**Recent Advances in the Extraction of Triazines from Water Samples**

José A. Rodríguez, Karina Aguilar-Arteaga, Cristina Díez and Enrique Barrado

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54962

### **1. Introduction**

[83] Stewart, P. A, & Knight, A. J. Trends affecting the next generation of U.S. agricultural biotechnology: Politics, policy, and plant-made pharmaceuticals. Technol. Forecast.

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[85] Tammes, T. The genetics of the genus *Linum*. bibl. genet. Bibl. Genet. (1928). , 4, 1-36. [86] Thompson, L. U, Chen, J. M, Li, T, Straaser, W. K, & Goss, P. E. Dietary flaxseed al‐ ters tumor biological markers in post menopausal breast cancer. Clin Cancer Res

[87] Vaisey-genser, M, & Morris, D. H. History of the cultivation and uses of flaxseed. In: Muir,A. D. and N. D. Wescott (eds.), The Genus *Linum*. Taylor and Francis Ltd, New

[88] Vromans, J. Molecular genetic studies in flax (*Linum usitatissimum* L.). Wageningen

[89] Wall, D. A. Fluazifop-P tank-mixtures with clethodim for annual grass control in flax

[90] Warwick, S. I, Beckie, H. J, & Hall, L. M. Gene flow, invasiveness, and ecological im‐ pact of genetically modified crops. Annals of New York Academy of Science (2009). ,

[91] Winkler, H. Linaceae, trib I. linoideae- eulineae. In: Engler,A. (ed.), Die Nature-Li‐ chen Pfanzenfamilien Nebst Ihren Gattungen Und Wichtigeren Arten, Insbesondere

[92] Yermanos, D. M, & Gill, K. S. Induction of polyploidy in *Linum* species. Crop Science

[93] Zhang, W. Q, Linscombe, S. D, Webster, E, Tan, S. Y, & Oard, J. Risk assessment of the transfer of imazethapyr herbicide tolerance from clearfield rice to red rice (*Oryza*

[94] Zhao, G, Etherton, T. D, Martin, K. R, West, S. G, Gillies, P. J, & Etherton, K. M. Diet‐ ary α-linolenic acid reduces inflammatory and lipid cardiovascular risk factors in hy‐ percholesterolemic men and women. Journal of Nutrition (2004). , 134, 2991-2997.

University, Wageningen, The Netherlands, (2006). , 22-39.

(*Linum usitatissimum* L.). Weed Technololy (1994). , 8, 673-678.

Den Nutzpflanzen. W. Engelmann, Leipzig, (1931). , 111-120.

*sativa* L.). Euphytica (2006). , 152, 75-86.

Soc. Change, (2005). , 72, 521-534.

2007;. Accessed:12/ 15, (2009).

York New York, USA, (2003).

(2005). , 11, 3828-3835.

254 Herbicides - Advances in Research

1168, 72-99.

(1967).

The use of herbicides in agriculture has helped to improve crop quality and yield. However, the presence of such substances has also caused serious environmental pollution problems. Triazine herbicides are a group of herbicides applied in agriculture for pre- and post-emer‐ gence weed control. The first report of the use of triazine derivatives was in 1952 by J.R. Geigy from Switzerland but it was not until 1954 that chlorazine was used as a herbicide, followed by simazine in 1955 [1]. During subsequent years the amount of commercially available triazines increased. The main triazine herbicides are derived from s-triazine, a six member heterocycle with symmetrically located atoms in which positions 2, 4 and 6 are substituted. The stereochemical stability of s-triazines is large enough to persist in environmental samples from several months to many years [2].

A list of common *s-*triazines and some of their properties are given in Table 1. The two most common *s-*triazines analyzed in waters are atrazine and simazine. The chemical common name depends on the substituent in position 2 (or R1 in Table 1), when a –Cl group is contained the names end with –azine, while –SCH3 and –OCH3 end with –tryn and –ton, respectively. The thermodynamical properties also depend on the substitutes, the acidity decreases according the following order -OCH3<-SCH3<-Cl and the solubility in water are higher in acidic condi‐ tions. The *s-*triazines which contain –SCH3 group are more polar than the –Cl and –OCH3 compounds according to the partition coefficient between n-octanol and water Kow (log P) [3]. The toxicity of these substances has promoted the development of new analytical methodol‐ ogies to evaluate their impact to the environment and human health.

© 2013 Rodríguez et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Rodríguez et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Maximum residue limits for *s-*triazines in water samples are in the µg l-1 order. This fact demands better quality and accurate analytical methodologies. Moreover, these concentration levels require performing an initial stage of concentration and purification of the analytes prior to their analysis. The analytical procedure usually is comprised of five steps: sampling, sample preparation, separation, detection and data analysis, but sampling and sample preparation are the critical steps of the analytical process. Over 80% of the analysis time is spent on these two steps. If one of these steps is not followed adequately, the performance of the procedure will be affected and the results will be inconsistent [4].

Matrix effects are the main problem in extracting analytes. A matrix effect can be defined as the influence of a property of the sample, independent of the presence of the analyte, on the recovery efficiency. In water samples a pre-concentration step is required prior to measure‐ ment of triazines. A pre-concentration factor of several orders of magnitude (200-1000 fold) is mandatory to reach the low detection limits necessary for identification and analysis of these herbicides, especially in highly organic matter content samples such as wastewaters. The common extraction methods used for isolation of polar compounds from water matrices are

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LLE has been used in the past for the extraction of triazines from environmental water samples [5]. LLE is based on the partition coefficient of the analytes between two liquid phases of low solubility. In the case of triazines the common sample volume used is 1 l (pH adjusted to 7) and it is mixed with an organic solvent, such as methylene chloride (at least 2x50 ml). The aqueous layer is then discarded, the organic phase evaporated and concentrated to a volume of 5 ml and the solvent is exchanged. The obtained extract is analyzed by a separation technique (gas or liquid chromatography). The main disadvantages of this procedure are: the use of large volumes of organic solvents, limited pre-concentration factors and tedious procedures.

In recent years, the scientific community has shown an increased interest in the development of environmentally friendly laboratory activities. Green analytical chemistry pursues the aim of replacing toxic reagents by clean ones. Also the development and improvement of new sample preparation techniques is a fast growing trend in analytical chemistry. In this context, liquid phase microextraction techniques have evolved from the use of tens of ml of solvent to the use of drop-based (µl) systems. The different approaches employed for the liquid micro‐ extraction of triazine herbicides from environmental matrices are mainly: hollow fiber liquid phase microextraction (HFLME) and dispersive liquid-liquid microextraction (DLLME).

HFLME is a membrane based separation technique. It can be sub-classified into two-phase and three-phase systems. The two-phase system is the most used system in the extraction of trazines from aqueous samples. The two-phase systems are also referred as microporous membrane extraction. It is comprised of an aqueous phase and a hydrophobic porous mem‐ brane impregnated with a suitable organic solvent (Figure 1). The aqueous phase usually contains the analyte and it is called the donor phase while the organic solvent is the receiving/

The extraction process involves partitioning of the analyte from the aqueous sample into the organic solvent which impregnates the hollow fiber (HF) and the diffusion through the membrane into the bulk receptor/acceptor phase. These systems are suitable for extraction of compounds with large partitioning coefficients in the organic phase. Polypropylene is the

material commonly used for triazines using two-phase HFLME systems.

the liquid-liquid extraction (LLE) and the solid phase extraction (SPE).

**2.1. Liquid-liquid extraction**

*2.1.1. Hollow fiber liquid phase microextraction*

acceptor phase [6].


**Table 1.** pKa and Kow (log P) of common *s-*triazines herbicides

Adequate sample preparation is a requisite for analytical techniques. Analysts have responded to this challenge, so in this work recent sample extraction techniques for analysis of *s-*triazines in water samples are overviewed.

### **2. Sample preparation**

The sample preparation concept is based on converting a real matrix into a sample suitable for analysis. This process involves a change in the chemical environment of the sample. An initial step in the design of an extraction method is the knowledge of the physical and chemical properties such as lipophilicity and the predominance of acid-basic species neutral or ionic.

Matrix effects are the main problem in extracting analytes. A matrix effect can be defined as the influence of a property of the sample, independent of the presence of the analyte, on the recovery efficiency. In water samples a pre-concentration step is required prior to measure‐ ment of triazines. A pre-concentration factor of several orders of magnitude (200-1000 fold) is mandatory to reach the low detection limits necessary for identification and analysis of these herbicides, especially in highly organic matter content samples such as wastewaters. The common extraction methods used for isolation of polar compounds from water matrices are the liquid-liquid extraction (LLE) and the solid phase extraction (SPE).

### **2.1. Liquid-liquid extraction**

Maximum residue limits for *s-*triazines in water samples are in the µg l-1 order. This fact demands better quality and accurate analytical methodologies. Moreover, these concentration levels require performing an initial stage of concentration and purification of the analytes prior to their analysis. The analytical procedure usually is comprised of five steps: sampling, sample preparation, separation, detection and data analysis, but sampling and sample preparation are the critical steps of the analytical process. Over 80% of the analysis time is spent on these two steps. If one of these steps is not followed adequately, the performance of the procedure will

**Compound R1 R2 R3 pKa Kow (log P)**

N

N H

N

N H N

Adequate sample preparation is a requisite for analytical techniques. Analysts have responded to this challenge, so in this work recent sample extraction techniques for analysis of *s-*triazines

The sample preparation concept is based on converting a real matrix into a sample suitable for analysis. This process involves a change in the chemical environment of the sample. An initial step in the design of an extraction method is the knowledge of the physical and chemical properties such as lipophilicity and the predominance of acid-basic species neutral or ionic.

Atrazine Cl C2H5 CH(CH3)2 1.68 2.7 Propazine Cl CH(CH3)2 CH(CH3)2 1.85 2.9 Simazine Cl C2H5 C2H5 1.65 2.3 Terbutylazine Cl C2H5 C(CH3)3 1.88 3.1 Terbutemon OCH3 C2H5 C(CH3)3 4.20 3.6 Ametryn SCH3 C2H5 CH(CH3)2 4.00 3.1 Prometryn SCH3 CH(CH3)2 CH(CH3)2 4.05 3.3 Simetryn SCH3 C2H5 C2H5 4.00 2.8 Terbutryn SCH3 C2H5 C(CH3)3 4.40 3.7

R3 R2

R1

be affected and the results will be inconsistent [4].

256 Herbicides - Advances in Research

**Table 1.** pKa and Kow (log P) of common *s-*triazines herbicides

in water samples are overviewed.

**2. Sample preparation**

LLE has been used in the past for the extraction of triazines from environmental water samples [5]. LLE is based on the partition coefficient of the analytes between two liquid phases of low solubility. In the case of triazines the common sample volume used is 1 l (pH adjusted to 7) and it is mixed with an organic solvent, such as methylene chloride (at least 2x50 ml). The aqueous layer is then discarded, the organic phase evaporated and concentrated to a volume of 5 ml and the solvent is exchanged. The obtained extract is analyzed by a separation technique (gas or liquid chromatography). The main disadvantages of this procedure are: the use of large volumes of organic solvents, limited pre-concentration factors and tedious procedures.

In recent years, the scientific community has shown an increased interest in the development of environmentally friendly laboratory activities. Green analytical chemistry pursues the aim of replacing toxic reagents by clean ones. Also the development and improvement of new sample preparation techniques is a fast growing trend in analytical chemistry. In this context, liquid phase microextraction techniques have evolved from the use of tens of ml of solvent to the use of drop-based (µl) systems. The different approaches employed for the liquid micro‐ extraction of triazine herbicides from environmental matrices are mainly: hollow fiber liquid phase microextraction (HFLME) and dispersive liquid-liquid microextraction (DLLME).

### *2.1.1. Hollow fiber liquid phase microextraction*

HFLME is a membrane based separation technique. It can be sub-classified into two-phase and three-phase systems. The two-phase system is the most used system in the extraction of trazines from aqueous samples. The two-phase systems are also referred as microporous membrane extraction. It is comprised of an aqueous phase and a hydrophobic porous mem‐ brane impregnated with a suitable organic solvent (Figure 1). The aqueous phase usually contains the analyte and it is called the donor phase while the organic solvent is the receiving/ acceptor phase [6].

The extraction process involves partitioning of the analyte from the aqueous sample into the organic solvent which impregnates the hollow fiber (HF) and the diffusion through the membrane into the bulk receptor/acceptor phase. These systems are suitable for extraction of compounds with large partitioning coefficients in the organic phase. Polypropylene is the material commonly used for triazines using two-phase HFLME systems.

Following the new tendencies, a polypropylene HF (3.0 cm X 0.6 mm i.d.) was impregnated with a suspension composed by n-octanol and multiwalled carbon nanotubes, intended for simazine, simetryn, propazine, and prometryn isolation. The HF was immersed into the sample (15.0 ml) containing 30 µl of chlorobenzene and 2.25 g of NaCl for 20 minutes. Then, the HF was washed with water and immersed in 50 µl of methanol for elution of the analytes. The analysis of the methanolic solution by HPLC with ultraviolet detection (HPLC-UV) gives

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DLLME is a miniaturized LLE technique based on a ternary component solvent system composed of a certain amount of the sample, a disperser solvent and an extraction solvent. The extraction steps involved on the DLLME are (Figure 2): a) a volume of the sample is placed in a tube with conic bottom, b) the disperser and extraction samples are injected into the sample, c) the mixture is then mixed and a cloudy solution is formed in the test tube. A higher contact area between the organic-aqueous phases is obtained due to the formation and dispersion of micro-drops of organic phase. Subsequently, equilibrium state is achieved quickly, resulting in a reduction in the extraction time. The final step is the centrifugation and depending on the density of the extraction solvent, d) it sediments at the bottom of the test tube or e) floats at the top of the solution. Finally, a definite volume of the pre-concentrated

**Centrifugation** 

**(d)**

**(e)**

a LOD of in the range of 0.08-0.15 µg l-1 [9].

*2.1.2. Dispersive liquid-liquid microextraction*

sample is recovered and analyzed.

**(a) (b) (c)**

**Figure 2.** Representation of the dispersive liquid-liquid microextraction methodology

Chlorobenzene is a suitable extraction solvent for triazine isolation, and it was applied for DLLME of atrazine, simazine, prometryn, propazine and simetryn from water samples. The

**Figure 1.** Representation of a hollow fiber liquid phase microextraction system. (a) donor phase, (b) hollow fiber with organic phase, (c) acceptor phase.

The common steps included on the HFLME are: a) cleaning of the HF, b) conditioning of the HF impregnating it with the extraction solvent, c) adding a specific volume of the solvent into the HF, d) immersing the HF into the sample for a definite time, e) aspiring the preconcentrated sample for its analysis.

A system based on the use a polypropylene HF (1.5 cm Χ 0.6 mm i.d.) containing 3 µl of toluene as organic solvent was used for the extraction of simazine, atrazine, propazine, simetryn and prometryn from 3.0 ml of water samples. The organic phase was analyzed by gas chromatog‐ raphy-mass spectrometry (GC-MS). The effect of salt addition, agitation, pH and exposure time were evaluated. The most critical variable was the pH value required (>4.0) which determines the formation of the suitable extractable analyte form. The method described provides good enrichment factors (<150), good precision (<3.5%, expressed as relative standard deviation, RSD) with limits of detection (LODs) in the range of 0.007-0.063 µg l-1[7].

The use of phosphorus-oxygen compounds as co-extraction solvents has been proposed for isolation of pesticides from water samples including triazine herbicides. Atriazine, simazine and propazine were extracted using a polypropylene HF (3.3 cm Χ 0.3 mm i.d.) filled with a *n-*dihexylether solution containing 10% of tri-*n-*octylphosphine oxide ((C8H17)3PO) and 10% of tri-n-butylphosphonate ((C4H9O)3PO). The dipolar moment from the P-O bond increases the polarity of the extraction solvent, allowing the isolation of the triazines and the other pesticides evaluated. The extraction was optimal when the donor pH was fixed to 8.0, using the organic phase, above mentioned and a contact time of 4 h in a 250 ml of a water sample. The system was coupled to high performance liquid chromatography- mass spectrometry (HPLC-MS) as separation and detection technique. Under these conditions LODs from 0.061 to 0.26 were obtained for triazine compounds [8].

Over the past decades, carbon nanotubes have elicited interest due to their chemical and physical properties. At the nanoscale, an increase of the contact surface area is observed. Following the new tendencies, a polypropylene HF (3.0 cm X 0.6 mm i.d.) was impregnated with a suspension composed by n-octanol and multiwalled carbon nanotubes, intended for simazine, simetryn, propazine, and prometryn isolation. The HF was immersed into the sample (15.0 ml) containing 30 µl of chlorobenzene and 2.25 g of NaCl for 20 minutes. Then, the HF was washed with water and immersed in 50 µl of methanol for elution of the analytes. The analysis of the methanolic solution by HPLC with ultraviolet detection (HPLC-UV) gives a LOD of in the range of 0.08-0.15 µg l-1 [9].

### *2.1.2. Dispersive liquid-liquid microextraction*

The common steps included on the HFLME are: a) cleaning of the HF, b) conditioning of the HF impregnating it with the extraction solvent, c) adding a specific volume of the solvent into the HF, d) immersing the HF into the sample for a definite time, e) aspiring the preconcentrated

**Figure 1.** Representation of a hollow fiber liquid phase microextraction system. (a) donor phase, (b) hollow fiber with

A system based on the use a polypropylene HF (1.5 cm Χ 0.6 mm i.d.) containing 3 µl of toluene as organic solvent was used for the extraction of simazine, atrazine, propazine, simetryn and prometryn from 3.0 ml of water samples. The organic phase was analyzed by gas chromatog‐ raphy-mass spectrometry (GC-MS). The effect of salt addition, agitation, pH and exposure time were evaluated. The most critical variable was the pH value required (>4.0) which determines the formation of the suitable extractable analyte form. The method described provides good enrichment factors (<150), good precision (<3.5%, expressed as relative standard deviation,

The use of phosphorus-oxygen compounds as co-extraction solvents has been proposed for isolation of pesticides from water samples including triazine herbicides. Atriazine, simazine and propazine were extracted using a polypropylene HF (3.3 cm Χ 0.3 mm i.d.) filled with a *n-*dihexylether solution containing 10% of tri-*n-*octylphosphine oxide ((C8H17)3PO) and 10% of tri-n-butylphosphonate ((C4H9O)3PO). The dipolar moment from the P-O bond increases the polarity of the extraction solvent, allowing the isolation of the triazines and the other pesticides evaluated. The extraction was optimal when the donor pH was fixed to 8.0, using the organic phase, above mentioned and a contact time of 4 h in a 250 ml of a water sample. The system was coupled to high performance liquid chromatography- mass spectrometry (HPLC-MS) as separation and detection technique. Under these conditions LODs from 0.061 to 0.26 were

Over the past decades, carbon nanotubes have elicited interest due to their chemical and physical properties. At the nanoscale, an increase of the contact surface area is observed.

