**3. Pre-emergent application technology of glyphosate**

Glyphosate, exerting global herbicidal action, has originally been intended to pre-emergent weed control treatments of field vegetation and weed control of orchards and ruderal areas.

Forty Years with Glyphosate 253

Dow AgroSciences IPA Dominator; Durango; Glyphomax; Glyphomax Plus;

K DupliKator

IPA + AMM Showdown

IPA + MA Credit Duo

Makhteshim-Agan IPA Eraser, Gladiator; Glyphogan; Hardflex; Herbolex; Taifun

UltraMAX

VisionMAX

AMM Medallon Premium DMA Touchdown IQ

TRI Coloso; Ouragan

a AMM = ammonium; DMA = dimethylamine; IPA = isopropylamine; K = potassium; TRI = trimesium b Formulations containing only glyphosate salts as active ingredient are listed, herbicide combinations

Table 1. Formulated herbicide preparations containing glyphosate as active ingredient.

Helm Agro US Inc. IPA Glyphosate 41%; Helosate Plus

IPA

Nufarm IPA Amega; Credit; Credit Extra

DMA Durango DMA; Duramax

GlyPro; Panzer; Ripper; Rodeo; Vantage

Accord; Aquamaster; Azural; Clinic; Glialka; Honcho; Ranger Pro, Roundup Bioforce / Classic / Original /

<sup>K</sup>Roundup Forte / Mega / PowerMAX / WeatherMAX;

K Refuge; Touchdown HiTech / Total; Traxion

**Manufacturer a.i. salt** <sup>a</sup>**Product** <sup>b</sup>

Albaugh Inc./Agri Star IPA Aqua Star, Gly Star Original

Technologies LLC IPA ClearOut; ClearOut Plus Cheminova IPA Glyfos; Glyfos X-tra

AAKO B.V. IPA Akosate Agriliance LLC IPA Cornerstone Agro-Chemie Ltd. IPA Fozát

Astrachem Ltd. IPA Tiller Barclay Chem. Mfg. Ltd. IPA Gallup Calliope S.A. IPA Kapazin

Control Solutions Inc. IPA Spitfire Crystal Chem. Inter-America IPA Glifonox

Drexel Chem. Co. IPA Imitator

Helena Chemical Co. IPA Rattler

Micro Flo IPA Gly-Flo

Oxon Italia S.p.A. AMM Buggy Pinus TKI d.d. IPA Boom Efekt Sinon Corporation IPA Glyfozat; Total

Tenkoz Inc. IPA Buccaneer UAP IPA Makaze

Alliance LLC IPA Gly-4 Winfield Solutions LLC IPA Cornerstone

FarmerSaver.com LLC IPA Glyphosate 4 Griffin LLC IPA Glyphosate Original Growmark Inc. IPA FS Glyxphosate Plus

Loveland Products Inc. IPA Mad Dog; Mirage

Chemical Products

Monsanto Co.

Syngenta AG

are not included

Universal Crop Protection

Post-emergent applications are impossible solely with glyphosate-based herbicide formulations due to the phytotoxicity of the compound to the crop as well.

Common first visible phytotoxicity effects of glyphosate include rapid (within 2-10 days upon application) chlorosis, usually followed by necrosis (Suwannamek & Parker, 1975; Putnam, 1976; Campbell et al., 1976; Fernandez & Bayer, 1977; Marriage & Khan, 1978; Segura et al., 1978; Abu-Irmaileh & Jordan, 1978), possibly accompanied with morphological leaf deformities (Marriage & Khan, 1978), root and rhizome damage (Suwannamek & Parker, 1975; Fernandez & Bayer, 1977). Glyphosate accumulation has been reported in the meristems (Haderlie et al., 1978). It is rather surprising that although glyphosate inhibits seedling growth as well, it did not exert significant effect on the germination of various species (Haderlie et al., 1978; Egley & Williams, 1978).

