative stress, in mouse bone marrow (Mañaset al., 2009a). Roundup caused the induction of micronuclei in mouse bone marrow. Both glyphosate and Roundup caused DNA strand breaks in mouse liver and kidney cells (Bolognesiet al., 1997). Roundup, but not glyphosate, caused dose-dependent formation of DNA adducts in mouse liver and kidney cells (Pelusoet al., 1998). Glyphosate caused chromosomal aberrations and sister chromatid exchange in bovine lymphocytes (Lioiet al., 1998b). Glyphosate caused sister chromatid exchange in bovine lymphocytes at concentrations of 56 to 1120 uM (Siviková and Dianovskỳ 2006).
Developmental exposure to glyphosate caused mutations in fruit flies (Drosophila melanogaster) (Kaya et al., 2000). Roundup caused DNA damage and micronucleus induction in the gill cells of the neotropical fish Prochiloduslineatus, and DNA damage but not micronucleus induction in its erythrocytes, at 10 mg/l concentration (Cavalcanteet al., 2008). Roundup caused dose-dependent DNA damage and micronucleus induction in the erythrocytes of newborn broad-snouted caiman (Caiman latirostris) after exposure in ovo to concentrations of Roundup of 500 ug/egg or higher (Polettaet al., 2009). Roundup caused micronucleus induction in the erythrocytes of the fish Tilapia rendalli, but not in mouse erythrocytes (Grisolia 2002). Roundup caused dose-dependent micronuclei induction, nuclear abnormalities, and DNA strand breaks in the erythrocytes of goldfish (Carassiusauratus) at concentrations of 5, 10 and 15 mg/l (Cavaş and Könen, 2007). Roundup caused DNA damage in blood, liver and gill tissue of the European eel (Anguilla anguilla) at 3.6 mg/l concentration (Guilhermeet al., 2009). Roundup caused DNA damage in bullfrog tadpoles (Ranacatesbeiana) at concentrations of 6.75 and 27 mg/l, but not at 1.69 mg/l (Clements et al., 1997). Roundup caused DNA damage in sea urchin embryos (Belléet al., 2007). Roundup and Pondmaster, another formulation of glyphosate, “induced a very high frequency of lethal sex-linked, recessive lethal mutations] in larval spermatocytes and in spermatogonia” of fruit flies (Kale et al., 1995). Roundup, but not glyphosate, caused chromosomal damage in root-tips cells of onions (Allium cepa) (Rank et al., 1993). Roundup was weakly mutagenic in the bacterium Salmonella typhimurium at a concentration of 360 ug/plate (Rank et al., 1993). AMPA caused the induction of micronuclei in mice (Mañaset al., 2009b).
Other studies have shown that both glyphosate and glyphosate products are mutagenic. Glyphosate-containing products are more potent mutagens than glyphosate. (Bolognesiet al., 1997) The studies include the following: In fruit flies, Roundup and Pondmaster (an aquatic herbicide consisting of glyphosate and a trade secret surfactantboth increased the frequency of sexlinked, recessive lethal mutations. (These mutations are usually visible only in males. Only a single concentration was tested in this study (Kale, 1995). A study of human lymphocytes (a type of white blood cell showed an increase in the frequency of sister chromatid exchanges following exposure to the lowest dose tested of Roundup. (Vigfussonet al., 1980) (Sister Chromatid exchanges are exchanges of genetic material during cell division between members of a chromosome pair. They result from point mutations). A 1997 study of human lymphocytes found similar results with Roundup (at both doses tested and with glyphosate (at all but the lowest dose tested) (Bolognesi, 1997). In Salmonella bacteria, Roundup was weakly mutagenic at two concentrations. In onion root cells, Roundup caused an increase in chromosome aberrations, (Rank et al., 1993).