RSD) with limits of detection (LODs) in the range of 0.007-0.063 µg l-1[7].

sample for its analysis.

organic phase, (c) acceptor phase.

258 Herbicides - Advances in Research

(a) (b) (c)

obtained for triazine compounds [8].

DLLME is a miniaturized LLE technique based on a ternary component solvent system composed of a certain amount of the sample, a disperser solvent and an extraction solvent. The extraction steps involved on the DLLME are (Figure 2): a) a volume of the sample is placed in a tube with conic bottom, b) the disperser and extraction samples are injected into the sample, c) the mixture is then mixed and a cloudy solution is formed in the test tube. A higher contact area between the organic-aqueous phases is obtained due to the formation and dispersion of micro-drops of organic phase. Subsequently, equilibrium state is achieved quickly, resulting in a reduction in the extraction time. The final step is the centrifugation and depending on the density of the extraction solvent, d) it sediments at the bottom of the test tube or e) floats at the top of the solution. Finally, a definite volume of the pre-concentrated sample is recovered and analyzed.

**Figure 2.** Representation of the dispersive liquid-liquid microextraction methodology

Chlorobenzene is a suitable extraction solvent for triazine isolation, and it was applied for DLLME of atrazine, simazine, prometryn, propazine and simetryn from water samples. The methodology uses 5.0 ml of water samples containing 4% (w/v) of sodium chloride mixed with 12.0 µl of chlorobenzene and 1.0 ml of acetone (disperser solvent). The mixture was centrifuged and a 2 µl sample was analyzed by GC-MS. The method proposed has a LODs between 0.021 and 0.12 µg l-1 with a precision <5%, expressed as RSD [10].

**2.2. Solid phase extraction**

precisions < 10% [16].

Solid phase extraction (SPE) is the main separation technique used for trace enrichment of triazines from aqueous samples. The use of cartridges or disk forms, allows a high degree of flexibility. In the last years, there has been a considerable interest in designing new selective and sensitive stationary phases for extracting triazine compounds. Selectivity is related with the extraction mechanism used during isolation of the analytes. The most important interac‐

Si

R

SPE using polar solid phases (Fig 3.a) has been applied for isolation of atrazine, ametryn, prometryn, terbuthylazine, terbutryn and simazine. Initially, a solution of 10 ml of water sample, 50 ml of acetonitrile and 10 g of NaCl was prepared. Then a 25 ml portion of the mixture was evaporated to dryness in a vacumm evaporator at 50 ºC. The residue was dissolved in hexane and it was then subjected to SPE clean-up with Florisil cartridges using hexane and acetone/hexane (80:20) as condition and elution solvents. The sample extract was analyzed by HPLC-MS, achieving limits of quantification (LOQs) in the range of 0.02-0.05 mg l-1 with

SPE, based on C18 and polymeric phases (Fig 3.b and c), has been widely used for determi‐ nation of triazine in water samples [17-23]. The common amount of water sample passing through the cartridge is 100 ml, followed by a drying step. Triazine compounds are eluted with a few ml of a solvent such as acetonitrile, ethyl acetate or methanol. The LODs achieved depend

R

SO3 <sup>R</sup> - <sup>+</sup>

N O R2

Non-polar interaction

Polar interaction

R1

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261

N

H O

Si <sup>R</sup>

**(e) Solid phase**

H

tions between the solid phase and the analytes are represented in Figure 3.

**(a)**

**(b)**

**(c)**

**(d)**

**(f)**

**Figure 3.** Representative interactions mode between the solid phase and the analytes during SPE

The search for new extraction solvents is a key trend in the solvent extraction evolution. In this sense, ionic liquid, which is an ionic media resulting from the combination of organic cations with various anions has attracted attention for its special features such as: low-vapour pressure, high viscosity, dual polarity and a wide range of miscibility with water and other organic solvents [11].

The ionic liquid 1-hexyl-3-methylimidazolium hexafluorophosphate has been evaluated as extraction solvent of atrazine, prometryn, and simazine. The proposed methodology prepared a solution mixing water sample (10 ml) and the ionic liquid (60 µl) in a conical test tube. The test tube was heated in water bath at 70 °C for 5 min and was thereafter cooled with ice for 30 min until the solution became turbid. The dispersion was centrifuged for 10 min at 3800 rpm, the upper aqueous phase was removed and the ionic liquid phase was dissolved in 100 µl of methanol for HPLC-UV analysis. Under optimal condition the LODs of the reported method are in the range from 0.46 to 0.89 µg l-1, with precisions below 10% (RSD) [12].

Following the same tendency, a DLLME method coupled to microwave assisted extraction (MAE) was design for extracting ametryne, prometryne, and terbutryn. A 10 ml microwave tube was filled with 5.0 ml of water sample. Then, 40 µl of 1-butyl-3- methylimidazolium tetrafluoroborate and 500 µl of a 0.2 g ml-1 disperser solution of lithium bis[(tri-fluorome‐ thane)sulfonyl]imide were added. The suspension was irradiated under microwave power of 30 W during 90 seconds. After cooling, the suspension was centrifuged at 5000 rpm for 6 minutes. The aqueous phase was removed and the ionic liquid phase was stored for HPLC-UV analysis. The LODs were between 0.52 and 1.30 µg l-1 with precisions <10%, as RSD [13].

In the case of DLLME using extraction solvents with lower density than water, there has been reported the use of DLLME based on the solidification of a floating organic droplet for analysis of atrazine and simazine in water samples. The conditions proposed for the sample treatment are: 10 µl of 1-undecanol (ρ=0.83 g ml-1) as extraction solvent, 100 µl of acetonitrile as disperser solvent, NaCl 5% (w/v) and 5ml of water sample. The mixture was then centrifuged for 3 min at 400 rpm and then transferred into an ice bath. After 5 minutes the extraction solvent solidified and was transferred into a clean conical tube, where it melts quickly at room temperature. The extract was then analyzed by GC-MS. The LODs reported were in the range of 0.52-1.30 µg l-1 with precisions <5.0%, as RSD [14].

Simultaneous DLLME and microwave assisted extraction was also applied in the analysis of cereal samples. The method involved the use of 1-dodecanol, methanol and water in order to extract the solid sample. Although the technique reported does not involve the analysis of water samples, it is an interesting example of coupled techniques which generates a dynamic and simple methodology for extraction of triazines from complex samples [15].

### **2.2. Solid phase extraction**

methodology uses 5.0 ml of water samples containing 4% (w/v) of sodium chloride mixed with 12.0 µl of chlorobenzene and 1.0 ml of acetone (disperser solvent). The mixture was centrifuged and a 2 µl sample was analyzed by GC-MS. The method proposed has a LODs between 0.021

The search for new extraction solvents is a key trend in the solvent extraction evolution. In this sense, ionic liquid, which is an ionic media resulting from the combination of organic cations with various anions has attracted attention for its special features such as: low-vapour pressure, high viscosity, dual polarity and a wide range of miscibility with water and other

The ionic liquid 1-hexyl-3-methylimidazolium hexafluorophosphate has been evaluated as extraction solvent of atrazine, prometryn, and simazine. The proposed methodology prepared a solution mixing water sample (10 ml) and the ionic liquid (60 µl) in a conical test tube. The test tube was heated in water bath at 70 °C for 5 min and was thereafter cooled with ice for 30 min until the solution became turbid. The dispersion was centrifuged for 10 min at 3800 rpm, the upper aqueous phase was removed and the ionic liquid phase was dissolved in 100 µl of methanol for HPLC-UV analysis. Under optimal condition the LODs of the reported method

Following the same tendency, a DLLME method coupled to microwave assisted extraction (MAE) was design for extracting ametryne, prometryne, and terbutryn. A 10 ml microwave tube was filled with 5.0 ml of water sample. Then, 40 µl of 1-butyl-3- methylimidazolium tetrafluoroborate and 500 µl of a 0.2 g ml-1 disperser solution of lithium bis[(tri-fluorome‐ thane)sulfonyl]imide were added. The suspension was irradiated under microwave power of 30 W during 90 seconds. After cooling, the suspension was centrifuged at 5000 rpm for 6 minutes. The aqueous phase was removed and the ionic liquid phase was stored for HPLC-UV analysis. The LODs were between 0.52 and 1.30 µg l-1 with precisions <10%, as RSD [13].

In the case of DLLME using extraction solvents with lower density than water, there has been reported the use of DLLME based on the solidification of a floating organic droplet for analysis of atrazine and simazine in water samples. The conditions proposed for the sample treatment are: 10 µl of 1-undecanol (ρ=0.83 g ml-1) as extraction solvent, 100 µl of acetonitrile as disperser solvent, NaCl 5% (w/v) and 5ml of water sample. The mixture was then centrifuged for 3 min at 400 rpm and then transferred into an ice bath. After 5 minutes the extraction solvent solidified and was transferred into a clean conical tube, where it melts quickly at room temperature. The extract was then analyzed by GC-MS. The LODs reported were in the range

Simultaneous DLLME and microwave assisted extraction was also applied in the analysis of cereal samples. The method involved the use of 1-dodecanol, methanol and water in order to extract the solid sample. Although the technique reported does not involve the analysis of water samples, it is an interesting example of coupled techniques which generates a dynamic

and simple methodology for extraction of triazines from complex samples [15].

are in the range from 0.46 to 0.89 µg l-1, with precisions below 10% (RSD) [12].

and 0.12 µg l-1 with a precision <5%, expressed as RSD [10].

of 0.52-1.30 µg l-1 with precisions <5.0%, as RSD [14].

organic solvents [11].

260 Herbicides - Advances in Research

Solid phase extraction (SPE) is the main separation technique used for trace enrichment of triazines from aqueous samples. The use of cartridges or disk forms, allows a high degree of flexibility. In the last years, there has been a considerable interest in designing new selective and sensitive stationary phases for extracting triazine compounds. Selectivity is related with the extraction mechanism used during isolation of the analytes. The most important interac‐ tions between the solid phase and the analytes are represented in Figure 3.

**Figure 3.** Representative interactions mode between the solid phase and the analytes during SPE

SPE using polar solid phases (Fig 3.a) has been applied for isolation of atrazine, ametryn, prometryn, terbuthylazine, terbutryn and simazine. Initially, a solution of 10 ml of water sample, 50 ml of acetonitrile and 10 g of NaCl was prepared. Then a 25 ml portion of the mixture was evaporated to dryness in a vacumm evaporator at 50 ºC. The residue was dissolved in hexane and it was then subjected to SPE clean-up with Florisil cartridges using hexane and acetone/hexane (80:20) as condition and elution solvents. The sample extract was analyzed by HPLC-MS, achieving limits of quantification (LOQs) in the range of 0.02-0.05 mg l-1 with precisions < 10% [16].

SPE, based on C18 and polymeric phases (Fig 3.b and c), has been widely used for determi‐ nation of triazine in water samples [17-23]. The common amount of water sample passing through the cartridge is 100 ml, followed by a drying step. Triazine compounds are eluted with a few ml of a solvent such as acetonitrile, ethyl acetate or methanol. The LODs achieved depend on the type of separation technique (GC or HPLC) and the detector used (MS, UV, etc.). In some cases it is reported limits in the ng l-1 when sensitive detectors are used.

Strong cation exchange SPE (SCX-SPE, Fig 3.d) has been proposed for ametryn, atrazine, propazine, prometryn, simazine, simetryn and terbutryn isolation. The positive charge of the acid form of triazines allows the isolation of the analytes. Elution was performed by adding a 0.07 M KCl aqueous-methanolic solution. The obtained extract was analyzed by HLPC-UV. The LODs reported using spikes water samples was 0.01 µgl-1 [24].

In order to increase the selectivity of extraction, the use of immunosorbents was reported (Fig. 3.e). The production of polyclonal antibodies was done by immunization of rabbits with caprolyl-atrazine. The immunosorbent was applied in the analysis of ametryn, atrazine, propazine, prometryn, simazine, simetryn and terbutryn in water samples. The system could be used in off- or on-line modes. The pre-concentration of 50 ml water samples and the use of methanol 70% (v/v) as elution solvent prior to HPLC-UV analysis provided LODs of 1-2 µg l -1 [25-26].

A highly specific method for atrazine isolation was developed using a moleculary imprinted polymer (MIP, Fig 3.f). The MIP was synthesized using atrazine : methacrylic acid: ethylene glycol dimethyl methacrylate in a molar ratio of 1:4:20. The polymer exhibited a high selectivity to atrazine isolation, achieving LOD of 0.5 µg l-1 when it is coupled to HPLC-UV [27].

Multiwalled carbon nanotubes have been evaluated as adsorbent for atrazine and simazine isolation. The atrazines were retained on the solid phase in their neutral form and they were eluted from the solid using acetonitrile or acetone. The methodology was tested using different geometries (disk and cartridge) and volumes (200 and 500 ml) and also different detection methods (GC-MS and HPLC-DAD). The LODs were in the µg l-1 order [28-29].

and inter-day below 10 and 20%, respectively. The absence of organic solvent during sample

to GC column

**(d)**

to HPLC injector

**(c)**

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Carbon solids are one of the most important adsorption materials since they exhibit high isolation capacity for organic compounds. Graphene is a novel carbon material with large delocalized π-electron system that can form strong π-stacking interaction with the aromatic ring presented in the triazine structure. Atrazine, ametryn, prometron and prometryn were extracted using iron fibers coated with graphene. The fiber was immersed into 10 ml water sample solution for 30 min with stirring at 950 rpm. Afterwards, the extracted analytes on the SPME fiber were desorbed with 50 µl of acetone. The extract was then analyzed by HPLC with diode array detection (HPLC-DAD). The LODs of the method were in the range of 0.05-0.2 µg

SBSE is a relatively new technique. It has been used with success for the extraction of organic compounds from aqueous, food, biological and environmental samples. In SBSE the sample is stirred for a given time with a stir bar coated with a sorbent (Fig. 5.a), until the analyte reaches equilibrium between the polymer and the aqueous phase according to their distribution constant. The sorbed analytes are then desorbed by high temperatures into the injector port of the GC (Fig. 5b) or by liquid removal for HPLC analysis (Fig. 5.c). The main disadvantage of

preparation was the main advantage of the proposed method [30].

**Figure 4.** Representation of the solid phase microextraction methodology

**(a) (b)**

SBSE is the high extraction time required during sample treatment.

l


*2.2.2. Stir bar sorptive extraction*

During the last decades, different techniques have been proposed to improve the SPE. Extraction of triazines has been usually carried out by solid phase microextraction (SPME), stir bar sorptive extraction (SBSE) and dispersive solid phase extraction (DSPE).

### *2.2.1. Solid phase microextraction*

SPME (Figure 4) has become popular for the analysis of organic compounds because it combines sampling and pre-concentration in a single step. In this technique a fused silica fiber coated with a polymeric film is immersed into the sample (Fig. 4.a and b). The analytes are adsorbed into the stationary phase and later desorbed for its ulterior analysis (Fig. 4.c and d). SPME has the following advantages: (i) the extraction time is reduced, (ii) it provides good results over a wide range of analyte concentrations and (iii) it can be easily automated. Obviously, the composition of the fibers is a great importance in this methodology.

Atrazine, simazine, terbuthylazine and terbutryn have been extracted from water and soil samples using SPME with a carbowax-divynilbenzene fiber. Extraction was carried out by direct immersion of the fiber into the sample (3.0 ml) containing 10% of NaCl to adjust the ionic force. The mixture was stirred for 30 min and desorption of the herbicides was carried out at 240 ºC in the hot GC-MS injector. The LODs were below 0.1% µg l-1 with precision intra-

**Figure 4.** Representation of the solid phase microextraction methodology

and inter-day below 10 and 20%, respectively. The absence of organic solvent during sample preparation was the main advantage of the proposed method [30].

Carbon solids are one of the most important adsorption materials since they exhibit high isolation capacity for organic compounds. Graphene is a novel carbon material with large delocalized π-electron system that can form strong π-stacking interaction with the aromatic ring presented in the triazine structure. Atrazine, ametryn, prometron and prometryn were extracted using iron fibers coated with graphene. The fiber was immersed into 10 ml water sample solution for 30 min with stirring at 950 rpm. Afterwards, the extracted analytes on the SPME fiber were desorbed with 50 µl of acetone. The extract was then analyzed by HPLC with diode array detection (HPLC-DAD). The LODs of the method were in the range of 0.05-0.2 µg l -1 with precision <5%, as RSD [31].

#### *2.2.2. Stir bar sorptive extraction*

on the type of separation technique (GC or HPLC) and the detector used (MS, UV, etc.). In

Strong cation exchange SPE (SCX-SPE, Fig 3.d) has been proposed for ametryn, atrazine, propazine, prometryn, simazine, simetryn and terbutryn isolation. The positive charge of the acid form of triazines allows the isolation of the analytes. Elution was performed by adding a 0.07 M KCl aqueous-methanolic solution. The obtained extract was analyzed by HLPC-UV.

In order to increase the selectivity of extraction, the use of immunosorbents was reported (Fig. 3.e). The production of polyclonal antibodies was done by immunization of rabbits with caprolyl-atrazine. The immunosorbent was applied in the analysis of ametryn, atrazine, propazine, prometryn, simazine, simetryn and terbutryn in water samples. The system could be used in off- or on-line modes. The pre-concentration of 50 ml water samples and the use of methanol 70% (v/v) as elution solvent prior to HPLC-UV analysis provided LODs of 1-2 µg

A highly specific method for atrazine isolation was developed using a moleculary imprinted polymer (MIP, Fig 3.f). The MIP was synthesized using atrazine : methacrylic acid: ethylene glycol dimethyl methacrylate in a molar ratio of 1:4:20. The polymer exhibited a high selectivity

Multiwalled carbon nanotubes have been evaluated as adsorbent for atrazine and simazine isolation. The atrazines were retained on the solid phase in their neutral form and they were eluted from the solid using acetonitrile or acetone. The methodology was tested using different geometries (disk and cartridge) and volumes (200 and 500 ml) and also different detection

During the last decades, different techniques have been proposed to improve the SPE. Extraction of triazines has been usually carried out by solid phase microextraction (SPME), stir

SPME (Figure 4) has become popular for the analysis of organic compounds because it combines sampling and pre-concentration in a single step. In this technique a fused silica fiber coated with a polymeric film is immersed into the sample (Fig. 4.a and b). The analytes are adsorbed into the stationary phase and later desorbed for its ulterior analysis (Fig. 4.c and d). SPME has the following advantages: (i) the extraction time is reduced, (ii) it provides good results over a wide range of analyte concentrations and (iii) it can be easily automated.