#### **3.1 Formulated glyphosate-based herbicides**

Glyphosate-based formulations such as Roundup®, Accord® and Touchdown® represent the most common types used for agricultural purposes (Franz et al., 1997). These formulated herbicides can be used for weed control in agricultural practice, including in no-till agriculture to prepare fields before planting, during crop development and after crop harvest; as well as in silvicultural, urban and, lately, aquatic environments. The main herbicide products currently distributed are listed in Table 1. These preparations contain glyphosate as formulated in form of its ammonium (AMM), dimethylammonium (DMA), isopropylammonium (IPA), potassium (K) or trimesium (TRI) salts. The very first formulations containing IPA, sodium and ammonium salts were patented by Monsanto in 1974. A unique form is the trimesium salt of outstanding water solubility, patented by ICI Agrochemicals (later Zeneca Agricultural Products Inc, then Novartis CP, and after 2000 Syngenta) in 1989 (Tomlin, 2000).

As the actual active ingredients of the formulations are salts, differing from each other in the cation(s) and consequently the molecular mass of the salts, active ingredient concentrations are specified as glyphosate equivalent, in other term acid equivalent (a.i.) referring to the free acid form of glyphosate. This provides instant comparability among various formulations. Moreover, the use of a.i. units is common practice in residue analysis of glyphosate as well.

#### **3.2 Formulating agents**

Formulated glyphosate-based herbicides contain various non-ionic surfactants to facilitate their uptake by the plants (Riechers et al., 1995). These components, as all other pesticide additives and diluents, are assumed to be inert, which as it turns out, is not the case for several such ingredients. The most common surfactant applied in combination with glyphosate is polyethyloxylated tallowamine (POEA), which itself has been found to exert ecotoxicity, also in synergy with glyphosate, causing the formulated herbicide (e.g., Roundup) more toxic than its technical grade active ingredient (Folmar et al., 1979; Atkinson, 1985; Wan et al., 1989; Powell et al., 1991; Giesy et al., 2000; Tsui & Chu, 2003; Marc et al., 2005; Benachour et al., 2007; Benachour & Séralini, 2009).

The apparent synergistic toxic effects of the assumedly inert ingredients with glyphosate triggered a legal case between Monsanto and the New York Attorney General's Office in

Post-emergent applications are impossible solely with glyphosate-based herbicide

Common first visible phytotoxicity effects of glyphosate include rapid (within 2-10 days upon application) chlorosis, usually followed by necrosis (Suwannamek & Parker, 1975; Putnam, 1976; Campbell et al., 1976; Fernandez & Bayer, 1977; Marriage & Khan, 1978; Segura et al., 1978; Abu-Irmaileh & Jordan, 1978), possibly accompanied with morphological leaf deformities (Marriage & Khan, 1978), root and rhizome damage (Suwannamek & Parker, 1975; Fernandez & Bayer, 1977). Glyphosate accumulation has been reported in the meristems (Haderlie et al., 1978). It is rather surprising that although glyphosate inhibits seedling growth as well, it did not exert significant effect on the germination of various

Glyphosate-based formulations such as Roundup®, Accord® and Touchdown® represent the most common types used for agricultural purposes (Franz et al., 1997). These formulated herbicides can be used for weed control in agricultural practice, including in no-till agriculture to prepare fields before planting, during crop development and after crop harvest; as well as in silvicultural, urban and, lately, aquatic environments. The main herbicide products currently distributed are listed in Table 1. These preparations contain glyphosate as formulated in form of its ammonium (AMM), dimethylammonium (DMA), isopropylammonium (IPA), potassium (K) or trimesium (TRI) salts. The very first formulations containing IPA, sodium and ammonium salts were patented by Monsanto in 1974. A unique form is the trimesium salt of outstanding water solubility, patented by ICI Agrochemicals (later Zeneca Agricultural Products Inc, then Novartis CP, and after 2000

As the actual active ingredients of the formulations are salts, differing from each other in the cation(s) and consequently the molecular mass of the salts, active ingredient concentrations are specified as glyphosate equivalent, in other term acid equivalent (a.i.) referring to the free acid form of glyphosate. This provides instant comparability among various formulations. Moreover, the use of a.i. units is common practice in residue analysis of