In mice injected with Roundup, the frequency of DNA adducts (the binding to genetic material of reactive molecules that lead to mutations) in the liver and kidney increased at all three doses tested (Pelusoet al., 1998). In another study of mice injected with glyphosate and Roundup, the frequency of chromosome damage and DNA damage increased in bone marrow, liver, and kidney (Bolognesi, 1997).In summary, some toxicological studies show glyphosate, Roundup, and/or the metabolite AMPA to be genotoxic and some do not. Using a precautionary approach, the conclusion should be reached that glyphosate appears to have the ability to cause genetic damage that can lead to cancer. This conclusion is supported by the epidemiological reports of DNA damage in people exposed to glyphosate, and cases of lymphocytic cancer, particularly non-Hodgkin’s lymphoma, reported in the section ‘Health Effects and Poisonings’. Colombian researchers further confirmed the plausibility of the link between glyphosate and haematological cancers such as non-Hodgkin’s lymphoma when they studied its effects on human peripheral blood mononuclear cells. They found that both glyphosate and Roundup decreased cell viability in a dose-dependent manner (Reyes et al., 2006; Martinez et al., 2007), and altered gene expression in the cells (Reyes et al., 2006).
188.8.131.52 Chronic toxicity of Glyphosate on the Mammalian Enzymes
A number of studies have shown adverse effects of glyphosate, and/or formulations of it, on mammalian enzymes: Glyphosate (Demerdashet al., 2001) and Roundup (Caglar and Kolankaya, 2008) have inhibitory effects on the enzymes lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) in human serum and rat liver. Activities of these enzymes are considered bioindicators for toxicity; and decreased activity of serum AST and ALT are indicators of liver damage, so the studies indicates that glyphosate and Roundup causes liver damage. Benedetti et al., (2004) showed that brief exposure to a Brazilian formulation of glyphosate caused liver damage in rats (fibrosis and leakage of liver enzymes AST and ALP) and they regarded this to be indicative of irreversible damage to liver cells. Sublethal doses of Roundup modified liver enzymatic activity (inhibited monoxygenases) in rats (Hietanenet al., 1983). Glyphosate inhibited acetylcholinesterase (AChE) in human serum, a hallmark of organophosphate toxicity (El Demerdashet al., 2001). Maternal exposure to glyphosate caused functional abnormalities in the specific activity of three enzymes found inside cells—isocitrate dehydrogenase, malic dehydrogenase, and glucose-6-phosphate dehydrogenase (G6PD)—in liver, heart, and brain of pregnant rats and their foetuses (Daruichet al., 2001). All enzymes are involved in the generation of NADPH (nicotinamide adenine dinucleotide phosphate), which has many essential roles in metabolism. (Ho and Ching, 2003) asserted that glyphosate has the potential to disrupt many important enzyme systems that utilisephosphoenol pyruvate, including energy metabolism and the synthesis of key membrane lipids required in nerve cells. Glyphosate acts in plants by preventing the binding of phosphoenol pyruvate to the active site of the enzyme 5-enolpyruvoyl-shikimate-3-phosphate synthetase (EPSPS) and, although this enzyme is specific to plants, phosphoenol pyruvate is a core metabolite in all organisms. Roundup, at low concentrations of 1-10 mM, 1 damages rat liver cells, including the mitochondrial membranes and nuclei (Malatestaet al., 2008). Higher, sublethal, doses of Roundup depress mitochondrial respiratory activity in rat liver cells (Peixoto, 2005).
184.108.40.206 Chronic Toxicity of Glyphosate on Endocrine System
The US EPA (2006) reported that “potential estrogen, androgen, and/or thyroid mediated toxicity was not indicated in any test on glyphosate”. However, a number of studies since then have demonstrated that both glyphosate and the Roundup formulation do disrupt both oestrogens and androgens. US researchers, Walsh et al., (2000) demonstrated that Roundup, but not glyphosate, significantly disrupted the production of the hormone progesterone in mouse cells, by disrupting expression of the steroidogenic acute regulatory (StAR) protein. The authors concluded that, as the StAR protein is also indispensable for steroidogenesis in the adrenal glands, a disruption in StAR protein expression may potentially affect carbohydrate metabolism, immune system function, and water balance, as well as fertility. It may have an impact on reproduction in humans, other mammals, birds, and amphibians.