Atrazine, simazine, terbuthylazine and terbutryn have been extracted from water and soil samples using SPME with a carbowax-divynilbenzene fiber. Extraction was carried out by direct immersion of the fiber into the sample (3.0 ml) containing 10% of NaCl to adjust the ionic force. The mixture was stirred for 30 min and desorption of the herbicides was carried out at 240 ºC in the hot GC-MS injector. The LODs were below 0.1% µg l-1 with precision intra-

Obviously, the composition of the fibers is a great importance in this methodology.

to atrazine isolation, achieving LOD of 0.5 µg l-1 when it is coupled to HPLC-UV [27].

methods (GC-MS and HPLC-DAD). The LODs were in the µg l-1 order [28-29].

bar sorptive extraction (SBSE) and dispersive solid phase extraction (DSPE).

some cases it is reported limits in the ng l-1 when sensitive detectors are used.

The LODs reported using spikes water samples was 0.01 µgl-1 [24].

l


262 Herbicides - Advances in Research

*2.2.1. Solid phase microextraction*

SBSE is a relatively new technique. It has been used with success for the extraction of organic compounds from aqueous, food, biological and environmental samples. In SBSE the sample is stirred for a given time with a stir bar coated with a sorbent (Fig. 5.a), until the analyte reaches equilibrium between the polymer and the aqueous phase according to their distribution constant. The sorbed analytes are then desorbed by high temperatures into the injector port of the GC (Fig. 5b) or by liquid removal for HPLC analysis (Fig. 5.c). The main disadvantage of SBSE is the high extraction time required during sample treatment.

**Figure 5.** Representation of the solid phase microextraction methodology

A stir bar of 10 mm length and 0.5 mm polydimethylsiloxane was used to extract ten triazines from water samples. The SBSE step was carried out by introducing the stir bar into a vial containing 20 ml of the sample, 30% of NaCl and stirred (31.4 s-1) during 60 min. The stir bar was washed with water and then thermally desorbed and analyzed by GC-MS. The LODs obtained were in the range from 0.2 to 3.4 ng l-1 [32].

The use of thermal desorption requires a cold trap during elution process. The hyphenation of SBSE with HPLC-DAD using solvent desorption was applied to atrazine, simazine and terbuthylazine in environmental water samples. A polyethylene bar impregnated with activated carbon (15 mm length and 0.5 mm thickness) was used to extract 10 ml of water samples. The extraction time required was 16 h, followed by desorption of the analytes using acetonitrile as solvent. The reported LODs were around 0.1 µg l-1 with precision <15%, as RSD. This method was an alternative to the analysis of analytes with polar characteristics [33].

in 5 ml of sample solution for 1 hour. Once the extraction was concluded, the solid phase was collected in a membrane filter. The solid was washed with methanol and then the atrazine was eluted with 5 ml of desorption solvent (methanol/acetic acid, 9:1 v/v). The extract was dried and redissolved in acetonitrile before its analysis by HPLC-UV. The LOD reported was 2.8 µg

**(a) (b) (c)**

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**Figure 6.** Representation of the dispersive solid phase extraction methodology

Recovery of the solid phase in DSPE requires the use of filtration or centrifugation techniques that may lead to a solid phase loss and the subsequent decrease in precision and accuracy. The use of magnetic solids is an alternative for the selective preconcentration of different chemical species. It offers adequate surface area, the possibility of functionalization and paramagnetic properties. Their application as dispersed sorbents in liquid samples is so-called magnetic solid phase extraction (MSPE, Fig. 7). This technique has demonstrated several advantages such as: the decrease in sample treatment time, the decrease in solvent use, and the easy treatment of high volume samples. MSPE has been applied for the selective separation of many organic contaminants including antibiotics, anti-inflammatory drugs, pesticides, etc. which are present

MSPE has been applied for the extraction of atrazine, prometon, propazine and prome‐ tryn in environmental water samples using graphene-Fe3O4 nanoparticles. The effect of the amount of magnetic solid, extraction time and pH of the sample were evaluated. 20 mg of the magnetic support was dispersed into a 250 ml aqueous sample solution for 20

l


in different matrices [35].

*2.2.4. Magnetic solid phase extraction*

#### *2.2.3. Dispersive solid phase extraction*

DSPE involves a sorbent addition to a water sample to form a dispersion (Fig. 6.a and b). The solid used has been derivatized to produce a bound organic phase (e.g., octadecyl, MIP, etc.) on its the surface similar to those used for packing SPE columns. The contact between the analytes and the support is higher than in traditional SPE, increasing the equilibrium rate and providing higher extraction yields. After centrifuging the suspension, the solid phase sedi‐ ments are at the bottom of the test tube. An appropriate organic solvent is then used to elute the analytes from the solid sorbent prior to the organic extract analysis (Fig. 6.c).

DSPE has been applied to determine atrazine, prometryn, simazine, terbumeton and terbu‐ thylazine in lettuce and corn acetonitrile extracts. Although this method is not used in water matrices, it is a good example for the isolation of triazine herbicides by DSPE. A MIP was synthesised using methacrylic acid, ethylenglycol, dimethacrylate, dithioester compounds and atrazine. The retention mechanism is based on the electrostatic interaction between the acid monomer and the basic properties of the target molecule. 100 mg of the MIP were dispersed

**Figure 6.** Representation of the dispersive solid phase extraction methodology

in 5 ml of sample solution for 1 hour. Once the extraction was concluded, the solid phase was collected in a membrane filter. The solid was washed with methanol and then the atrazine was eluted with 5 ml of desorption solvent (methanol/acetic acid, 9:1 v/v). The extract was dried and redissolved in acetonitrile before its analysis by HPLC-UV. The LOD reported was 2.8 µg l -1 with precisions <10% [34].

#### *2.2.4. Magnetic solid phase extraction*

**(b)**

0 0

**(c)**

**(a)**

264 Herbicides - Advances in Research

**Figure 5.** Representation of the solid phase microextraction methodology

obtained were in the range from 0.2 to 3.4 ng l-1 [32].

*2.2.3. Dispersive solid phase extraction*

A stir bar of 10 mm length and 0.5 mm polydimethylsiloxane was used to extract ten triazines from water samples. The SBSE step was carried out by introducing the stir bar into a vial containing 20 ml of the sample, 30% of NaCl and stirred (31.4 s-1) during 60 min. The stir bar was washed with water and then thermally desorbed and analyzed by GC-MS. The LODs

The use of thermal desorption requires a cold trap during elution process. The hyphenation of SBSE with HPLC-DAD using solvent desorption was applied to atrazine, simazine and terbuthylazine in environmental water samples. A polyethylene bar impregnated with activated carbon (15 mm length and 0.5 mm thickness) was used to extract 10 ml of water samples. The extraction time required was 16 h, followed by desorption of the analytes using acetonitrile as solvent. The reported LODs were around 0.1 µg l-1 with precision <15%, as RSD. This method was an alternative to the analysis of analytes with polar characteristics [33].

DSPE involves a sorbent addition to a water sample to form a dispersion (Fig. 6.a and b). The solid used has been derivatized to produce a bound organic phase (e.g., octadecyl, MIP, etc.) on its the surface similar to those used for packing SPE columns. The contact between the analytes and the support is higher than in traditional SPE, increasing the equilibrium rate and providing higher extraction yields. After centrifuging the suspension, the solid phase sedi‐ ments are at the bottom of the test tube. An appropriate organic solvent is then used to elute

DSPE has been applied to determine atrazine, prometryn, simazine, terbumeton and terbu‐ thylazine in lettuce and corn acetonitrile extracts. Although this method is not used in water matrices, it is a good example for the isolation of triazine herbicides by DSPE. A MIP was synthesised using methacrylic acid, ethylenglycol, dimethacrylate, dithioester compounds and atrazine. The retention mechanism is based on the electrostatic interaction between the acid monomer and the basic properties of the target molecule. 100 mg of the MIP were dispersed

the analytes from the solid sorbent prior to the organic extract analysis (Fig. 6.c).

Recovery of the solid phase in DSPE requires the use of filtration or centrifugation techniques that may lead to a solid phase loss and the subsequent decrease in precision and accuracy. The use of magnetic solids is an alternative for the selective preconcentration of different chemical species. It offers adequate surface area, the possibility of functionalization and paramagnetic properties. Their application as dispersed sorbents in liquid samples is so-called magnetic solid phase extraction (MSPE, Fig. 7). This technique has demonstrated several advantages such as: the decrease in sample treatment time, the decrease in solvent use, and the easy treatment of high volume samples. MSPE has been applied for the selective separation of many organic contaminants including antibiotics, anti-inflammatory drugs, pesticides, etc. which are present in different matrices [35].

MSPE has been applied for the extraction of atrazine, prometon, propazine and prome‐ tryn in environmental water samples using graphene-Fe3O4 nanoparticles. The effect of the amount of magnetic solid, extraction time and pH of the sample were evaluated. 20 mg of the magnetic support was dispersed into a 250 ml aqueous sample solution for 20

**3.2. Equipment**

microphotographs.

**3.3. Analytical method**

*3.3.2. Sampling and sample treatment*

rate of 1.0 ml min-1 was established at 25 °C.

*3.3.1. Synthesis and characterization of magnetic supports*

Magnetic solids were characterized by X-ray diffraction in a PHILIPS PW1710 (Almelo, The Netherlands) instrument equipped with a Cu anode, automatic divergence slit and a graphite monochrometer under the following experimental conditions: CuKα radiation, 1.54 Å; generator tension, 40 kV; generator current, 30 mA; intensity ratio (α2/α1), 0.500; divergence slit, 1°; receiving slit, 0.1; start angle (2Ѳ°), 5; end angle (2Ѳ°), 70. A JEOL JSM-820 (Tokio, Japan) scanning electron microscope (SEM) was used for obtaining the magnetic solid

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The separation and analysis were performed using HPLC equipment consisting of a Gilson (Middleton, WI, USA) model 302 pressure pump, a Rheodyne mod. 7525 injection valve and a UV–VIS diode array HP8453 spectrophotometer (Hewlett Packard, Palo Alto, CA, USA). The absorbance of atrazine and simazine was monitored at 220 nm [37]. The quantification of triazines was made by comparison of peak height with those of the standards. The chromato‐ graphic separation was achieved with a Scharlau C18 column (5 µm; 150 mm×4.6 mm i.d.) (Barcelona, Spain). The mobile phase consisted of methanol-deionized water (2:1, v/v). A flow

The magnetic solids were synthesized by emulsion polymerization. Magnetite particles Fe3O4 were synthesized by a co-precipitation method [38] (Figure 8.a). The magnetite obtained was washed three times with 50 ml portions of deionized water and added to a flask containing a mixture methanol/water 3:1, Triton X-100 2%, CTAB 0.05% and the precursors indicated in Table 2. The mixture was heated and refluxed at 120 °C for 16 h with stirring (Figure 8.b). The solids were washed with two portions of 20 ml of deionized water and then a portion of 20 ml of ethanol then were dried at 60 ° C. In order to block superficial silanol groups (–Si–OH), the solids obtained in the previous process were derivatized using a mixture of 0.9 g of chlorotri‐ methylsilane (CTMS), and 1 ml of pyridine per gram of support in 50 ml of toluene (Figure 8.c). The supports were then washed with 20 ml portions of each of the following solvents: toluene, ethanol and deionized water until the washing liquid was colorless. The obtained magnetic particles were dried at 60 °C for 24 h [39]. Subsequently, all the magnetic solids were characterized by different techniques like SEM, X-ray diffraction and infrared spectroscopy.

Surface water samples were collected from an agricultural area in Zamora, Spain in October 2009. Polypropylene bottles previously washed with deionized water and H2SO4 solution of 2% (v/v) were used. Once at the sampling site, the bottles were rinsed several times with the water to be collected and the temperature was measured. The samples were stored at 4°C before analysis. They were filtered through 0.45 µm cellulose acetate filters (Sartorius,

**Figure 7.** Representation of the magnetic solid phase extraction methodology

minutes. The solid was isolated from the sample solution using a magnet. The liquid phase was discarded and the solid was vortexed with acetone to desorb the analytes pri‐ or to its analysis by HPLC-DAD. The LODs of the method ranged between 0.025 and 0.040 µg l-1 with reproducilities <5.2% [36].

In this study, the effect of polarity of the solid phase on the interaction between triazines with magnetic supports used as part of a MSPE system coupled to HPLC-UV was evaluated. The developed methodology was used to determine atrazine and simazine in surface water samples.

### **3. Experimental conditions**

### **3.1. Reagents and solutions**

Ferrous sulfate heptahydrate (FeSO4 7H2O), ammonium solution (NH3 25%, w/w), sodium hydroxide 99%, and hydrochloric acid 36% were purchased from J.T. Baker (Phillipsburg, NJ, USA). Triton X-100, cetyltrimethylammonium bromide (CTAB), tetramethoxysilane (TMOS), phenyltrimethoxysilane (PTMS), octyltriethoxysilane (C8-TEOS), chlorotrimethylsilane (CTMS), and methanol (HPLC grade) were obtained from Sigma-Aldrich (St. Louis, MO, USA). 1-chloro-3-ethylamino-5-isopropylamino-2,4,6-triazine standard 99.5% (atrazine) and 6 chloro-N,N'-diethyl-1,3,5-triazine-2,4-diamine standard 98.5% (simazine) were provided by Dr. Ehrenstorfer GmbH (Augsburg, Germany).

Stock solutions of triazines were prepared at a concentration of 250 mg l-1 in deionized water. These solutions were protected from light and stored under refrigeration (4 °C) until use to avoid possible decomposition. Calibration standards were prepared at con‐ centrations of 5-1000 µg l-1 by mixing adequate volumes of each standard solution in de‐ ionized water.

### **3.2. Equipment**

Magnetic solids were characterized by X-ray diffraction in a PHILIPS PW1710 (Almelo, The Netherlands) instrument equipped with a Cu anode, automatic divergence slit and a graphite monochrometer under the following experimental conditions: CuKα radiation, 1.54 Å; generator tension, 40 kV; generator current, 30 mA; intensity ratio (α2/α1), 0.500; divergence slit, 1°; receiving slit, 0.1; start angle (2Ѳ°), 5; end angle (2Ѳ°), 70. A JEOL JSM-820 (Tokio, Japan) scanning electron microscope (SEM) was used for obtaining the magnetic solid microphotographs.

The separation and analysis were performed using HPLC equipment consisting of a Gilson (Middleton, WI, USA) model 302 pressure pump, a Rheodyne mod. 7525 injection valve and a UV–VIS diode array HP8453 spectrophotometer (Hewlett Packard, Palo Alto, CA, USA). The absorbance of atrazine and simazine was monitored at 220 nm [37]. The quantification of triazines was made by comparison of peak height with those of the standards. The chromato‐ graphic separation was achieved with a Scharlau C18 column (5 µm; 150 mm×4.6 mm i.d.) (Barcelona, Spain). The mobile phase consisted of methanol-deionized water (2:1, v/v). A flow rate of 1.0 ml min-1 was established at 25 °C.

### **3.3. Analytical method**

minutes. The solid was isolated from the sample solution using a magnet. The liquid phase was discarded and the solid was vortexed with acetone to desorb the analytes pri‐ or to its analysis by HPLC-DAD. The LODs of the method ranged between 0.025 and

**(e)**

**(a) (b) (c) (d)**

**Figure 7.** Representation of the magnetic solid phase extraction methodology

In this study, the effect of polarity of the solid phase on the interaction between triazines with magnetic supports used as part of a MSPE system coupled to HPLC-UV was evaluated. The developed methodology was used to determine atrazine and simazine in surface water

Ferrous sulfate heptahydrate (FeSO4 7H2O), ammonium solution (NH3 25%, w/w), sodium hydroxide 99%, and hydrochloric acid 36% were purchased from J.T. Baker (Phillipsburg, NJ, USA). Triton X-100, cetyltrimethylammonium bromide (CTAB), tetramethoxysilane (TMOS), phenyltrimethoxysilane (PTMS), octyltriethoxysilane (C8-TEOS), chlorotrimethylsilane (CTMS), and methanol (HPLC grade) were obtained from Sigma-Aldrich (St. Louis, MO, USA). 1-chloro-3-ethylamino-5-isopropylamino-2,4,6-triazine standard 99.5% (atrazine) and 6 chloro-N,N'-diethyl-1,3,5-triazine-2,4-diamine standard 98.5% (simazine) were provided by

Stock solutions of triazines were prepared at a concentration of 250 mg l-1 in deionized water. These solutions were protected from light and stored under refrigeration (4 °C) until use to avoid possible decomposition. Calibration standards were prepared at con‐ centrations of 5-1000 µg l-1 by mixing adequate volumes of each standard solution in de‐

0.040 µg l-1 with reproducilities <5.2% [36].

Dr. Ehrenstorfer GmbH (Augsburg, Germany).

**3. Experimental conditions**

**3.1. Reagents and solutions**

samples.

266 Herbicides - Advances in Research

ionized water.

### *3.3.1. Synthesis and characterization of magnetic supports*

The magnetic solids were synthesized by emulsion polymerization. Magnetite particles Fe3O4 were synthesized by a co-precipitation method [38] (Figure 8.a). The magnetite obtained was washed three times with 50 ml portions of deionized water and added to a flask containing a mixture methanol/water 3:1, Triton X-100 2%, CTAB 0.05% and the precursors indicated in Table 2. The mixture was heated and refluxed at 120 °C for 16 h with stirring (Figure 8.b). The solids were washed with two portions of 20 ml of deionized water and then a portion of 20 ml of ethanol then were dried at 60 ° C. In order to block superficial silanol groups (–Si–OH), the solids obtained in the previous process were derivatized using a mixture of 0.9 g of chlorotri‐ methylsilane (CTMS), and 1 ml of pyridine per gram of support in 50 ml of toluene (Figure 8.c). The supports were then washed with 20 ml portions of each of the following solvents: toluene, ethanol and deionized water until the washing liquid was colorless. The obtained magnetic particles were dried at 60 °C for 24 h [39]. Subsequently, all the magnetic solids were characterized by different techniques like SEM, X-ray diffraction and infrared spectroscopy.

### *3.3.2. Sampling and sample treatment*

Surface water samples were collected from an agricultural area in Zamora, Spain in October 2009. Polypropylene bottles previously washed with deionized water and H2SO4 solution of 2% (v/v) were used. Once at the sampling site, the bottles were rinsed several times with the water to be collected and the temperature was measured. The samples were stored at 4°C before analysis. They were filtered through 0.45 µm cellulose acetate filters (Sartorius,

of the beaker, and using a syringe the extract was isolated, dried, redisolved in 50 µl of

The SPE procedure for comparison was performed as described [40]: C18 SPE cartridges (500 mg, Bound Elut, Varian, Netherlands) were conditioned using 5 ml of ethyl acetate, 5 ml of methanol and 5 ml of deionized water at a flow rate around 2 ml min-1. Water samples (1 l) were flowed through the cartridges with a flow rate between 10-15 ml min-1 under vacuum and the loaded cartridges were rinsed with 3 ml of methanol:water (5:95, v/v). The elution was performed with three aliquots (1 ml) of ethyl acetate at a flow-rate of about 1 mL min-1. The combined aliquots were evaporated to dryness by a gentle stream of nitrogen and the residues

Figure 9 shows the effect of the polarity of the magnetic solid and the pH value of the aqueous phase on the recovery of each analyte. The results obtained demonstrate the high affinity achieved by phenyl based supports. The best extractions were observed when using the PH1 solid at pH 5 with recoveries of 90% and 100% for simazine and atrazine, respectively. Based

on this performance, solid PH1 and pH 5 were selected for further experiments.