Formulated glyphosate-based herbicides contain various non-ionic surfactants to facilitate their uptake by the plants (Riechers et al., 1995). These components, as all other pesticide additives and diluents, are assumed to be inert, which as it turns out, is not the case for several such ingredients. The most common surfactant applied in combination with glyphosate is polyethyloxylated tallowamine (POEA), which itself has been found to exert ecotoxicity, also in synergy with glyphosate, causing the formulated herbicide (e.g., Roundup) more toxic than its technical grade active ingredient (Folmar et al., 1979; Atkinson, 1985; Wan et al., 1989; Powell et al., 1991; Giesy et al., 2000; Tsui & Chu, 2003;

The apparent synergistic toxic effects of the assumedly inert ingredients with glyphosate triggered a legal case between Monsanto and the New York Attorney General's Office in

Marc et al., 2005; Benachour et al., 2007; Benachour & Séralini, 2009).

formulations due to the phytotoxicity of the compound to the crop as well.

species (Haderlie et al., 1978; Egley & Williams, 1978).

**3.1 Formulated glyphosate-based herbicides** 

Syngenta) in 1989 (Tomlin, 2000).

glyphosate as well.

**3.2 Formulating agents** 


a AMM = ammonium; DMA = dimethylamine; IPA = isopropylamine; K = potassium; TRI = trimesium b Formulations containing only glyphosate salts as active ingredient are listed, herbicide combinations are not included

Table 1. Formulated herbicide preparations containing glyphosate as active ingredient.

Forty Years with Glyphosate 255

(part of Sanofi-Aventis) Cotton *GHB614 2mepsps* 

Bayer CropSience

Monsanto Co.

Pioneer Hi-Bred (part of DuPont)

Union.

**Variety owner Crop Genetical event Transgene introduced** <sup>a</sup>

Cotton *MON 1445 cp4 epsps*, *nptII*, *aad*  Cotton *MON 88913 cp4 epsps*  Maize *MON 88017 cp4 epsps*, *cry3Bb1*  Maize *NK603 cp4 epsps*  Rape *GT 73 cp4 epsps*, *gox*  Soybean *MON40-3-2 cp4 epsps*  Soybean *MON 87705 cp4 epsps*, *FAD2-1A*, *FATB1-A*  Soybean *MON 89788 cp4 epsps*  Sugar-beet b *A5-15 cp4 epsps*, *nptII*, Sugar-beet c *H7-1 cp4 epsps* 

Maize *DP-98140 Gat4601*, *als*  Soy *DP-356043 gat4601* 

Syngenta Maize *GA21 mepsps* 

<sup>a</sup> *aad* – gene of *Escherchia coli* origin, encoding resistance against aminoglycoside antibotics (streptomycin and spectinomycin); *als* – gene (*zm-hra*) of maize origin, enhancing tolerance of ALS inhibiting herbicides (e.g., chlorimuron and thifensulfuron); *cry3Bb1* – gene of *Bacillus thuringiensis* origin, encoding Cry3 toxin; *FAD2-1A* – gene of soy origin, encoding fatty acid desaturease enzyme, silencing of which enhances the proportion of monounsaturated fatty acids; *FATB1-A* – gene of soy origin, encoding medium-chain fatty acid thioesterase, silencing of which reduces the proportion of saturated fatty acids; *cp4 epsps* – *epsps* gene of *Agrobacterium* sp.; *mepsps* – *epsps* gene of maize origin; *2mepsps* – double mutated *epsps* gene of Mexican black, sweet maize origin; *gat4601* – gene of *Bacillus lichiformis* origin, encoding glyphosate acetyltransferase enzyme; *gox* – gene of *Ochrobactrum anthropi* origin, encoding glyphosate oxidase enzyme; *nptII* – gene of *Escherichia coli* K12 origin, encoding