Lin and Garry (2000) found that both Roundup and glyphosate caused the proliferation of MCF-7 human breast cancer cells, but not via an oestrogenic mechanism. Subsequent studies by Richard et al. (2005) and Hokansonet al. (2007), reported below, may explain the mechanisms by which this could have occurred.
In 2005, a research team from Caen University in France (Richard et al., 2005) demonstrated that glyphosate and Roundup, at non-toxic concentrations, affected the enzyme aromatase which is responsible for the synthesis of oestrogen. They found that glyphosate, at dilutions 100 times lower than agricultural rates, inhibited aromatase activity, interacted with the active site of the enzyme, and decreased aromatase mRNA levels. The effects were greater with the Roundup formulation than glyphosate alone. However, there appears to be a differential effect on aromatase: although both glyphosate and Roundup reduced aromatase activity once it had entered the cells, prior to this entry into the cells the Roundup (but not glyphosate) actually caused a 40% increase in aromatase activity. A similar differential effect has been observed with lindane and bisphenol-A. The authors concluded that glyphosate has endocrine disrupting effects in mammals, and that the presence of Roundup adjuvants enhances glyphosate bioavailability and/or bioaccumulation in cells, and that these effects could explain premature births and miscarriages observed in epidemiological studies involving women farmers using glyphosate.
Hokansonet al. (2007) demonstrated that a commercial glyphosate formulation (“a 15% home use preparation”) dysregulated 680 out of 1,550 genes in MCF-7 human breast cancer cells. They also identified a synergistic effect with oestrogen (17B-estradiol). Oestrogen-regulated gene expression is a major factor in the regulation of a number of physiological functions, and the genes affected in this study have implications for tumour formation and growth, immune function,
Benachouret al. (2007) also demonstrated glyphosate-dependent endocrine disruption via aromatase inhibition in human embryonic and placental, and equine testis cells, at non-toxic levels of glyphosate. As little as 0.01% Roundup provoked a significant reduction of 19% of oestrogen production in exposed cells. The authors linked the effects to glyphosate itself, with a synergistic effect provoked by the adjuvants in the formulation. The embryonic cells, which are the most sensitive cells, showed evidence of either bioaccumulation or time-delayed effect, suggesting a cumulative impact of very low doses of glyphosate approximating the Acceptable Daily Intake (0.3 mg/kg). The authors expressed concern that significant levels of glyphosate are likely to reach the placenta and embryo in exposed pregnant women, given that little protective equipment is usually worn with this herbicide. A study by Moseet al. (2008) confirmed that glyphosate does cross the placenta: they found 15% of glyphosate in maternal circulation crossed to fetal circulation, although this figure could be higher as 32% of the glyphosate was unaccounted for after the experiment.
Using human liver HepG2 cells, Gasnieret al. (2009), found that Roundup (but not glyphosate) inhibited the conversion of androgens to oestrogen, with a non-linear biphasic effect on the aromatase mRNA levels: the greatest effect occurring at medium doses. Effects occurred at 10 mg/l. Lower doses caused linear and dose-dependent disruption of oestrogen- and androgen- dependent transcriptional activity: androgen receptors at 0.5 ppm, 2 and oestrogen receptors at 2 mg/l). Glyphosate alone had no anti-oestrogenic effect but “was clearly anti-androgenic at sub-agricultural and non-cytotoxic dilutions”. Most formulations were more anti-androgenic than anti-oestrogenic. The doses used in this study were described by the authors as far below agricultural doses, and the effects occurred within 24 hours of exposure.
The implications of the endocrine-disrupting effects reported above can be profound and far-reaching, involving a range of developmental