3 5 8 11

**Figure 9.** Effect of magnetic solid polarity and sample pH value on the triazine recoveries in spiked surface water (10

The adsorption of analytes on the solid surface depends on acid-base interactions (hydrogen bonds), π-π interactions, and Van der Waals forces (hydrophobic interactions). However, it has been observed that solid absorbent with aromatic groups improve the adsorption of triazines, due to π-π interactions, improving significantly recovery percentages [41-43]. This type of interaction has been reported for adsorption of triazines on mineral oxides coated with surfactants, the hydrophobic interaction between adsorbents and analytes improve the adsorption [44]. On the other hand, the acid-base equilibrium has an important role during

% Recovery

3 5 8

0 11

Simazine

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P1 P2 PH1 PH2 C8-1 C8-2

Magnetic support

methanol and injected into the HPLC system for their separation and analysis.

were dissolved in 50 µl of methanol and injected into the HPLC system.

**4. Results and discussion**

**4.1. Triazine extraction by MSPE**

P1 P2 PH1 PH2 C8-1 C8-2

Atrazine

Magnetic support

ng ml-1)

% Recovery

**Figure 8.** Synthetic methodology for magnetic supports preparation; a) magnetite preparation by co-precipitation method, b) silica polymerization onto magnetite particles by emulsion polymerization, c) silanol block reaction.


**Table 2.** SiO2/Fe3O4 ratio (w/w) and functionalized monomers for magnetic supports used such as adsorbents in MSPE for atrazine and simazine isolation

Göttingen, Germany) in a glass filtration device connected to a hand-operated vacuum pump (Sartorius, Göttingen, Germany).

Parameters such as the water samples' pH and the nature of the functionalized magnetic solids (mainly polarity) were modified to find the adequate extraction conditions. All the experiments were carried out with five replicates.

The optimal developed MSPE procedure involves the following steps: First, 1 ml of methanol is added to 0.1 g of the magnetic support (PH1) for activation and the magnetic solid is washed with 5 ml of deionized water. After addition of a known volume of water sample (0.2, 0.5 or 1.0 l) and pH adjustment to a value of 5 with HCl 1 M, the mixture is dispersed in an ultrasonic bath for 10 min. Then, a neodymium magnet is placed on the bottom of the beaker providing the isolation of the magnetic supports with the adsorbed analytes from the solution. The water sample is then eliminated by decantation. After the adsorption process, the solid is rinsed twice with 10 ml of deionized water. Finally, 1 ml of methanol was added to the magnetic solid and dispersed in an ultrasonic bath for 10 min. A neodymium magnet was placed on the bottom -SiOH Cl-Si(CH3)3 |-Si-O-Si(CH3)3 + C5H6N+ - <sup>+</sup> Cl Toluene, Pyridine, 60°C of the beaker, and using a syringe the extract was isolated, dried, redisolved in 50 µl of methanol and injected into the HPLC system for their separation and analysis.

> The SPE procedure for comparison was performed as described [40]: C18 SPE cartridges (500 mg, Bound Elut, Varian, Netherlands) were conditioned using 5 ml of ethyl acetate, 5 ml of methanol and 5 ml of deionized water at a flow rate around 2 ml min-1. Water samples (1 l) were flowed through the cartridges with a flow rate between 10-15 ml min-1 under vacuum and the loaded cartridges were rinsed with 3 ml of methanol:water (5:95, v/v). The elution was performed with three aliquots (1 ml) of ethyl acetate at a flow-rate of about 1 mL min-1. The combined aliquots were evaporated to dryness by a gentle stream of nitrogen and the residues were dissolved in 50 µl of methanol and injected into the HPLC system.

### **4. Results and discussion**

### **4.1. Triazine extraction by MSPE**

Göttingen, Germany) in a glass filtration device connected to a hand-operated vacuum pump

**Table 2.** SiO2/Fe3O4 ratio (w/w) and functionalized monomers for magnetic supports used such as adsorbents in

Fe2+ (ac) + O2 Fe3++ Fe2+ + H2O Fe(OH)x

NH4OH, MeOH/H2O, Triton X-100 2% CTAB 0.05%, 120°C, 16h

> 6h 6h

**Figure 8.** Synthetic methodology for magnetic supports preparation; a) magnetite preparation by co-precipitation method, b) silica polymerization onto magnetite particles by emulsion polymerization, c) silanol block reaction.

> **Magnetic Solid Name Ratio SiO2 monomer-Fe3O4 (w/w)** Polar 1 (P1) 1:1 / TMOS Polar 2 (P2) 1:2 / TMOS Phenyl 1 (PH1) 1:1 / PTMS Phenyl 2 (PH2) 1:2 / PTMS Octyl 1 (C8-1) 1:1 / C8-TEOS Octyl 2 (C8-2) 1:2 / C8-TEOS

+ Si(OCH3)3R

3.

**c)**

2.

**b)**

**a)**

268 Herbicides - Advances in Research

R= CH3 (methyl), C6H6 (phenyl) and C8H17 (Octyl)

3-x + Fe(OH)y

Si O

S

R


R

6h 1.

i

O

O

R

Si

> O

Si

R

Si

R

2-y Fe3O4

R

Si

O

R

O

R

Si

O

Si

O

Parameters such as the water samples' pH and the nature of the functionalized magnetic solids (mainly polarity) were modified to find the adequate extraction conditions. All the experiments

The optimal developed MSPE procedure involves the following steps: First, 1 ml of methanol is added to 0.1 g of the magnetic support (PH1) for activation and the magnetic solid is washed with 5 ml of deionized water. After addition of a known volume of water sample (0.2, 0.5 or 1.0 l) and pH adjustment to a value of 5 with HCl 1 M, the mixture is dispersed in an ultrasonic bath for 10 min. Then, a neodymium magnet is placed on the bottom of the beaker providing the isolation of the magnetic supports with the adsorbed analytes from the solution. The water sample is then eliminated by decantation. After the adsorption process, the solid is rinsed twice with 10 ml of deionized water. Finally, 1 ml of methanol was added to the magnetic solid and dispersed in an ultrasonic bath for 10 min. A neodymium magnet was placed on the bottom

(Sartorius, Göttingen, Germany).

MSPE for atrazine and simazine isolation

were carried out with five replicates.

Figure 9 shows the effect of the polarity of the magnetic solid and the pH value of the aqueous phase on the recovery of each analyte. The results obtained demonstrate the high affinity achieved by phenyl based supports. The best extractions were observed when using the PH1 solid at pH 5 with recoveries of 90% and 100% for simazine and atrazine, respectively. Based on this performance, solid PH1 and pH 5 were selected for further experiments.

**Figure 9.** Effect of magnetic solid polarity and sample pH value on the triazine recoveries in spiked surface water (10 ng ml-1)

The adsorption of analytes on the solid surface depends on acid-base interactions (hydrogen bonds), π-π interactions, and Van der Waals forces (hydrophobic interactions). However, it has been observed that solid absorbent with aromatic groups improve the adsorption of triazines, due to π-π interactions, improving significantly recovery percentages [41-43]. This type of interaction has been reported for adsorption of triazines on mineral oxides coated with surfactants, the hydrophobic interaction between adsorbents and analytes improve the adsorption [44]. On the other hand, the acid-base equilibrium has an important role during adsorption, in this case is evident that the better adsorption onto the surface solid is presented at pH 5. In these conditions the triazines are neutral without electric charges increasing the hydrophobic interactions. At pH values >7.0 the remaining surface silanol groups acquire a negative charge, increasing the repulsions between the surface of magnetic solid and the triazines decreasing the percentage recoveries for both analytes [45].

According to the results from Table 3, it is possible to observe that the LODs decrease when higher initial sample volumes are used. The lowest limits of detection were reached between 0.01 and 0.02 µg l-1 using 1 l of initial sample. LOD and LOQ obtained by the method are

**Repeatability Reproducibility**

Recent Advances in the Extraction of Triazines from Water Samples

4.8 3.2

http://dx.doi.org/10.5772/54962

2.5 4.2

1.5 1.2 2.8

271

2.9

2.0

6.2 5.1

6.7 5.6

2.8 3.9

**LOQ (µg l-1)**

> 100 µg l-1

> > 2.3

1.6

1.6

2.6 Atrazine <sup>3</sup> <sup>9</sup>

2.2 Atrazine <sup>1</sup> <sup>3</sup>

1.5 Atrazine 0.02 0.06

**Table 3.** Analytical parameters for different sample volumes, repeatability and reproducibility (%RSD, n = 5) for two

The precision of method expressed as the repeatability and reproducibility values (%RSD < 5%) and the high recoveries obtained make the proposed method a viable alternative to be routinely implemented in the analysis of simazine and atrazine in water samples, without the

The developed method was applied to the determination of triazines in surface water samples from agricultural lands in Zamora, Spain. Only one of the four samples analyzed showed contamination by triazines, being this water sample was collected in a waterhole near a corn

The concentrations found with the MSPE-HPLC (mean and %RSD, n=5) method were 9.9(3.0) and 12.2(2.5) µg l-1 for simazine and atrazine, respectively. The concentrations determined using the SPE-HPLC were 9.8(3.0) and 11.8(2.3 µg l-1. The average of each analyte (determined by both methods) was compared by a t-test for comparison of means, assuming comparable variances (verified by a F-test). Calculated t values were compared with the tabulated t value for 8 degrees of freedom and a significance level of α= 0.05 (t = 2.30). Thus, the null hypothesis was accepted meaning that there were no significant differences between the results provided

Figure 11 showed the chromatogram of surface water sample collected from Zamora, Spain, extracted with MSPE and SPE and a standard chromatogram. The clean-up process results for

field, which shows that pesticides applied to crops, migrate to nearby water bodies.

comparable to those reported by other methods.

**Analyte LOD**

100 µg l-1 **(µg l-1)**

50 µg l-1

0.2 Simazine 4 12 2.8

0.5 Simazine 1 3 2.4

1.0 Simazine 0.01 0.03 1.4

necessity of expensive or difficult access equipment.

**4.4. Analysis of superficial water samples**

**Sample volume (l)**

> 50 µg l-1

concentration levels.

by both methods.

#### **4.2. Characterization of magnetic solids**

The magnetic solid characterization has been previously reported [39]. In this paper, we focus on the characterization of the most adequate solid for the selected triazine preconcentration. The magnetic particle morphology is spherical with core-shell type, where the core particle is magnetite, with super paramagnetic properties (20-50 emu g-1) [46]. On the other hand, the shell is formed by silica phase functionalized with phenyl groups. The micrograph shown in Figure 10 confirms the spherical morphology of magnetic particles, with an approximated diameter of 2 µm. The diffraction pattern shows the magnetite line diffraction (m) and a broadband signal between 2Ѳ° of 10° and 40°, corresponding to the amorphous silica phase. The physiochemical and magnetic properties of the magnetic particles were adequate for their application as adsorbents in MSPE.

**Figure 10.** Microphotography and diffraction pattern of PH1 support

### **4.3. Analytical parameters of the MSPE**

In the optimized conditions, the analytical parameters and precision data were determined using spiked tap water samples. Different volumes of spiked surface water samples (0.2, 0.5 and 1.0 l) with an interval concentration of 10-1000 µg l-1 were used. The results obtained are listed in Table 3. The limits of detection (LODs) were calculated for a signal/noise relation equal to (S/N = 3.29). The limits of quantification (LOQ) were determined using a signal/noise relation equal to 10 (S/N = 10). The calibration curves were constructed from the high signal versus concentration µg l-1.

According to the results from Table 3, it is possible to observe that the LODs decrease when higher initial sample volumes are used. The lowest limits of detection were reached between 0.01 and 0.02 µg l-1 using 1 l of initial sample. LOD and LOQ obtained by the method are comparable to those reported by other methods.


**Table 3.** Analytical parameters for different sample volumes, repeatability and reproducibility (%RSD, n = 5) for two concentration levels.

The precision of method expressed as the repeatability and reproducibility values (%RSD < 5%) and the high recoveries obtained make the proposed method a viable alternative to be routinely implemented in the analysis of simazine and atrazine in water samples, without the necessity of expensive or difficult access equipment.

### **4.4. Analysis of superficial water samples**

adsorption, in this case is evident that the better adsorption onto the surface solid is presented at pH 5. In these conditions the triazines are neutral without electric charges increasing the hydrophobic interactions. At pH values >7.0 the remaining surface silanol groups acquire a negative charge, increasing the repulsions between the surface of magnetic solid and the

The magnetic solid characterization has been previously reported [39]. In this paper, we focus on the characterization of the most adequate solid for the selected triazine preconcentration. The magnetic particle morphology is spherical with core-shell type, where the core particle is magnetite, with super paramagnetic properties (20-50 emu g-1) [46]. On the other hand, the shell is formed by silica phase functionalized with phenyl groups. The micrograph shown in Figure 10 confirms the spherical morphology of magnetic particles, with an approximated diameter of 2 µm. The diffraction pattern shows the magnetite line diffraction (m) and a broadband signal between 2Ѳ° of 10° and 40°, corresponding to the amorphous silica phase. The physiochemical and magnetic properties of the magnetic particles were adequate for their

In the optimized conditions, the analytical parameters and precision data were determined using spiked tap water samples. Different volumes of spiked surface water samples (0.2, 0.5 and 1.0 l) with an interval concentration of 10-1000 µg l-1 were used. The results obtained are listed in Table 3. The limits of detection (LODs) were calculated for a signal/noise relation equal to (S/N = 3.29). The limits of quantification (LOQ) were determined using a signal/noise relation equal to 10 (S/N = 10). The calibration curves were constructed from the high signal versus

**Counts**

5 15 25 35 45 55 65

m

m

**2**

m m m

m

triazines decreasing the percentage recoveries for both analytes [45].

**4.2. Characterization of magnetic solids**

270 Herbicides - Advances in Research

application as adsorbents in MSPE.

**Figure 10.** Microphotography and diffraction pattern of PH1 support

**4.3. Analytical parameters of the MSPE**

concentration µg l-1.

The developed method was applied to the determination of triazines in surface water samples from agricultural lands in Zamora, Spain. Only one of the four samples analyzed showed contamination by triazines, being this water sample was collected in a waterhole near a corn field, which shows that pesticides applied to crops, migrate to nearby water bodies.

The concentrations found with the MSPE-HPLC (mean and %RSD, n=5) method were 9.9(3.0) and 12.2(2.5) µg l-1 for simazine and atrazine, respectively. The concentrations determined using the SPE-HPLC were 9.8(3.0) and 11.8(2.3 µg l-1. The average of each analyte (determined by both methods) was compared by a t-test for comparison of means, assuming comparable variances (verified by a F-test). Calculated t values were compared with the tabulated t value for 8 degrees of freedom and a significance level of α= 0.05 (t = 2.30). Thus, the null hypothesis was accepted meaning that there were no significant differences between the results provided by both methods.

Figure 11 showed the chromatogram of surface water sample collected from Zamora, Spain, extracted with MSPE and SPE and a standard chromatogram. The clean-up process results for both preconcentration methods are similar, showing that MSPE can be used as an alternative method for the determination of atrazine and simazine.

**Author details**

José A. Rodríguez1

(SCAV), Switzerland

**References**

17-31

2003.

3 Universidad de Valladolid, Spain

, Karina Aguilar-Arteaga1

1 Universidad Autonoma del Estado de Hidalgo, Mexico

tionizing Agriculture. Hungary: Elsevier; 2008

ical Chemistry 2011; 30(11) 1781-1792

ods 2012; 5 540-550

Herbicides. Analytical Chemistry 2002; 74 648-654

cal and Bioanalytical Chemistry 2010; 397 2233-2243

, Cristina Díez2

2 University of Geneva (Analytical Pharmaceutical Chemistry) and Food Authority Control

[1] Crafts AS. The Chemistry and Mode of Action of Herbicides. New York: Wiley; 1965 [2] LeBaron HM, McFarland, Burnside, OC. The Triazine Herbicides. 50 Years Revolu‐

[3] Pacakova V, Stulik K, Jiskra J. High-Performance Separations in the Determination of Triazine Herbicides and Their Residues. Journal of Chromatography A 1996;754

[4] Mitra S. Sample Preparation Techniques in Analytical Chemistry. New Jersey: Wiley;

[5] U.S. Environmental Protection Agency, EPA 500 Series, Method 507 (revision 2.0). Determination of Nitrogen- and Phosphorus-Containing Pesticides in Water by Gas

[6] Chimuka L, Michel M, Cukrowska E. Buszewski. Advances in Sample Preparation Using Membrane-Based Liquid-Phase Microextraction Techniques. Trends in Analyt‐

[7] Shen G, Lee HK. Hollow Fiber-Protected Liquid Phase Microextraction of Triazine

[8] Trtic-Petrovic T, Dordevic J, Dujakovic N, Kumric K, Vasiljevic T, Lausevic M. Deter‐ mination of Selected Pesticides in Environmental Water by Employing Liquid-Phase Microextraction and Liquid Chromatography-Tandem Mass Spectrometry. Analyti‐

[9] Wu C, Liu Y, Wu Q, Wang C, Wang Z. Combined Use of Liquid-Liquid Microextrac‐ tion and Carbon Nanotube Reinforced Hollow Fiber Microporous Membrane Solid-Phase Microextraction for the Determination of Triazine Herbicides in Water and Milk Samples by High-Performance Liquid Chromatography. Food Analytical Meth‐

chromatography with a Nitrogen-Phosphorus Detector. 1989

and Enrique Barrado3

http://dx.doi.org/10.5772/54962

273

Recent Advances in the Extraction of Triazines from Water Samples

**Figure 11.** Chromatograms obtained at the optimized conditions: (a) surface water sample obtained by MSPE preconcentration method, (b) surface water sample obtained by SPE pre-concentration method and (c) simazine and atrazine standard solution 20 µg l-1. S: simazine and A: atrazine.

### **5. Conclusions**

Due to the wide application of herbicides, it is necessary to develop fast and reliable methods for their determination in different analytical matrices providing a correct risk assessment to human health and the environment.

The results obtained by the optimized and validated MSPE method are comparable with other reported methods, concluding that the developed MSPE-HPLC procedure can be used for the screening and quantification of atrazine and simazine in water samples.