Table 2. Glyphosate tolerant crop variety groups under registration process in the European

seven locations, as well as regular or directed post-emergent applications of 10 formulated glyphosate preparations (ClearOut 41 Plus™, Gly Star™, Glyfos®, Glyfos® X-tra, Glyphomax™, Roundup Original™, Roundup UltraMAX®, Roundup WeatherMAX™, Touchdown® and Touchdown Total™) containing isopropylamine or potassium salts of glyphosate found no herbicide efficacy or produce quality differences, no phytotoxicity to maize and medium phytotoxicity to cotton at high doses in some instances, and therefore proposed post-emergent glyphosate applications. As a result, the use of glyphosate has

Another impact of GT crops on agricultural practices is the spread of no-till agriculture. As the crop tolerates the active ingredient, intensive herbicide treatments are possible to be carried out, instead of former tillage practices, to eradicate vegetation in the field. This has greatly increased herbicide use and consequent chemical pressure on the environment. Notill practice is particularly common in GT crop cultivating areas in South America, including

An interesting detail is that in parallel to industrial development of GT crops, illegal genetic modification projects are also being carried out to achieve "crops" that are resistant to

neomycin phosphostransferase, causing neomycin and kanamycin resistance.

expanded almost 20-fold by 2007 in the United States (Pérez et al., 2011).

Brazil, Argentina, Paraguay and Uruguay (Altieri & Pengue, 2006).

b together with Danisco Seeds and DLF Trifolium as variety owners c together with KWS Saat Ag. as variety owners

1996 (Attorney General of the State of New York, 1996). The toxicological basis of the legal claim was that Monsanto inaccurately implied toxicity data of the active ingredient glyphosate on the formulated product Roundup. As a result of the lawsuit, Monsanto was fined, and agreed to drop description of being "environmentally friendly" and "biodegradable" from the advertisements of the herbicide.

Concerns about application safety, triggered by the above studies and findings on teratogenic effects (see 6.3 Teratogenic activity of glyphosate), have brought re-registration of glyphosate and its formulated products in focus in the European Union, as part of the regular pesticide revision process due to take place in 2012. Nonetheless, the EU Commission dismissed these findings, based on a rebuttal by the EU "rapporteur" member state for glyphosate, Germany, provided by the German Federal Office for Consumer Protection and Food Safety (BVL), and postponed the review of glyphosate and 38 other pesticides until 2015 (European Commission, 2010). To protest against such delay in reevaluation of these 39 pesticides, the Pesticides Action Network Europe and Greenpeace brought a lawsuit against the EU Commission, and the dismissal of the reported teratogenicity data from the official current evaluation has been judged by several researchers as irresponsible act (Antoniou et al., 2011).

#### **4. Post-emergent application technology of glyphosate**

A group so far of the highest financial importance within GM crops has been modified to be tolerant to this active ingredient, outstandingly broadening its application possibilities.

#### **4.1 Glyphosate-tolerant crops**

Upon pre-emergent applications of the global herbicide glyphosate, the majority of the weeds decays, perishes, and does not get consumed by wild animals. This situation has been changed tremendously by the appearance of GT crops, leading to increasing environmental herbicide loads due to approved post-emergent treatments (2-3 applications in total). Of these crops, the varieties of Monsanto became most publicised, under the trade mark Roundup Ready® (RR), indicating that these plants can be treated with the herbicide preparation of Monsanto, Roundup® containing glyphosate as active ingredient even, after the emergence of the crop seedlings. Similar varieties by Bayer CropScience, Pioneer Hi-Bred and Syngenta AG are termed Gly-TolTM, Optimum® GAT® and Agrisure® GT, respectively. Two strategies have been followed by plant gene technology in the development of GT varieties: either the genes (*cp4 epsps*, *mepsps*, *2mepsps*) of mutant forms of the target enzyme less sensitive to glyphosate or genes (*gat*, *gox*) of enzymes metabolizing glyphosate have been transferred into the GM plant varieties (Table 2). The genetically created tolerance to glyphosate does not alter the mode of action of the compound: the molecular mechanism of glyphosate tolerance has been elucidated (Funke et al., 2006), and the sole mechanism of inhibition remains blocking of the shikimate pathway when applied at very high doses on GT soybean and canola (Nandula et al., 2007).