Although there are more sensitive methods, they require expensive and inaccessible instru‐ mentation such as mass spectrometry, representing the MSPE-HPLC-DAD a rapid and low cost determination method of atrazine and simazine in water samples.

### **Acknowledgements**

The authors wish to thank the CONACyT (project 61310) and Consejería de Educación, Junta de Castilla y León (project VA023A10-2) and MCINN project AIB2010PT-00234 for the financial support.

### **Author details**

both preconcentration methods are similar, showing that MSPE can be used as an alternative

S A

0 1 2 3 4 5 6 7 8 9

**Time (minutes)**

**Figure 11.** Chromatograms obtained at the optimized conditions: (a) surface water sample obtained by MSPE preconcentration method, (b) surface water sample obtained by SPE pre-concentration method and (c) simazine and

Due to the wide application of herbicides, it is necessary to develop fast and reliable methods for their determination in different analytical matrices providing a correct risk assessment to

The results obtained by the optimized and validated MSPE method are comparable with other reported methods, concluding that the developed MSPE-HPLC procedure can be used for the

Although there are more sensitive methods, they require expensive and inaccessible instru‐ mentation such as mass spectrometry, representing the MSPE-HPLC-DAD a rapid and low

The authors wish to thank the CONACyT (project 61310) and Consejería de Educación, Junta de Castilla y León (project VA023A10-2) and MCINN project AIB2010PT-00234 for the financial

screening and quantification of atrazine and simazine in water samples.

cost determination method of atrazine and simazine in water samples.

method for the determination of atrazine and simazine.

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

human health and the environment.

**Acknowledgements**

support.

atrazine standard solution 20 µg l-1. S: simazine and A: atrazine.

**Abs (AU)**

272 Herbicides - Advances in Research

**5. Conclusions**

José A. Rodríguez1 , Karina Aguilar-Arteaga1 , Cristina Díez2 and Enrique Barrado3

1 Universidad Autonoma del Estado de Hidalgo, Mexico

2 University of Geneva (Analytical Pharmaceutical Chemistry) and Food Authority Control (SCAV), Switzerland

3 Universidad de Valladolid, Spain

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[29] Katsumata H, Kojima H, Kaneco S, Suzuki T, Ohta K. Preconcentration of Atrazine and Simazine with Miltiwalled Carbon Nanotubes as Solid-Phase Extraction Disk.

[30] Hernandez F, Beltran J, Lopez FJ, Gaspar JV. Use of Solid-Phase Microextraction for the Quantitative Determination of Herbicides in Soil and Water Samples. Analytical

[31] Wu Q, Feng C, Zhao G, Wand C, Wang Z. Graphene-Cated Fiber for Solid-Phase Mi‐ croextraction of Triazine Herbicides in Water Samples. Journal of Separation Science

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**Chapter 14**

**Floral Biology and Africanized Honeybee Behaviour in**

**Transgenic (Roundup ReadyTM var. BR-245 RR) and**

**Merrill) Flowers**

Wainer César Chiari,

Clara Beatriz Hoffmann-Campo,

Emerson Dechechi Chambó,

http://dx.doi.org/10.5772/55847

**1. Introduction**

Carlos Arrabal Arias, Tais da Silva Lopes,

Maria Claudia Ruvolo-Takasusuki and Vagner de Alencar Arnaut de Toledo

Additional information is available at the end of the chapter

Tiago Cleiton Simões de Oliveira Arnaut De Toledo,

**Conventional (var. BRS-133) Soybean (Glycine max L.**

Therefore, this research was carried out to evaluate the Africanized honeybee effect on soybean production, mainly, over genetically modified organisms (GMOs). This chapter presents experimental data about floral biology of soybean *Glycine max* L. Merrill, BR-245 RR (transgenic soy – Roundup Read™) and BRS-133 (conventional soy) with and without application of

Soybean (*Glycine max* L. Merrill) is one of the main commodities in the world. Currently, more than 70% of its production is genetically modified. Brazil has a highlighted place concerning about this crop and is the second place in world production. During the 2010/2011 harvest season, Brazil produced 75 million tons of soybean on 24.2 million hectares of planted area with 3,106 kg.ha-1 of productivity. The soybean crop represents the main worldwide oleagi‐ nous crop produced consumed by animals and human beings. After several decades of searching for alternatives to control weed pests, genotypes of genetically modified (GM)

> © 2013 Chiari et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 Chiari et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

Glyphosate, as well as the honeybee behaviour in those flowers of these varieties.


**Floral Biology and Africanized Honeybee Behaviour in Transgenic (Roundup ReadyTM var. BR-245 RR) and Conventional (var. BRS-133) Soybean (Glycine max L. Merrill) Flowers**

Wainer César Chiari, Clara Beatriz Hoffmann-Campo, Carlos Arrabal Arias, Tais da Silva Lopes, Tiago Cleiton Simões de Oliveira Arnaut De Toledo, Emerson Dechechi Chambó, Maria Claudia Ruvolo-Takasusuki and Vagner de Alencar Arnaut de Toledo

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55847

### **1. Introduction**

[33] Neng NR, Mestre AS, Carvalho AP, Nogueira JMF. Powdered Activated Carbons as Effective Phases for Bar Sorptive Micro-Extraction (BSµE) to monitor levels of triazin‐

[34] Xu S, Li J, Chen L. Moleculary Imprinted Polymers by Reversible Addition-Fragmen‐ tation Chain Transfer Precipitation Polymerization for Preconcentration of Atrazine

[35] Aguilar-Artega K, Rodriguez JA, Barrado E. Magnetic Solids in Analytical Chemis‐

[36] Zhao G, Song S, Wang C, Wu Q, Wang Z. Determination of Triazine Herbicides in Environmental Water Samples by High-Performance Liquid Chromatography using Graphene-Coated Magnetic Nanoparticles as adsorbent. Analytica Chimica Acta

[37] Katsumata H, Kaneco S, Suzuki T, Ohta K. Determination of Atrazine and Simazine in Water Samples by High-Performance Liquid Chromatography After Preconcentra‐ tion with Heat-Treated Diatomaceous Earth. Analytica Chimica Acta 2006; 577

[38] Barrado E, Rodríguez JA, Prieto F, Medina J. Characterization of Iron Oxides Embed‐ ded in Silica Gel Obtained by Two Different Methods. Journal of Non-Crystalline

[39] Aguilar-Arteaga K, Rodriguez JA, Miranda JM, Medina J, Barrado E. Determination of Non-Steroidal Anti-Inflammatory Drugs in Wastewaters by Magnetic Matrix Solid

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Therefore, this research was carried out to evaluate the Africanized honeybee effect on soybean production, mainly, over genetically modified organisms (GMOs). This chapter presents experimental data about floral biology of soybean *Glycine max* L. Merrill, BR-245 RR (transgenic soy – Roundup Read™) and BRS-133 (conventional soy) with and without application of Glyphosate, as well as the honeybee behaviour in those flowers of these varieties.

Soybean (*Glycine max* L. Merrill) is one of the main commodities in the world. Currently, more than 70% of its production is genetically modified. Brazil has a highlighted place concerning about this crop and is the second place in world production. During the 2010/2011 harvest season, Brazil produced 75 million tons of soybean on 24.2 million hectares of planted area with 3,106 kg.ha-1 of productivity. The soybean crop represents the main worldwide oleagi‐ nous crop produced consumed by animals and human beings. After several decades of searching for alternatives to control weed pests, genotypes of genetically modified (GM)

soybean were developed to be resistant to the herbicide glyphosate, of the substituted glycine chemical group.

metabolites inplants,fungi, andmicroorganisms [3].Glyphosate competes withPEPandforms a stable ternary complex with the enzyme and s3p [4, 5]. The safety and efficacy of glyphosate, togetherwiththewidelyusedglyphosate-resistantcropplantscontainingatransgeneforEPSPS,

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The consequences of what this gene can provoke on plant biology is not known; some studies show that the abortion of conventional soybean flowers can reach up to 80%, depending on the cultivar and from the environmental conditions [6]. [7] found on cultivar BRS-133, an average of 53.31 and 82.90% of flowers aborted on the areas in which Africanized honeybees

The correct use of pesticides can arise the productivity of all crops in the world, mainly because pesticides contain biologically active compounds developed for the purpose of protecting plants. Insecticides control pest insect populations; herbicides control weeds, while fungicides are used to control fungal plant diseases [28]. According to Regulation (EC) No.1107/2009, a plant protection product (pesticides) shall be approved only when pesticides "…has no unacceptable acute or chronic effects on colony survival and development, taking into account effects on honeybee larvae and honeybee behaviour" [29]. Neonicotinoid residues in plants and plant parts only become dangerous for bees once they are exposed. Many lethal and sublethal effects of neonicotinoid insecticides on bees have been described in laboratory studies; however, no effects were observed in field studies [34]. Neurotoxic compounds such as neonicotinoids also interfere with the orientation process of honeybees [35]. The toxicity of neonicotinoids may, however, increase by synergistic effects with other compounds as

The sugar concentration of nectar in the flowers determines the frequency of visitors, while the volume determines the quantity of honeybee foragers that will visit [8]. Besides that,

In the central region of the USA, the production of nectar and the visit of honeybees to soybean flowers occur among 9h00 and 15h00 every day. The peak of these activities and the time in which the flowers will remain open vary between the cultivar and the local environmental condition. The contents of total sugar in nectar varied from 37 to 45%, and the sugar of soybean flowers increased and the volume decreased according to the time of day and temperature [9, 46]. Most likely, the sugar concentration in nectar is associated with the intensity of foraging by honeybees, which is directly related to the nectar quantity and quality [47, 48] or to its sugar

The volume of nectar on each flower, which is increased in warm climates, varied significantly between cultivars from 0.2 to 0.5 µL. In another experiment, performed by [10], in Haiti observing the same parameters, the authors verified that the most significant change occurred on the output rate of nectar per flower, which varied from 0.022 to 0.127 µL between cultivars. A study in state of Parana (Brazil), with cultivar BRS-133 [7] showed that the Africanized honeybees searched the soybean culture to collect nectar and pollen. Just a small amount of pollen was from some other areas; however, more than 50% of the total amount of collected

have combined to make glyphosate the most used herbicide in the world [6].

*A. mellifera* were present, and were not present, respectively.

flowers have a period of viability for pollination and fecundation.

pollen by some colonies could have been from soybean [11].

reported by [36].

composition [49, 50, 51].

Brazil increased soybean hectares more than any other country, with an impressive increase of four million hectares in 2009. Brazil is now second in the commercial cultivation of GM plants, 25.4 million hectares, in the whole word [54]. GM soybean, in 2010, occupied 50% of cultivated global area among genetically modified organisms, reaching 73.3 million ha. Since the beginning of commercialization in 1996 until 2010, the tolerance to herbicides is the dominant attribute [54].

The current thought of scientists is increasing the productivity to meet the demand for soybean meal and soybean oil for animal and human consumption, respectively. Despite intensive research in this area with advances in genetically modified genotypes, new techniques in cultivation, irrigation, machine production, and agricultural implements with excellent performance are available. However, pollination, especially that performed by honeybees is little studied.

Around 70% of the world's most produced crop species depends, to some extent, on insect pollination [30], contributing an estimated €153 billion to the global economy, according to [31], and accounting for approximately 6 to 9% of agricultural production [27, 32]. Some authors [30] claim that about one third of the global food production depends on biotic pollination. However, the generally accepted figures are considerably lower [31, 46]. The estimated value of insect pollination for European agriculture is €22 Billion [33].

The scarcity of information about Africanized honeybees in cross-pollination of soybean indicates the need for more research, and for instance, the soybean crop can benefit greatly from biotic pollination with increased profits and production. Therefore, studies in pollination ecology, with special attention to pollinator-flower interaction may contribute to the success in this crop. Although soybean is auto compatible with auto pollination, auto incompatibility occurs too; the increase of production is possible to achieve with pollinator introduction in agricultural areas. A study in floral biology and honeybee behaviour in soybean is necessary to elucidate the effective importance of honeybees for this crop.

Transgenic can aid in genetic improvement of plants, with an aim on food production, fibers and oil, as well as the production of medicines and other industrialized products [1]. One of the main concerns with these plants is that an unanticipated and cumulative effect of contam‐ ination from crossing among genetically modified and conventional plants may occur. The instability and risk of propagating one gene to wild species are critical for environmental conservation.

TheMonsantoCorporationhasdevelopedRoundupReady™soybeancultivarswithatolerance to glyphosate,the active ingredient ofthe weedkillerRoundup®, by the production of enolpyr‐ uvylshikimate -3- phosphate synthase (EPSPS; EC 2.5.1.19) [2]. This enzyme is a carboxyvinyl transferase that catalyzes the transfer ofthe enolpyruvylmoiety ofphosphoenolpyruvate (PEP) to the 5-hydroxyl of shikimate-3-phosphate (S3P) and forms enolpyruvylshikimate-3-phos‐ phate. It is the sixth key enzyme in the shikimate pathway, which is essential for the produc‐ tion of aromatic amino acids Phe, Tyr, and Trp, as well as chorismate-derived secondary metabolites inplants,fungi, andmicroorganisms [3].Glyphosate competes withPEPandforms a stable ternary complex with the enzyme and s3p [4, 5]. The safety and efficacy of glyphosate, togetherwiththewidelyusedglyphosate-resistantcropplantscontainingatransgeneforEPSPS, have combined to make glyphosate the most used herbicide in the world [6].

soybean were developed to be resistant to the herbicide glyphosate, of the substituted glycine

Brazil increased soybean hectares more than any other country, with an impressive increase of four million hectares in 2009. Brazil is now second in the commercial cultivation of GM plants, 25.4 million hectares, in the whole word [54]. GM soybean, in 2010, occupied 50% of cultivated global area among genetically modified organisms, reaching 73.3 million ha. Since the beginning of commercialization in 1996 until 2010, the tolerance to herbicides is the

The current thought of scientists is increasing the productivity to meet the demand for soybean meal and soybean oil for animal and human consumption, respectively. Despite intensive research in this area with advances in genetically modified genotypes, new techniques in cultivation, irrigation, machine production, and agricultural implements with excellent performance are available. However, pollination, especially that performed by honeybees is

Around 70% of the world's most produced crop species depends, to some extent, on insect pollination [30], contributing an estimated €153 billion to the global economy, according to [31], and accounting for approximately 6 to 9% of agricultural production [27, 32]. Some authors [30] claim that about one third of the global food production depends on biotic pollination. However, the generally accepted figures are considerably lower [31, 46]. The

The scarcity of information about Africanized honeybees in cross-pollination of soybean indicates the need for more research, and for instance, the soybean crop can benefit greatly from biotic pollination with increased profits and production. Therefore, studies in pollination ecology, with special attention to pollinator-flower interaction may contribute to the success in this crop. Although soybean is auto compatible with auto pollination, auto incompatibility occurs too; the increase of production is possible to achieve with pollinator introduction in agricultural areas. A study in floral biology and honeybee behaviour in soybean is necessary

Transgenic can aid in genetic improvement of plants, with an aim on food production, fibers and oil, as well as the production of medicines and other industrialized products [1]. One of the main concerns with these plants is that an unanticipated and cumulative effect of contam‐ ination from crossing among genetically modified and conventional plants may occur. The instability and risk of propagating one gene to wild species are critical for environmental

TheMonsantoCorporationhasdevelopedRoundupReady™soybeancultivarswithatolerance to glyphosate,the active ingredient ofthe weedkillerRoundup®, by the production of enolpyr‐ uvylshikimate -3- phosphate synthase (EPSPS; EC 2.5.1.19) [2]. This enzyme is a carboxyvinyl transferase that catalyzes the transfer ofthe enolpyruvylmoiety ofphosphoenolpyruvate (PEP) to the 5-hydroxyl of shikimate-3-phosphate (S3P) and forms enolpyruvylshikimate-3-phos‐ phate. It is the sixth key enzyme in the shikimate pathway, which is essential for the produc‐ tion of aromatic amino acids Phe, Tyr, and Trp, as well as chorismate-derived secondary

estimated value of insect pollination for European agriculture is €22 Billion [33].

to elucidate the effective importance of honeybees for this crop.

chemical group.

278 Herbicides - Advances in Research

dominant attribute [54].

little studied.

conservation.

The consequences of what this gene can provoke on plant biology is not known; some studies show that the abortion of conventional soybean flowers can reach up to 80%, depending on the cultivar and from the environmental conditions [6]. [7] found on cultivar BRS-133, an average of 53.31 and 82.90% of flowers aborted on the areas in which Africanized honeybees *A. mellifera* were present, and were not present, respectively.

The correct use of pesticides can arise the productivity of all crops in the world, mainly because pesticides contain biologically active compounds developed for the purpose of protecting plants. Insecticides control pest insect populations; herbicides control weeds, while fungicides are used to control fungal plant diseases [28]. According to Regulation (EC) No.1107/2009, a plant protection product (pesticides) shall be approved only when pesticides "…has no unacceptable acute or chronic effects on colony survival and development, taking into account effects on honeybee larvae and honeybee behaviour" [29]. Neonicotinoid residues in plants and plant parts only become dangerous for bees once they are exposed. Many lethal and sublethal effects of neonicotinoid insecticides on bees have been described in laboratory studies; however, no effects were observed in field studies [34]. Neurotoxic compounds such as neonicotinoids also interfere with the orientation process of honeybees [35]. The toxicity of neonicotinoids may, however, increase by synergistic effects with other compounds as reported by [36].

The sugar concentration of nectar in the flowers determines the frequency of visitors, while the volume determines the quantity of honeybee foragers that will visit [8]. Besides that, flowers have a period of viability for pollination and fecundation.

In the central region of the USA, the production of nectar and the visit of honeybees to soybean flowers occur among 9h00 and 15h00 every day. The peak of these activities and the time in which the flowers will remain open vary between the cultivar and the local environmental condition. The contents of total sugar in nectar varied from 37 to 45%, and the sugar of soybean flowers increased and the volume decreased according to the time of day and temperature [9, 46]. Most likely, the sugar concentration in nectar is associated with the intensity of foraging by honeybees, which is directly related to the nectar quantity and quality [47, 48] or to its sugar composition [49, 50, 51].

The volume of nectar on each flower, which is increased in warm climates, varied significantly between cultivars from 0.2 to 0.5 µL. In another experiment, performed by [10], in Haiti observing the same parameters, the authors verified that the most significant change occurred on the output rate of nectar per flower, which varied from 0.022 to 0.127 µL between cultivars.

A study in state of Parana (Brazil), with cultivar BRS-133 [7] showed that the Africanized honeybees searched the soybean culture to collect nectar and pollen. Just a small amount of pollen was from some other areas; however, more than 50% of the total amount of collected pollen by some colonies could have been from soybean [11].

The structure of the soybean flower assured the foraging of *A. mellifera,* favoring the pollen transfer [12, 46]. The soybean autogamy and that the self-pollination would guarantee good productivity to the agriculturists, not needing insects to do the pollination [13].

Based on the lack of information of the effect of honeybees on soybean production and, mainly, on genetically modified organisms, this research was carried out to study the floral biology of the *Glycine max*, cultivar BR-245 RR (transgenic soy - Roundup Read™) and BRS-133 (conven‐ tional soybean) and to evaluate the Africanized honeybee *A. mellifera* behaviour in the flowers of these cultivars.

### **2. Materials and methods**

This research was carried out in the experimental area of Empresa Brasileira de Pesquisa Agropecuária (Embrapa Soja), located in Londrina city (23° 08'47" S and 51º 19'11" W), which is situated in the North region of state of Paraná, Brazil. The planting season, the cultivation, and management of the culture and crop occurred in appropriated time, and followed technical recommendations for soybean plants [27].

A completely randomized design was used with three treatments and six replications each. Three treatments were evaluated: covered soybean area with a colony of honeybees inside during the flowering; covered soybean area without a honeybee colony; and an uncovered soybean area, free for insect visitation. In each area, of 24 m2 each, soybean planting was in eight lines, of 6 m, interlaced two by two, with cultivars BR-245 RR and BRS-133. The stand used was 0.5 m between lines and 30 seeds by linear metre (Figure 1).

For covered areas, pollination cages were installed, made with nylon screen (two mm), supported by PVC tubes (¾ inch), and iron (3/8 inch), forming cages in a semi-arch four metres wide, six metres long and two metres high, covering an area of 24 m2 (Figure 2) to prohibit the passage of insects [14].

Each treatment was six parcels, with 24 m2 for each pollination cage and the planting was carried through in eight intercalated lines, two by two, with cultivars BR-245 RR (transgenic soybean - Roundup Ready™), with and without an application of glyphosate (32 days after the germination) and BRS-133 (conventional soybean). Therefore, in each treatment, there were three parcels with an application of herbicide and three parcels without an application. The culture of soybean was monitored during all periods, with particular attention at bloom, which began on December 31 2003 and extended up to January 28 2004. Harvesting was carried out separately line-by-line on the 18 parcels (Figure 2).

In each line, five floral buttons were randomly marked with labels numbered and followed by comments made periodically, during all anthesis period. The stigma receptivity to the pollen grains was evaluated in five flowers collected at 8h00, 11h00 and at 17h00 in five days during the flowering, from January 6 to January 10 2004, in the transgenic and conventional lines of all parcels, following the method of [15].

The stigma of each flower was separated and immersed in hydrogen peroxide (20 volumes) and the air bubbles detached observed and evaluated with scores that varied from zero for non-receptive, one for moderate receptivity and two for high receptivity [7, 40]. For the verification of pollen grain viability, withdrawal of the pollen grains was made from the same flowers used to evaluate receptivity; these were deposited in blades, stained with acetic carmine and stored for posterior analysis, following the technique of [16, 17]. The viability of

**Figure 1.** Pollination cage model used in the experiment with dimensions of 24 m2, below the Africanized honeybee

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colony inside the cage.

Floral Biology and Africanized Honeybee Behaviour in Transgenic (Roundup ReadyTM var. BR-245 RR) and… http://dx.doi.org/10.5772/55847 281

The structure of the soybean flower assured the foraging of *A. mellifera,* favoring the pollen transfer [12, 46]. The soybean autogamy and that the self-pollination would guarantee good

Based on the lack of information of the effect of honeybees on soybean production and, mainly, on genetically modified organisms, this research was carried out to study the floral biology of the *Glycine max*, cultivar BR-245 RR (transgenic soy - Roundup Read™) and BRS-133 (conven‐ tional soybean) and to evaluate the Africanized honeybee *A. mellifera* behaviour in the flowers

This research was carried out in the experimental area of Empresa Brasileira de Pesquisa Agropecuária (Embrapa Soja), located in Londrina city (23° 08'47" S and 51º 19'11" W), which is situated in the North region of state of Paraná, Brazil. The planting season, the cultivation, and management of the culture and crop occurred in appropriated time, and followed technical

A completely randomized design was used with three treatments and six replications each. Three treatments were evaluated: covered soybean area with a colony of honeybees inside during the flowering; covered soybean area without a honeybee colony; and an uncovered

eight lines, of 6 m, interlaced two by two, with cultivars BR-245 RR and BRS-133. The stand

For covered areas, pollination cages were installed, made with nylon screen (two mm), supported by PVC tubes (¾ inch), and iron (3/8 inch), forming cages in a semi-arch four metres wide, six metres long and two metres high, covering an area of 24 m2 (Figure 2) to prohibit the

carried through in eight intercalated lines, two by two, with cultivars BR-245 RR (transgenic soybean - Roundup Ready™), with and without an application of glyphosate (32 days after the germination) and BRS-133 (conventional soybean). Therefore, in each treatment, there were three parcels with an application of herbicide and three parcels without an application. The culture of soybean was monitored during all periods, with particular attention at bloom, which began on December 31 2003 and extended up to January 28 2004. Harvesting was carried out

In each line, five floral buttons were randomly marked with labels numbered and followed by comments made periodically, during all anthesis period. The stigma receptivity to the pollen grains was evaluated in five flowers collected at 8h00, 11h00 and at 17h00 in five days during the flowering, from January 6 to January 10 2004, in the transgenic and conventional lines of

each, soybean planting was in

for each pollination cage and the planting was

productivity to the agriculturists, not needing insects to do the pollination [13].

of these cultivars.

280 Herbicides - Advances in Research

**2. Materials and methods**

passage of insects [14].

recommendations for soybean plants [27].

Each treatment was six parcels, with 24 m2

separately line-by-line on the 18 parcels (Figure 2).

all parcels, following the method of [15].

soybean area, free for insect visitation. In each area, of 24 m2

used was 0.5 m between lines and 30 seeds by linear metre (Figure 1).

**Figure 1.** Pollination cage model used in the experiment with dimensions of 24 m2, below the Africanized honeybee colony inside the cage.

The stigma of each flower was separated and immersed in hydrogen peroxide (20 volumes) and the air bubbles detached observed and evaluated with scores that varied from zero for non-receptive, one for moderate receptivity and two for high receptivity [7, 40]. For the verification of pollen grain viability, withdrawal of the pollen grains was made from the same flowers used to evaluate receptivity; these were deposited in blades, stained with acetic carmine and stored for posterior analysis, following the technique of [16, 17]. The viability of

the maturation phase, the string beans of each plant were counted to get the percentage of the

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The frequency of the insect visits, during the day, was obtained by counting them, observing during 10 minutes in each schedule (8h00 to 17h00) in the transgenic and conventional lines of the covered area by pollination cages with honeybees and in the area of free insect visitation. The insects most frequent were photographed, filmed, collected with an entomological net,

The time of nectar and/or pollen collection was evaluated with a chronometer, having followed the honeybee forager in its activity. In addition, the time of permanence of the honeybee in the

flower and the number of visits per minute were counted and recorded.

**Figure 3.** A transgenic soybean plant in flowering, and in zoom showing the abortion of the flowers.

To observe the type of collection (nectar and/or pollen) carried through by the *A. mellifera*, six honeybees were collected each hour (9h00 to 16h00) in all the parcels for three days in the area with honeybees and in the area of free insect visitation. Corbicula content and honey stomach content was recorded with scores that varied from zero for empty (honey stomach and corbicula), one for moderate, two for full and three for maximum load. Evaluation of the total

aborted flowers (Figure 3).

fixed and later, identified by specialists.

**Figure 2.** The soybean harvesting carried out separately line-by-line on the 18 parcels in the experimental area of Em‐ brapa – Soja in Londrina-PR, Brazil.

the pollen grains removed from the honeybee forager corbicula was also verified using this same technique.

The percentage of abortion in the soybean flowers was measured by counting all floral blossoms of three marked plants of each line with ribbons of different colors, in all parcels. In the maturation phase, the string beans of each plant were counted to get the percentage of the aborted flowers (Figure 3).

The frequency of the insect visits, during the day, was obtained by counting them, observing during 10 minutes in each schedule (8h00 to 17h00) in the transgenic and conventional lines of the covered area by pollination cages with honeybees and in the area of free insect visitation. The insects most frequent were photographed, filmed, collected with an entomological net, fixed and later, identified by specialists.

The time of nectar and/or pollen collection was evaluated with a chronometer, having followed the honeybee forager in its activity. In addition, the time of permanence of the honeybee in the flower and the number of visits per minute were counted and recorded.

**Figure 3.** A transgenic soybean plant in flowering, and in zoom showing the abortion of the flowers.

the pollen grains removed from the honeybee forager corbicula was also verified using this

**Figure 2.** The soybean harvesting carried out separately line-by-line on the 18 parcels in the experimental area of Em‐

The percentage of abortion in the soybean flowers was measured by counting all floral blossoms of three marked plants of each line with ribbons of different colors, in all parcels. In

same technique.

brapa – Soja in Londrina-PR, Brazil.

282 Herbicides - Advances in Research

To observe the type of collection (nectar and/or pollen) carried through by the *A. mellifera*, six honeybees were collected each hour (9h00 to 16h00) in all the parcels for three days in the area with honeybees and in the area of free insect visitation. Corbicula content and honey stomach content was recorded with scores that varied from zero for empty (honey stomach and corbicula), one for moderate, two for full and three for maximum load. Evaluation of the total sugar concentration was performed by placing the crop content in a manual refractometer and recording the brix value.

The anthesis period in hours was, on average, 8h04min greater (*P*=0.0001) in the pollination cages without honeybee colonies than in the pollination cages with honeybee colonies and then in the uncovered area for free insect visitation (*P*>0.05). In Table 1, there is a summary of the variance analysis and the average comparison of the anthesis period in the three treatments,

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The receptivity of the stigma was not influenced by cultivar (*P*>0.05) with a general average of 87.43%, and was not influenced by treatments either (P>0.05) which had a general mean of 88.71%. There was no difference (*P*> 0.05) in the percentage of viable pollen grains in the soybean flowers between treatments and the general average was 89.82% ±7.27 (Table 2).

The percentages of viable pollen grains removed from corbicula honeybee forager in the pollination cages with honeybee colonies and in the areas of free insect visitation were not

In the analysis of the removed pollen grains from corbicula of honeybee forager, 100% were

**Variation source Anthesis period in hours**

different (*P*> 0.05) and the average was 99.95% (±0.14).

CV % of cultivar 10.24

CV % of treatments 15.27

of soybean, showing a high constancy of the honeybees to this crop.

Cultivar 0.13 *P* = 0.7243

Treatments 3.26 *P*= 0.0104

Transgenic soy 42.37 a\* (±6.83) Conventional soy 42.89 a (±6.36) Covered area with honeybees 37.90 b (±4.05) Covered area without honeybees 48.00 a (±3.72) Uncovered area 41.98 b (±6.95)

Means followed by different letters, in the same column, are different by Tukey's test (*P*< 0.05)

and BRS-133 (conventional soybean), in experimental area of EMBRAPA Soja, in Londrina-PR, Brazil.

Cultivar 0.52 *P =* 0.9100 0.53 *P* = 0.4668

Treatments 0.87 *P* = 0.4816 0.62 *P* = 0.6167

Transgenic soy 87.65 a (±6.19) 90.14 a (±7.52) Conventional soy 87.20 a (±6.52) 89.51 a (±7.07) Covered area with honeybees 89.81 a\* (±7.04) 89.22 a (±7.70)

CV % of cultivar 8.48 8.19

CV % of treatments 7.17 8.11

**Table 1.** F values with respective probability (*P*), coefficient of variation (CV%), means and their standard deviation of anthesis period, in hours, for soybean flowers, *Glycine max*, var. BR-245 RR (transgenic soybean - Roundup Ready™)

**Variation source Stigma receptivity (%) Viable pollen grains (%)**

and two cultivars.

The statistical analyses of the variables were carried out with Statistical Analysis System [18] software using the following model:

Yijklm= µ + Bi + Hj + Tk + Cl + (BH)ij + (BT)ik + (BC)jl + (HT)jk + (HC)jl + (TC)kl + and ijklm, where,

Yijklm= Observed in reference to variants of Block i, Herbicide j, Treatment k, Cultivar l

µ is the effect of general average;

Bi is the effect of Block (i = 1, 2... 6);

Hj is the effect of herbicide (j = 1, 2);

Tk is the effect of treatment (1, 2, 3);

Cl is the effect of cultivar;

(BH)ij is the interaction of Block i and Herbicide J;

(BT)ik is the interaction of Block i and Treatment k;

(BC)jl is the interaction of Block i and Cultivar l;

(HT)jk is the interaction of Herbicide j and the Treatment k;

(HC)jl is the interaction of Herbicide i and the Cultivar l;

eijklm is the mistake associated to the observation ijklm.

The delineation used was of completely randomized blocks and the data submitted to the variance analysis. Tukey's test was used for comparison of the averages.

Frequency of stigma receptivity was analyzed by the NPAR1WAY Wilcoxon procedure and the viability was estimated by the Kruskal-Wallis test.

Data without normal distributions were analyzed using generalized linear models [19], assuming Poisson distribution with logarithmic link function.

For analysis of the type of collection, the model considers the treatment effect as a fixed variable, random for the effect of the interaction treatment versus day and the linear effect, squared and cubical for the hour collection.

### **3. Results**

There was no effect (*P*> 0.05) of cultivar, from the application of the herbicide and the inter‐ actions of these variables, therefore floral biology and behaviour of the insects in two cultivars were similar (*P*> 0.05), independent of the herbicide used, but the presence of insects influenced these parameters (Table 1).

The anthesis period in hours was, on average, 8h04min greater (*P*=0.0001) in the pollination cages without honeybee colonies than in the pollination cages with honeybee colonies and then in the uncovered area for free insect visitation (*P*>0.05). In Table 1, there is a summary of the variance analysis and the average comparison of the anthesis period in the three treatments, and two cultivars.

sugar concentration was performed by placing the crop content in a manual refractometer and

The statistical analyses of the variables were carried out with Statistical Analysis System [18]

The delineation used was of completely randomized blocks and the data submitted to the

Frequency of stigma receptivity was analyzed by the NPAR1WAY Wilcoxon procedure and

Data without normal distributions were analyzed using generalized linear models [19],

For analysis of the type of collection, the model considers the treatment effect as a fixed variable, random for the effect of the interaction treatment versus day and the linear effect,

There was no effect (*P*> 0.05) of cultivar, from the application of the herbicide and the inter‐ actions of these variables, therefore floral biology and behaviour of the insects in two cultivars were similar (*P*> 0.05), independent of the herbicide used, but the presence of insects influenced

Yijklm= Observed in reference to variants of Block i, Herbicide j, Treatment k, Cultivar l

+ (BH)ij + (BT)ik + (BC)jl + (HT)jk + (HC)jl + (TC)kl + and ijklm, where,

recording the brix value.

284 Herbicides - Advances in Research

+ Hj

µ is the effect of general average;

is the effect of Block (i = 1, 2... 6);

is the effect of herbicide (j = 1, 2);

(BH)ij is the interaction of Block i and Herbicide J; (BT)ik is the interaction of Block i and Treatment k;

(BC)jl is the interaction of Block i and Cultivar l;

(HT)jk is the interaction of Herbicide j and the Treatment k;

variance analysis. Tukey's test was used for comparison of the averages.

(HC)jl is the interaction of Herbicide i and the Cultivar l; eijklm is the mistake associated to the observation ijklm.

the viability was estimated by the Kruskal-Wallis test.

squared and cubical for the hour collection.

**3. Results**

these parameters (Table 1).

assuming Poisson distribution with logarithmic link function.

Tk is the effect of treatment (1, 2, 3);

is the effect of cultivar;

Yijklm= µ + Bi

Bi

Hj

Cl

software using the following model:

+ Tk + Cl

The receptivity of the stigma was not influenced by cultivar (*P*>0.05) with a general average of 87.43%, and was not influenced by treatments either (P>0.05) which had a general mean of 88.71%. There was no difference (*P*> 0.05) in the percentage of viable pollen grains in the soybean flowers between treatments and the general average was 89.82% ±7.27 (Table 2).

The percentages of viable pollen grains removed from corbicula honeybee forager in the pollination cages with honeybee colonies and in the areas of free insect visitation were not different (*P*> 0.05) and the average was 99.95% (±0.14).

In the analysis of the removed pollen grains from corbicula of honeybee forager, 100% were of soybean, showing a high constancy of the honeybees to this crop.


Means followed by different letters, in the same column, are different by Tukey's test (*P*< 0.05)

**Table 1.** F values with respective probability (*P*), coefficient of variation (CV%), means and their standard deviation of anthesis period, in hours, for soybean flowers, *Glycine max*, var. BR-245 RR (transgenic soybean - Roundup Ready™) and BRS-133 (conventional soybean), in experimental area of EMBRAPA Soja, in Londrina-PR, Brazil.



The nectar collection throughout the day varied and differed in the amount of honeybees in the different schedules (*P*=0.0001), but did not differ (*P*> 0.05) between the covered area by cages with honeybees present and of free insect visitation. In these two areas, the moment of greatest visitation of the *A. mellifera* was at 12h34min. The frequency of honeybees with a larger amount of nectar in the covered areas by cages with honeybees and of free insect visitation is

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In the area of free insect visitation, the time that *A. mellifera* spent collecting nectar was 2.59 ±0.52 seconds/flower which was less (*P*=0.0001) than the observed time in the pollination cages with honeybee colonies (2.99 ±0.54 seconds/flower). Regardless of treatment, honeybee *A. mellifera* spent, on average, 2.74 ±0.56 seconds/flower in nectar collection and 4.37 ±0.62 seconds/flower in pollen collection and this difference was significant (*P*=0.0001). *A. mellifera* visited 7.14 (±0.26) flowers/min. collecting nectar and 3.75 (±0.30) flowers/min in pollen

The average concentration of total sugar removed from the honey stomach (called crop) of the honeybees that collected nectar throughout the day was of 41.19% ±3.72, in pollination cages with honeybee colonies inside; this was greater (*P*=0.0008) than the 38.22% ±3.37 measured from the area of free insect visitation (Figure 7). In these two areas there were also a difference

represented in Figure 6.

(P=0.0001) between the collecting schedule.

**Figure 4.** Lepidoptera in soybean flower

collection.

**Table 2.** F values with respective probability (*P*), coefficient of variation (CV%), means and their standard deviation of stigma receptivity and viable pollen grains (%), in soybean flowers *Glycine max*, var. BR-245 RR (transgenic soybean - Roundup Ready™), and BRS-133 (conventional soybean) in experimental area of EMBRAPA Soja, in Londrina-PR, Brazil.

The percentage of aborted flowers was greater (*P*=0.0001) in the covered area by cages without honeybees in relation to the covered areas by cages with honeybee colony and of free insect visitation; these last treatments did not differ (*P*>0.05). The averages and the standard deviation of the abortion percentage in the soybean flowers in the treatments are presented in Table 3.