The first GT crop was RR soybean by Monsanto in 1996, followed by GT cotton, GT maize, GT canola, GT alfalfa and GT sugarbeet (Dill et al., 2008). GT crops allow a new form of technology, post-emergent application of glyphosate. The utilizability of post-emergent applications was systematically tested in 2002 and 2003 in field experiments in the United States (Parker et al., 2005). The extensive study involving GT maize and GT soybean sites at

1996 (Attorney General of the State of New York, 1996). The toxicological basis of the legal claim was that Monsanto inaccurately implied toxicity data of the active ingredient glyphosate on the formulated product Roundup. As a result of the lawsuit, Monsanto was fined, and agreed to drop description of being "environmentally friendly" and

Concerns about application safety, triggered by the above studies and findings on teratogenic effects (see 6.3 Teratogenic activity of glyphosate), have brought re-registration of glyphosate and its formulated products in focus in the European Union, as part of the regular pesticide revision process due to take place in 2012. Nonetheless, the EU Commission dismissed these findings, based on a rebuttal by the EU "rapporteur" member state for glyphosate, Germany, provided by the German Federal Office for Consumer Protection and Food Safety (BVL), and postponed the review of glyphosate and 38 other pesticides until 2015 (European Commission, 2010). To protest against such delay in reevaluation of these 39 pesticides, the Pesticides Action Network Europe and Greenpeace brought a lawsuit against the EU Commission, and the dismissal of the reported teratogenicity data from the official current evaluation has been judged by several

A group so far of the highest financial importance within GM crops has been modified to be tolerant to this active ingredient, outstandingly broadening its application possibilities.

Upon pre-emergent applications of the global herbicide glyphosate, the majority of the weeds decays, perishes, and does not get consumed by wild animals. This situation has been changed tremendously by the appearance of GT crops, leading to increasing environmental herbicide loads due to approved post-emergent treatments (2-3 applications in total). Of these crops, the varieties of Monsanto became most publicised, under the trade mark Roundup Ready® (RR), indicating that these plants can be treated with the herbicide preparation of Monsanto, Roundup® containing glyphosate as active ingredient even, after the emergence of the crop seedlings. Similar varieties by Bayer CropScience, Pioneer Hi-Bred and Syngenta AG are termed Gly-TolTM, Optimum® GAT® and Agrisure® GT, respectively. Two strategies have been followed by plant gene technology in the development of GT varieties: either the genes (*cp4 epsps*, *mepsps*, *2mepsps*) of mutant forms of the target enzyme less sensitive to glyphosate or genes (*gat*, *gox*) of enzymes metabolizing glyphosate have been transferred into the GM plant varieties (Table 2). The genetically created tolerance to glyphosate does not alter the mode of action of the compound: the molecular mechanism of glyphosate tolerance has been elucidated (Funke et al., 2006), and the sole mechanism of inhibition remains blocking of the shikimate pathway when applied

The first GT crop was RR soybean by Monsanto in 1996, followed by GT cotton, GT maize, GT canola, GT alfalfa and GT sugarbeet (Dill et al., 2008). GT crops allow a new form of technology, post-emergent application of glyphosate. The utilizability of post-emergent applications was systematically tested in 2002 and 2003 in field experiments in the United States (Parker et al., 2005). The extensive study involving GT maize and GT soybean sites at

"biodegradable" from the advertisements of the herbicide.

researchers as irresponsible act (Antoniou et al., 2011).

**4.1 Glyphosate-tolerant crops** 

**4. Post-emergent application technology of glyphosate** 

at very high doses on GT soybean and canola (Nandula et al., 2007).