Means followed by different letters, in the same column, are different by Tukey's test (*P*< 0.05)

**Table 3.** F values with respective probability (*P*), coefficient of variation (CV%), means and their standard deviation of abortion percentage of soybean flowers *Glycine max*, var. BR-245 RR (transgenic soybean - Roundup Ready™), and BRS-133 (conventional soybean) in experimental area of EMBRAPA Soja, in Londrina-PR, Brazil.

In the area of free insect visitation, *A. mellifera* was the species most abundant with 97.02% frequency. Other bee species with 1.65%10 frequency had been observed also, and, Lepidop‐ tera with 1.33% (Figure 4). Amongst the collected bees, there were nine species, six from the *Apidae* family, two from *Megachilidae* and one from *Halictidae*.

The behaviour for the type of collection observed in workers of *A. mellifera* foraging the soybean flowers was typical of nectar collecting, although, workers with unique behavior for pollen collection was observed on occasion (Figure 5).

In an accurate evaluation of the honey stomach contents and corbicula, it was observed that, throughout the day, 69.39% of the foragers had collected nectar, 37.05% had collected nectar and pollen and 2.56% had only collected pollen.

The nectar collection throughout the day varied and differed in the amount of honeybees in the different schedules (*P*=0.0001), but did not differ (*P*> 0.05) between the covered area by cages with honeybees present and of free insect visitation. In these two areas, the moment of greatest visitation of the *A. mellifera* was at 12h34min. The frequency of honeybees with a larger amount of nectar in the covered areas by cages with honeybees and of free insect visitation is represented in Figure 6.

In the area of free insect visitation, the time that *A. mellifera* spent collecting nectar was 2.59 ±0.52 seconds/flower which was less (*P*=0.0001) than the observed time in the pollination cages with honeybee colonies (2.99 ±0.54 seconds/flower). Regardless of treatment, honeybee *A. mellifera* spent, on average, 2.74 ±0.56 seconds/flower in nectar collection and 4.37 ±0.62 seconds/flower in pollen collection and this difference was significant (*P*=0.0001). *A. mellifera* visited 7.14 (±0.26) flowers/min. collecting nectar and 3.75 (±0.30) flowers/min in pollen collection.

The average concentration of total sugar removed from the honey stomach (called crop) of the honeybees that collected nectar throughout the day was of 41.19% ±3.72, in pollination cages with honeybee colonies inside; this was greater (*P*=0.0008) than the 38.22% ±3.37 measured from the area of free insect visitation (Figure 7). In these two areas there were also a difference (P=0.0001) between the collecting schedule.

**Figure 4.** Lepidoptera in soybean flower

**Variation source Stigma receptivity (%) Viable pollen grains (%)**

**Table 2.** F values with respective probability (*P*), coefficient of variation (CV%), means and their standard deviation of stigma receptivity and viable pollen grains (%), in soybean flowers *Glycine max*, var. BR-245 RR (transgenic soybean - Roundup Ready™), and BRS-133 (conventional soybean) in experimental area of EMBRAPA Soja, in Londrina-PR,

The percentage of aborted flowers was greater (*P*=0.0001) in the covered area by cages without honeybees in relation to the covered areas by cages with honeybee colony and of free insect visitation; these last treatments did not differ (*P*>0.05). The averages and the standard deviation of the abortion percentage in the soybean flowers in the treatments are presented in Table 3.

**Variation source Abortion percentage of flowers**

Covered area whithout honeybees 87.50 a (±4.81) 90.38 a (±6.64) Uncovered area 84.72 a (±6.28) 89.86 a (±7.46) Means followed by different letters, in the same column, are different by Tukey's test (*P*< 0.05)

Cultivar 2.55 *P=*0.1302

Treatments 13.21 *P*< 0.0001

Transgenic soy 73.51 a\* (±9.54) Conventional soy 80.21 a (±7.03) Covered area with honeybees 50.78 b\* (±8.30) Covered area without honeybees 71.10 A (±3.05) Uncovered area 55.12 B (±5.84)

Means followed by different letters, in the same column, are different by Tukey's test (*P*< 0.05)

BRS-133 (conventional soybean) in experimental area of EMBRAPA Soja, in Londrina-PR, Brazil.

*Apidae* family, two from *Megachilidae* and one from *Halictidae*.

collection was observed on occasion (Figure 5).

and pollen and 2.56% had only collected pollen.

**Table 3.** F values with respective probability (*P*), coefficient of variation (CV%), means and their standard deviation of abortion percentage of soybean flowers *Glycine max*, var. BR-245 RR (transgenic soybean - Roundup Ready™), and

In the area of free insect visitation, *A. mellifera* was the species most abundant with 97.02% frequency. Other bee species with 1.65%10 frequency had been observed also, and, Lepidop‐ tera with 1.33% (Figure 4). Amongst the collected bees, there were nine species, six from the

The behaviour for the type of collection observed in workers of *A. mellifera* foraging the soybean flowers was typical of nectar collecting, although, workers with unique behavior for pollen

In an accurate evaluation of the honey stomach contents and corbicula, it was observed that, throughout the day, 69.39% of the foragers had collected nectar, 37.05% had collected nectar

CV % of cultivar 6.55

CV % of treatments 18.08

Brazil.

286 Herbicides - Advances in Research

**Figure 7.** Regression curve obtained through the equation Y=41.19+0.9127(h-12.5)-0.3836(h-12.5)2 in covered area with honeybee colony and Y=38.22+0.9127(h-12.5)-0.3836(h-12.5)2 in uncovered area for free insect visitation show‐ ing the total sugar, in Brix, measured from worker crop content of captured honeybees, foraging soybean flowers *Glycine max*, var. BR-245 RR (transgenic soybean - Roundup Read™) and BRS-133 (conventional soybean) in experi‐

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289

The results showed that the genetically modified cultivar, regardless of receiving an applica‐ tion of the herbicide had a similar pattern as the conventional cultivar for the analyzed parameters. This showed that the transgenes did not interfere in the analyzed parameters, like floral biology, and the Africanized honeybee behaviour. The bees, mainly honeybees, are considered the most effective pollinator in nature, principally, in cultivated plants like soybean [39]. The honeybee has become a domesticated animal; they can be managed, moved to a new

When evaluating the outcome of Africanized honeybee presence on soybean culture, the parameters of anthesis and the percentage of aborted flowers decreased just as much on the transgenic cultivar as the conventional one. [37] describes an excellent protocol to be applied to focal crops at the farm-scale level to assess pollinator density and diversity for comparison purposes among different sites. It becomes an important tool for researchers and professors who work in pollination because in several areas, one of the most difficult things is to compare

The anthesis period was, on average, 20.49% longer in the area where insects had been prevented from visiting the flowers than in the pollination cages with honeybee colonies and the area for free insect visitation. Similar results have been observed by [7] on BRS-133, which

site, divided, mature, and stimulated to go to a specific crop for a specific time.

mental area of EMBRAPA Soja, in Londrina-PR, Brazil.

results that are obtained by different methods.

**4. Discussion**

**Figure 5.** The honeybee searching a reward in soybean flower

**Figure 6.** Regression curve obtained through the equation: Y=exp(-7.111+1.231xh-0.049xh2) of number of worker honeybees collecting nectar during the day in covered area with honeybee colony inside, and Y=exp(-7.111+1.318xh-0.049xh2) in the area for free insect visitation, in soybean *Glycine max*, var. BR-245 RR (transgenic soybean - Roundup Ready™) and BRS-133 (conventional soybean) in experimental area of EMBRAPA Soja, in Londrina-PR, Brazil.

**Figure 7.** Regression curve obtained through the equation Y=41.19+0.9127(h-12.5)-0.3836(h-12.5)2 in covered area with honeybee colony and Y=38.22+0.9127(h-12.5)-0.3836(h-12.5)2 in uncovered area for free insect visitation show‐ ing the total sugar, in Brix, measured from worker crop content of captured honeybees, foraging soybean flowers *Glycine max*, var. BR-245 RR (transgenic soybean - Roundup Read™) and BRS-133 (conventional soybean) in experi‐ mental area of EMBRAPA Soja, in Londrina-PR, Brazil.

### **4. Discussion**

**Figure 5.** The honeybee searching a reward in soybean flower

288 Herbicides - Advances in Research

**Figure 6.** Regression curve obtained through the equation: Y=exp(-7.111+1.231xh-0.049xh2) of number of worker honeybees collecting nectar during the day in covered area with honeybee colony inside, and Y=exp(-7.111+1.318xh-0.049xh2) in the area for free insect visitation, in soybean *Glycine max*, var. BR-245 RR (transgenic soybean - Roundup Ready™) and BRS-133

(conventional soybean) in experimental area of EMBRAPA Soja, in Londrina-PR, Brazil.

The results showed that the genetically modified cultivar, regardless of receiving an applica‐ tion of the herbicide had a similar pattern as the conventional cultivar for the analyzed parameters. This showed that the transgenes did not interfere in the analyzed parameters, like floral biology, and the Africanized honeybee behaviour. The bees, mainly honeybees, are considered the most effective pollinator in nature, principally, in cultivated plants like soybean [39]. The honeybee has become a domesticated animal; they can be managed, moved to a new site, divided, mature, and stimulated to go to a specific crop for a specific time.

When evaluating the outcome of Africanized honeybee presence on soybean culture, the parameters of anthesis and the percentage of aborted flowers decreased just as much on the transgenic cultivar as the conventional one. [37] describes an excellent protocol to be applied to focal crops at the farm-scale level to assess pollinator density and diversity for comparison purposes among different sites. It becomes an important tool for researchers and professors who work in pollination because in several areas, one of the most difficult things is to compare results that are obtained by different methods.

The anthesis period was, on average, 20.49% longer in the area where insects had been prevented from visiting the flowers than in the pollination cages with honeybee colonies and the area for free insect visitation. Similar results have been observed by [7] on BRS-133, which verified an average increase of 20.38% into the anthesis period in the area that insects were prevented from visiting in relation to the areas where the insects had access. In agriculture and horticulture pollination management, a good pollenizer is a plant that provides compatible, viable and plentiful pollen and blooms at the same time as the plant that is to be pollinated or has pollen that can be used and/or stored when needed to pollinate the desired flowers.

increase of productivity. The larger part of agricultural land consists of cultivated areas like fields. However, an amount of space remains that is often quantitatively underestimated, and could be managed to promote plant and biotope diversity. Natural or semi-natural habitat remnants provide nesting sites and reliable food sources for pollinators. Conserving these areas can benefit biodiversity and offer potential for improved crop productivity [28]. Soybean is largely used in Brazilian agriculture in extensive systems called monoculture. Therefore, in these areas it is easier to implant management programs for rational use of pesticides focusing

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291

The comments regarding the collecting behavior have shown that *A. mellifera* visits the soybean flowers intensely, mainly, for nectar collection and, in part of the morning, from 9h00 to 11h30, the nectar and pollen collection was intense. On occasion, the behavior of foragers collecting pollen was observed. These results are in accordance with [23] and [24], which observed a great number of worker honeybees visiting soybean flowers. Even with nectar collecting behavior, *A. mellifera* was efficient as a pollinator, strengthening the affirmations of [11] that the honeybee does not necessarily need to be pollen collectors to make an efficient pollinator since they are able to accomplish that as they collect nectar. [7] observed intense visits of *A. mellifera* in the cv. BRS-133 collecting, predominantly, nectar and had an increase of 58.86% in the areas with *A. mellifera* in relation to the area with insect restriction. Honeybee nectar foragers were more frequent (2.28 bees per chapter) than pollen foragers (0.40 bees per chapter) in sunflowers and seed yield was 43% higher (*P <* 0.05) from sunflower plants that were visited by pollinator-

The evaluation of amount of total sugars present in the crop content of the foragers showed that the harvested nectar awakened the interest of the *A. mellifera* for the visitation of these cultivars of soybean. The consistency of the honeybees to the soybean showed that the flowers of this species satisfies its nutritional requirements and they do not need to visit other species, even if there are other flowers available and some very attractive, such as sunflower (*Helianthus*

In the research of [41] and [42], the means of sucrose.hexose-1 (S/H) per flower for all treatments were: 0.91 µg.µL-1, for covered areas with Africanized honeybee colonies – rich in sucrose; 0.74 µg.µL-1, semi-covered areas with free insect visitation – rich in sucrose; 0.86 µg.µL-1, uncovered areas with free insect visitation – rich in sucrose; and 3.05 µg.µL-1, for covered areas without Africanized honeybee colonies – sucrose dominant. According to [43], this range suggests that sugar concentration in soybean nectar is influenced by other environmental factors independ‐ ently of pollinator action. [44] reported that edaphic and climatic factors affect the number of flowers and other floral characteristics during soybean growing. Therefore, the environmental conditions that generate an increase in number and size of flowers, longer anthesis period, more intense coloring, and greater nectar production are the factors responsible for making flowers more attractive to honeybees [42]. [45] suggested that the maximum nectar accumu‐

Technological innovations play an important role in pollinator protection. Application technologies allow for reductions in spray drift; this helps prevent pesticide residues in nontarget areas. This is achieved with application nozzles that create spray droplets large enough

on the reduction of negative effects on the bees [38].

insects than plants restricted to pollinators [25].

lation occurs before or at the beginning of pollination activity.

to be less affected by wind [28].

*annuus*).

An increase in the anthesis period in areas that insects were prevented from visiting has also occurred with other species of plants. [18] observed an increase of 43.5% into the anthesis period in flowers of siratro (*Macroptilium atropurpureum* Urb.) when they had been prevented from having insect visitation. The outcomes of the above mentioned authors show that the Africanized honeybees have been efficient in the pollination process, since that anthesis period was smaller in areas for free insect visitation than flowers protected from them, which presented a greater anthesis period and when the Africanized honeybees were present, the anthesis period of soybean and *M. atropurpureum* was smaller.

The stigma receptivity was similar between treatments. Similar results had been observed by [18] in the *M. atropurpureum* flowers which had 76.9% of the blossoms and 91.95% of the flowers opened in the receptive stigma to pollen grains.

The high viability of the pollen grains in all treatments, 99.6% (±0.02) on average, suggest that these do not depend on the conditions imposed on the flowers in this cultivar. [18] found 100% of viability in the pollen grains in *M. atropurpureum*. [7] on cultivar BRS-133 obtained 99.86% of viable pollen grains.

In the analysis of the removed pollen grains from corbicula of foragers in the area of free insect visitation, no pollen from other flowers was found. These results are in accordance with [7] that observed 93 to 100% of honeybee foragers collected pollen in one single species. These results have shown that the alimentary resource offered by the soybean flower satisfies the requirements of *A. mellifera* and they did not need to visit other cultures while the samples were being collected.

The number of aborted flowers was 40.02% greater in the covered area by cages without honeybees in relation to the covered area by cages with honeybees, and 28.99% greater than in the area of free insect visitation. These results match the results of [20] that observed high rates of abortion in soybean, varying from 43% to 87%, depending on the cultivar. [9] had 75% of the flowers aborted in some cultivars, [11] had superior rates of more than 75% of abortion and [7] found a rate of abortion 53.66% greater in areas protected of insect visitation to flowers compared with covered area of cages with *A. mellifera*. This biological response of the flower shows that, even if soybean is considered to have autogamy by researchers such as [13], [21], it probably also possesses a mechanism of genetic auto-incompatibility to prevent 100% of selffertilization, as observed on plants of the genus *Brassica* by [22]. This evidences the importance of honeybees for the reduction in the abortion percentage and, consequently, increases the amount of string beans and seeds in soybean.

The largest frequency of *A. mellifera* (97.02%) in soybean flowers in the area for free insect visitation was similar to the results obtained by [7], which found 95.18% of this species visiting soybean flowers on cultivar BRS-133. Therefore, in this cultivar of soybean, the presence of *A. mellifera* is important not only for honey production but also for pollination, and mainly for an increase of productivity. The larger part of agricultural land consists of cultivated areas like fields. However, an amount of space remains that is often quantitatively underestimated, and could be managed to promote plant and biotope diversity. Natural or semi-natural habitat remnants provide nesting sites and reliable food sources for pollinators. Conserving these areas can benefit biodiversity and offer potential for improved crop productivity [28]. Soybean is largely used in Brazilian agriculture in extensive systems called monoculture. Therefore, in these areas it is easier to implant management programs for rational use of pesticides focusing on the reduction of negative effects on the bees [38].

verified an average increase of 20.38% into the anthesis period in the area that insects were prevented from visiting in relation to the areas where the insects had access. In agriculture and horticulture pollination management, a good pollenizer is a plant that provides compatible, viable and plentiful pollen and blooms at the same time as the plant that is to be pollinated or has pollen that can be used and/or stored when needed to pollinate the desired flowers.

An increase in the anthesis period in areas that insects were prevented from visiting has also occurred with other species of plants. [18] observed an increase of 43.5% into the anthesis period in flowers of siratro (*Macroptilium atropurpureum* Urb.) when they had been prevented from having insect visitation. The outcomes of the above mentioned authors show that the Africanized honeybees have been efficient in the pollination process, since that anthesis period was smaller in areas for free insect visitation than flowers protected from them, which presented a greater anthesis period and when the Africanized honeybees were present, the

The stigma receptivity was similar between treatments. Similar results had been observed by [18] in the *M. atropurpureum* flowers which had 76.9% of the blossoms and 91.95% of the flowers

The high viability of the pollen grains in all treatments, 99.6% (±0.02) on average, suggest that these do not depend on the conditions imposed on the flowers in this cultivar. [18] found 100% of viability in the pollen grains in *M. atropurpureum*. [7] on cultivar BRS-133 obtained 99.86%

In the analysis of the removed pollen grains from corbicula of foragers in the area of free insect visitation, no pollen from other flowers was found. These results are in accordance with [7] that observed 93 to 100% of honeybee foragers collected pollen in one single species. These results have shown that the alimentary resource offered by the soybean flower satisfies the requirements of *A. mellifera* and they did not need to visit other cultures while the samples

The number of aborted flowers was 40.02% greater in the covered area by cages without honeybees in relation to the covered area by cages with honeybees, and 28.99% greater than in the area of free insect visitation. These results match the results of [20] that observed high rates of abortion in soybean, varying from 43% to 87%, depending on the cultivar. [9] had 75% of the flowers aborted in some cultivars, [11] had superior rates of more than 75% of abortion and [7] found a rate of abortion 53.66% greater in areas protected of insect visitation to flowers compared with covered area of cages with *A. mellifera*. This biological response of the flower shows that, even if soybean is considered to have autogamy by researchers such as [13], [21], it probably also possesses a mechanism of genetic auto-incompatibility to prevent 100% of selffertilization, as observed on plants of the genus *Brassica* by [22]. This evidences the importance of honeybees for the reduction in the abortion percentage and, consequently, increases the

The largest frequency of *A. mellifera* (97.02%) in soybean flowers in the area for free insect visitation was similar to the results obtained by [7], which found 95.18% of this species visiting soybean flowers on cultivar BRS-133. Therefore, in this cultivar of soybean, the presence of *A. mellifera* is important not only for honey production but also for pollination, and mainly for an

anthesis period of soybean and *M. atropurpureum* was smaller.

opened in the receptive stigma to pollen grains.

amount of string beans and seeds in soybean.

of viable pollen grains.