<sup>a</sup> *aad* – gene of *Escherchia coli* origin, encoding resistance against aminoglycoside antibotics (streptomycin and spectinomycin); *als* – gene (*zm-hra*) of maize origin, enhancing tolerance of ALS inhibiting herbicides (e.g., chlorimuron and thifensulfuron); *cry3Bb1* – gene of *Bacillus thuringiensis* origin, encoding Cry3 toxin; *FAD2-1A* – gene of soy origin, encoding fatty acid desaturease enzyme, silencing of which enhances the proportion of monounsaturated fatty acids; *FATB1-A* – gene of soy origin, encoding medium-chain fatty acid thioesterase, silencing of which reduces the proportion of saturated fatty acids; *cp4 epsps* – *epsps* gene of *Agrobacterium* sp.; *mepsps* – *epsps* gene of maize origin; *2mepsps* – double mutated *epsps* gene of Mexican black, sweet maize origin; *gat4601* – gene of *Bacillus lichiformis* origin, encoding glyphosate acetyltransferase enzyme; *gox* – gene of *Ochrobactrum anthropi* origin, encoding glyphosate oxidase enzyme; *nptII* – gene of *Escherichia coli* K12 origin, encoding neomycin phosphostransferase, causing neomycin and kanamycin resistance.

b together with Danisco Seeds and DLF Trifolium as variety owners c together with KWS Saat Ag. as variety owners

Table 2. Glyphosate tolerant crop variety groups under registration process in the European Union.

seven locations, as well as regular or directed post-emergent applications of 10 formulated glyphosate preparations (ClearOut 41 Plus™, Gly Star™, Glyfos®, Glyfos® X-tra, Glyphomax™, Roundup Original™, Roundup UltraMAX®, Roundup WeatherMAX™, Touchdown® and Touchdown Total™) containing isopropylamine or potassium salts of glyphosate found no herbicide efficacy or produce quality differences, no phytotoxicity to maize and medium phytotoxicity to cotton at high doses in some instances, and therefore proposed post-emergent glyphosate applications. As a result, the use of glyphosate has expanded almost 20-fold by 2007 in the United States (Pérez et al., 2011).

Another impact of GT crops on agricultural practices is the spread of no-till agriculture. As the crop tolerates the active ingredient, intensive herbicide treatments are possible to be carried out, instead of former tillage practices, to eradicate vegetation in the field. This has greatly increased herbicide use and consequent chemical pressure on the environment. Notill practice is particularly common in GT crop cultivating areas in South America, including Brazil, Argentina, Paraguay and Uruguay (Altieri & Pengue, 2006).

An interesting detail is that in parallel to industrial development of GT crops, illegal genetic modification projects are also being carried out to achieve "crops" that are resistant to

Forty Years with Glyphosate 257

Royer et al., 2000; Hudzin et al., 2002). Validated LC methods also resulting in similar analytical parameters (Cowell et al., 1986; Winfield et al., 1990; DFG, 1992) utilise washing with chloroform and hydrochloric acid, purification on ion exchange column, and upon neutralisation and derivatisation with o-phthalic aldehyde and mercaptoethanol, determination by high performance liquid chromatography (HPLC) with fluorescence detection. Yet the LOD of the official method (Method 547) established by the U.S. Environmental Protection Agency is as high as 6 g/l in reagent water and 9 g/l in surface water (Winfield et al., 1990). Ninhydrin or 9-methlyfluorenyl chloroformiate have also been applied as derivatising agents (Wigfield & Lanquette, 1991; Sancho et al., 1996; Nedelkoska & Low, 2004, Peruzzo et al., 2008). More recent LC procedures with somewhat simplified sample preparation steps offer rapid and more economic analytical methods than GC procedures always requiring complex, often several step derivatisation. As a result, GC methods remain being used solely due to their analytical parameters, including sensitivity. Nonetheless, LODs of LC and ion chromatographic methods were achieved to be lowered (Mallat & Barceló, 1998; Vreeken, 1998; Bauer et al., 1999; Grey et al., 2001; Patsias et al., 2001; Lee et al., 2002a; Nedelkoska & Low, 2004; Ibáñez et al., 2006; Laitinen et al., 2006; Hanke et al., 2008; Popp et al., 2008) to meet the strictening maximal residue levels (MRLs) in environmental and health regulations. The most recent LC-MS methods using electrospray ionisation (Granby et al., 2003; Martins-Júnior et al., 2011) easily meet the MRL by the EU for given pesticide residues in drinking water, 0.1 µg/l, but the instrumentation

Among novel innovative analytical methods for the detection of glyphosate, mostly capillary electrophoresis (CE) and immunoanalytical methods are to be mentioned. Initial drawbacks of the CE methods included relatively high LOD and the need for derivatisation or external fluorescent labeling (Cikalo et al., 1996; You et al., 2003; Kodama et al., 2008), later solved by coupling CE with MS (Goodwin et al., 2003) and microextraction techniques (Hsu and Whang 2009; See et al., 2010). Among various immunoanalytical techniques, enzyme-linked immunosorbent assays (ELISAs) gained the highest utility. While in the early nineties we considered yet that effective antibodies are not produced against glyphosate and similar zwitterioninc compounds due to their low immunogeneity (Hammock et al., 1990), difficulties in immunisation have been overcome within a decade, and sensitive ELISAs, also employing derivatisation, were developed (Clegg et al., 1999; Lee et al., 2002b; Rubio et al., 2003; Selvi et al., 2011), proven to be of great utility in environmental analytical studies for glyphosate (Mörtl et al., 2010; Kantiani et al., 2011). On the basis of the immunoassay principle, sensors using glyphosate-sensitive antibodies (González-Martínez et al., 2005) or

molecularly imprinted polymers (MIPs) (Zhao et al., 2011) were also developed.

Decomposition of glyphosate takes place mostly by two processes: decarboxylation or dephosphorylation, and the corresponding intermediate metabolites are AMPA or glycine, respectively. The first pathway is catalyzed by oxidoreductases, the second by C–P lyases cleaving the carbon-phosphorous bond. Both pathways occur in environmental matrices (water, soil) and plants, but the main metabolite in all cases is AMPA (Fig. 2). The environmental fate, behaviour and analysis of both AMPA and glyphosate has received

**5.2 Glyphosate and its decomposition products** 

considerable attention (Stalikas & Konidari, 2001).

demand of these methods is substantial.

glyphosate e.g, a new marijuana (*Canabis* sp.) hybrid that can be cultivated all year and cannot be controled with herbicides (Anonymous, 2006). The GT marijuana hybrid, first appeared in Mexico in 2004, allows 8-9-times higher yields than "conventional" varieties, and became the plant of choice for drug traffickers in Michoacan.

#### **4.2 The effect of glyphosate-tolerant crops on glyphosate residues**

As a result of the combined effect of the expiration of the patent protection of glyphosate (in 2000 in the United States) and the spread of cultivation of GT GM crops (since 1996 in the United States), the use of glyphosate products is again increasing (Woodburn, 2000). Besides GT GM crops, energy crop cultivation is also an and emerging source of glyphosate contamination (Love et al., 2011). Moreover, due to the modified metabolic pool in the GT GM crops, residues of the systemic glyphosate active ingredient are expected to occur in the surviving plants. In case of EPSP-mutant (RR and Agrisure GT) varieties, the residue composition is expected to be similar to those seen at regular glyphosate applications, while in the case of the boosted glyphosate metabolizing (regardless whether *epsps* or *gox* transgene based) varieties, increased amounts of *N*-acetylglyphosate (NAG) (Optimum GAT variety) or aminomethylphosphonic acid (AMPA*)* (RR and Agrisure GT varieties) are expected in the plants. In turn, residue patterns not yet seen in food and feed are to be expected. Summarizing the results of their studies in Argentina between 1997 and 1999, Arregui and co-workers (2004) reported glyphosate residue levels after 2-3 glyphosate applications as high as 0.3-5.2 mg glyphosate/kg and 0.3-5.7 mg AMPA/kg in the leaves and stem of RR soy during harvest, and 0.1-1.8 mg glyphosate/kg and 0.4-0.9 mg AMPA/kg in the produce. In turn, glyphosate occurred as surface water, soil and sediment contaminant in a GM soybean cultivating area in Argentina (Peruzzo et al., 2008).