290 Herbicides - Advances in Research

were being collected.

The comments regarding the collecting behavior have shown that *A. mellifera* visits the soybean flowers intensely, mainly, for nectar collection and, in part of the morning, from 9h00 to 11h30, the nectar and pollen collection was intense. On occasion, the behavior of foragers collecting pollen was observed. These results are in accordance with [23] and [24], which observed a great number of worker honeybees visiting soybean flowers. Even with nectar collecting behavior, *A. mellifera* was efficient as a pollinator, strengthening the affirmations of [11] that the honeybee does not necessarily need to be pollen collectors to make an efficient pollinator since they are able to accomplish that as they collect nectar. [7] observed intense visits of *A. mellifera* in the cv. BRS-133 collecting, predominantly, nectar and had an increase of 58.86% in the areas with *A. mellifera* in relation to the area with insect restriction. Honeybee nectar foragers were more frequent (2.28 bees per chapter) than pollen foragers (0.40 bees per chapter) in sunflowers and seed yield was 43% higher (*P <* 0.05) from sunflower plants that were visited by pollinatorinsects than plants restricted to pollinators [25].

The evaluation of amount of total sugars present in the crop content of the foragers showed that the harvested nectar awakened the interest of the *A. mellifera* for the visitation of these cultivars of soybean. The consistency of the honeybees to the soybean showed that the flowers of this species satisfies its nutritional requirements and they do not need to visit other species, even if there are other flowers available and some very attractive, such as sunflower (*Helianthus annuus*).

In the research of [41] and [42], the means of sucrose.hexose-1 (S/H) per flower for all treatments were: 0.91 µg.µL-1, for covered areas with Africanized honeybee colonies – rich in sucrose; 0.74 µg.µL-1, semi-covered areas with free insect visitation – rich in sucrose; 0.86 µg.µL-1, uncovered areas with free insect visitation – rich in sucrose; and 3.05 µg.µL-1, for covered areas without Africanized honeybee colonies – sucrose dominant. According to [43], this range suggests that sugar concentration in soybean nectar is influenced by other environmental factors independ‐ ently of pollinator action. [44] reported that edaphic and climatic factors affect the number of flowers and other floral characteristics during soybean growing. Therefore, the environmental conditions that generate an increase in number and size of flowers, longer anthesis period, more intense coloring, and greater nectar production are the factors responsible for making flowers more attractive to honeybees [42]. [45] suggested that the maximum nectar accumu‐ lation occurs before or at the beginning of pollination activity.

Technological innovations play an important role in pollinator protection. Application technologies allow for reductions in spray drift; this helps prevent pesticide residues in nontarget areas. This is achieved with application nozzles that create spray droplets large enough to be less affected by wind [28].

One of the greatest concerns of Brazilian farmers in relation to GM plants is the unexpected and cumulative effect of cross-contamination between GM plants and conventional plants. The instability and risk of propagation of a gene to wild species are critical to maintenance of the environment [52]. Genetic pollution is inevitable and the transgenic pollen may contaminate conventional or biological fields located several kilometres from GM plantations [55].

with a honeybee colony and in the area of free insect visitation. The average of the analyzed stigma receptivity was 87.35% and there was no difference between treatments. The average of viable pollen was 89.82% with no difference between treatments. In the analysis of the removed pollen grains from the honeybee forager corbicula, 100% observed belonging to soybean. The flower abortion rate was 71.10% in covered areas without honeybee colonies, which was greater by 50.78% and 55.12%, respectively, than the covered area with a honeybee colony and area of free insect visitation. *A. mellifera* was the insect that most frequently (97.02%) visited the soybean flowers. The time that *A. mellifera* spent to collect nectar was greater in the covered area with a honeybee colony than in the area of free insect visitation. The average time foragers spent for nectar collection was 2.74 second/flower and 4.37 second/flower for the pollen collection. *A. mellifera* visited, on average, 7.14 flowers/min, collecting nectar, and 3.75 flowers/min for pollen collection. The total sugar concentration in the honey stomach content was 41.19% in the covered area with a honeybee colony, greater than the 38.22% observed for the area of free insect visitation. In the uncovered areas for free insect visitation and covered areas with Africanized honeybee colony, the results were different from the covered area without honeybee colony. There was no difference in the evaluations of floral biology and behaviour of insects between the transgenic and the conventional soybean, independent of the

Floral Biology and Africanized Honeybee Behaviour in Transgenic (Roundup ReadyTM var. BR-245 RR) and…

To the National Council of Scientific and Technological Development (CNPq), process number 479868/01-8 for the financial support and granted scholarship. To the Coordination of Perfec‐ tioning of Staff of Superior Level (CAPES), for the granted financial support for execution of

, Tiago Cleiton Simões de Oliveira Arnaut De Toledo1

, Maria Claudia Ruvolo-Takasusuki3

1 Animal Science Department, Universidade Estadual de Maringá, Maringá, Paraná, Brazil

3 Biotechnology, Cell Biology and Genetics Department, Universidade Estadual de Maringá,

2 Empresa de Brasileira de Pesquisa Agropecuária - Soja, Londrina, Paraná, Brazil

, Carlos Arrabal Arias2

and

,

http://dx.doi.org/10.5772/55847

293

,

, Clara Beatriz Hoffmann-Campo2

application of the herbicide glyphosate in transgenic soybean.

**Acknowledgements**

this project.

**Author details**

Wainer César Chiari1

Tais da Silva Lopes1

Paraná, Brazil

Emerson Dechechi Chambó1

Vagner de Alencar Arnaut de Toledo1\*

\*Address all correspondence to: abelha.vagner@gmail.com

In another study [52] in transgenic and conventional soybean, it was reported that the estimate of grain production increased 37.84% in the area where honeybee visits were permitted. However, the cv. BRS-133, not GM [7, 14] was intensively visited by *A. mellifera* Africanized honeybees. The researchers reported an increase of 61.38% in number of pods, and 58.86% in seed production, when compared with plants not visited by insects. [26] reported that Africanized honeybees provided a considerable increase of gene flow from transgenic cv. BR-245 RR to conventional cv. BRS-133 soybean (1.57%). Since these cultivars of soybean were attractive to the honeybees, they performed the cross-pollination in the tested varieties.

The economic value of honeybee service in the USA reported by [57, 58, 59, and 60] was about US \$15 billion, and specifically for soybean, the value was US \$754 million. [53] using the same table of [57, 58, and 59], estimated the economic value of Africanized honeybee service for soybean culture, using data from [7, 14, and 52] obtained as medium value US \$3.561,2 million. These estimates are considerable in Brazil and in other parts of the world, and must not be disregarded.

The Green Revolution has reduced the percentage of the word-wide population that suffers from hunger from 50% in sixty years to 20% at present. In plantations free of pesticides, the loss of production is 10% to 40%. Without this technology, about two billion of the seven billion inhabitants of the planet would be starving. The use of transgenic crops may help to increase the productivity, avoiding more deforestation, and more erosion of the soils [56].

Floral biology and the behaviour of the honeybees in transgenic soybean cv. BR-245 RR did not depend on the application of herbicide and was similar to the verified one in the conven‐ tional cv. BRS-133. This implies that it is possible to have cross-pollination between transgenic and conventional soybean and gene flow between them like reported by [26].

### **General summary**

The experiment was carried out to study the floral biology of the *Glycine max* L. Merrill, cultivar BR-245 RR (transgenic - Roundup Ready™) and BRS-133 (conventional) and to evaluate the behavior of the Africanized honeybee *A. mellifera* in the flowers of these cultivars. Three treatments established included: a covered area with an Africanized honeybee colony, a covered area without a honeybee colony and area of free insect visitation. Each treatment was six parcels of 24 m2 each and the planting of soybean was carried through in this area in eight pared and intercalated lines with tested cultivars, being applied herbicide (glyphosate) in the plants of transgenic cultivars in half of the parcels in the three treatments. The anthesis period was 8h04min longer in the covered area without a honeybee colony than in the covered area with a honeybee colony and in the area of free insect visitation. The average of the analyzed stigma receptivity was 87.35% and there was no difference between treatments. The average of viable pollen was 89.82% with no difference between treatments. In the analysis of the removed pollen grains from the honeybee forager corbicula, 100% observed belonging to soybean. The flower abortion rate was 71.10% in covered areas without honeybee colonies, which was greater by 50.78% and 55.12%, respectively, than the covered area with a honeybee colony and area of free insect visitation. *A. mellifera* was the insect that most frequently (97.02%) visited the soybean flowers. The time that *A. mellifera* spent to collect nectar was greater in the covered area with a honeybee colony than in the area of free insect visitation. The average time foragers spent for nectar collection was 2.74 second/flower and 4.37 second/flower for the pollen collection. *A. mellifera* visited, on average, 7.14 flowers/min, collecting nectar, and 3.75 flowers/min for pollen collection. The total sugar concentration in the honey stomach content was 41.19% in the covered area with a honeybee colony, greater than the 38.22% observed for the area of free insect visitation. In the uncovered areas for free insect visitation and covered areas with Africanized honeybee colony, the results were different from the covered area without honeybee colony. There was no difference in the evaluations of floral biology and behaviour of insects between the transgenic and the conventional soybean, independent of the application of the herbicide glyphosate in transgenic soybean.

### **Acknowledgements**

One of the greatest concerns of Brazilian farmers in relation to GM plants is the unexpected and cumulative effect of cross-contamination between GM plants and conventional plants. The instability and risk of propagation of a gene to wild species are critical to maintenance of the environment [52]. Genetic pollution is inevitable and the transgenic pollen may contaminate

In another study [52] in transgenic and conventional soybean, it was reported that the estimate of grain production increased 37.84% in the area where honeybee visits were permitted. However, the cv. BRS-133, not GM [7, 14] was intensively visited by *A. mellifera* Africanized honeybees. The researchers reported an increase of 61.38% in number of pods, and 58.86% in seed production, when compared with plants not visited by insects. [26] reported that Africanized honeybees provided a considerable increase of gene flow from transgenic cv. BR-245 RR to conventional cv. BRS-133 soybean (1.57%). Since these cultivars of soybean were attractive to the honeybees, they performed the cross-pollination in the tested varieties.

The economic value of honeybee service in the USA reported by [57, 58, 59, and 60] was about US \$15 billion, and specifically for soybean, the value was US \$754 million. [53] using the same table of [57, 58, and 59], estimated the economic value of Africanized honeybee service for soybean culture, using data from [7, 14, and 52] obtained as medium value US \$3.561,2 million. These estimates are considerable in Brazil and in other parts of the world, and must not be

The Green Revolution has reduced the percentage of the word-wide population that suffers from hunger from 50% in sixty years to 20% at present. In plantations free of pesticides, the loss of production is 10% to 40%. Without this technology, about two billion of the seven billion inhabitants of the planet would be starving. The use of transgenic crops may help to increase

Floral biology and the behaviour of the honeybees in transgenic soybean cv. BR-245 RR did not depend on the application of herbicide and was similar to the verified one in the conven‐ tional cv. BRS-133. This implies that it is possible to have cross-pollination between transgenic

The experiment was carried out to study the floral biology of the *Glycine max* L. Merrill, cultivar BR-245 RR (transgenic - Roundup Ready™) and BRS-133 (conventional) and to evaluate the behavior of the Africanized honeybee *A. mellifera* in the flowers of these cultivars. Three treatments established included: a covered area with an Africanized honeybee colony, a covered area without a honeybee colony and area of free insect visitation. Each treatment was six parcels of 24 m2 each and the planting of soybean was carried through in this area in eight pared and intercalated lines with tested cultivars, being applied herbicide (glyphosate) in the plants of transgenic cultivars in half of the parcels in the three treatments. The anthesis period was 8h04min longer in the covered area without a honeybee colony than in the covered area

the productivity, avoiding more deforestation, and more erosion of the soils [56].

and conventional soybean and gene flow between them like reported by [26].

disregarded.

292 Herbicides - Advances in Research

**General summary**

conventional or biological fields located several kilometres from GM plantations [55].

To the National Council of Scientific and Technological Development (CNPq), process number 479868/01-8 for the financial support and granted scholarship. To the Coordination of Perfec‐ tioning of Staff of Superior Level (CAPES), for the granted financial support for execution of this project.

### **Author details**

Wainer César Chiari1 , Clara Beatriz Hoffmann-Campo2 , Carlos Arrabal Arias2 , Tais da Silva Lopes1 , Tiago Cleiton Simões de Oliveira Arnaut De Toledo1 , Emerson Dechechi Chambó1 , Maria Claudia Ruvolo-Takasusuki3 and Vagner de Alencar Arnaut de Toledo1\*

\*Address all correspondence to: abelha.vagner@gmail.com

1 Animal Science Department, Universidade Estadual de Maringá, Maringá, Paraná, Brazil

2 Empresa de Brasileira de Pesquisa Agropecuária - Soja, Londrina, Paraná, Brazil

3 Biotechnology, Cell Biology and Genetics Department, Universidade Estadual de Maringá, Paraná, Brazil

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**Chapter 15**

**Herbicidal Activity of Mimosine and Its Derivatives**

Mimosine [β-[N-(3-hydroxy-4-oxypyridyl)]-α-aminopropionic acid] is a non-protein amino acid, and is a major compound present in all plant parts of *Mimosaceae*, which includes Leucaena (*Leucaena leucocephala*), *Leucaena glauca*, and other legumes belonging to *Mimosa* spp.. Structurally, mimosine is an analog of dihydroxyphenylalanine with a 3-hydroxy-4-pyridone ring instead of a 3,4-dihydroxy-phenyl ring (Fig. 1). Although Leucaena has a rich protein content and high annual yield, the presence of mimosine has limited the wide use of this plant as animal feed. This compound causes alopecia, growth retardation, cataracts and infertility in animals [1]. Mimosine can be degraded to DHP [3-hydroxy-4(4H)-pyridone] (Fig. 2) by microorganisms in the rumen and bacteria in rhizome nodules of Leucaena, by endogenous enzymes in the Leucaena plants, or by HCl hydrolysis. Although DHP is also toxic, it exerts lower toxicity than mimosine [2]. Mimosine possesses antimitotic activity that blocks the cell cycle in the large G1 phase [3] and inhibits DNA synthesis, which prevents the formation of the replication fork by altering deoxyribonucleotide metabolism [4]. The amino acid may also act as a tyrosine analogue which incorporates biologically vital proteins and, in turn, causes

N

OH

O

© 2013 Xuan et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 Xuan et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

NH2

distribution, and reproduction in any medium, provided the original work is properly cited.

O

HO

Tran Dang Xuan, Shinkichi Tawata and

Additional information is available at the end of the chapter

Tran Dang Khanh

**1. Introduction**

hair loss [1].

**Figure 1.** Chemical structure of mimosine

http://dx.doi.org/10.5772/55845


## **Herbicidal Activity of Mimosine and Its Derivatives**

Tran Dang Xuan, Shinkichi Tawata and Tran Dang Khanh

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55845

### **1. Introduction**

[54] James, C. (2010). Global Status of Commercialized Biotech/GM Crops: 2010. lsaaa

[55] Scottish Crop Research Institute. (1999). Gene flow in agriculture: relevance for trans‐ genic crops conference, Proceedings of a Symposium Held at the University of Keele,

[56] Souza, L. (2006). Liberação da soja transgênica no Brasil, vantagem ou não? In: AN‐ Bio, 7/03/2006, Available from http://www.anbio.org.br/noticiasflucia.htm (accessed

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Brief No.42, ISBN 978-1-89245649-4, Ithaca, NY, USA

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298 Herbicides - Advances in Research

pp. 13-21, ISBN 190139672, Staffordshire, UK, April 12-14, 1999.

of U.S. crops. Part I. American Bee Journal, 1989a. 129, p.411-423.

of U.S. crops. Part II. American Bee Journal, 1989b. 129, n.7, p.477-487.

Mimosine [β-[N-(3-hydroxy-4-oxypyridyl)]-α-aminopropionic acid] is a non-protein amino acid, and is a major compound present in all plant parts of *Mimosaceae*, which includes Leucaena (*Leucaena leucocephala*), *Leucaena glauca*, and other legumes belonging to *Mimosa* spp.. Structurally, mimosine is an analog of dihydroxyphenylalanine with a 3-hydroxy-4-pyridone ring instead of a 3,4-dihydroxy-phenyl ring (Fig. 1). Although Leucaena has a rich protein content and high annual yield, the presence of mimosine has limited the wide use of this plant as animal feed. This compound causes alopecia, growth retardation, cataracts and infertility in animals [1]. Mimosine can be degraded to DHP [3-hydroxy-4(4H)-pyridone] (Fig. 2) by microorganisms in the rumen and bacteria in rhizome nodules of Leucaena, by endogenous enzymes in the Leucaena plants, or by HCl hydrolysis. Although DHP is also toxic, it exerts lower toxicity than mimosine [2]. Mimosine possesses antimitotic activity that blocks the cell cycle in the large G1 phase [3] and inhibits DNA synthesis, which prevents the formation of the replication fork by altering deoxyribonucleotide metabolism [4]. The amino acid may also act as a tyrosine analogue which incorporates biologically vital proteins and, in turn, causes hair loss [1].

**Figure 1.** Chemical structure of mimosine

© 2013 Xuan et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Xuan et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

yield mimosine in highly pure grade. Our group has already developed a simple method to purify mimosine at an industrial scale by the use of ion exchange resin. Although mimosine shows herbicidal and antifungal activity, the synthesis of mimosine-derived compounds is indispensable because of the need to find mimosine derivatives with stronger suppression against pests and fungi. Synthesized compounds exerting strong activity and require a simple process for synthesis will be selected for development of novel bioactive pesticides. However,

This paper describes the allelopathic interaction of mimosine as well as its efficacy in appli‐ cation for biological control of weeds and pests. The analytical and purified methods of this compound developed in our laboratory and the synthesis of its derivatives and their suppres‐

Mimosine was first isolated from the sap of *Mimosa pudica* by Renz [7] and was given the name "mimosine". Later, minosine was biologically characterized from *M. pudica* by Nienburg and Taubõck [8]. From the extraction of ground *Leucaena glauca* seeds, Mascré [9] successfully

formula was elucidated to be (C4H5O2N)χ, and further experimental research has shown that it contains an α-amino acid including a phenolic hydroxyl. Bickel and Wibaut [10] named leucaenol and concluded the formula of mimosine was C8H10N2O4; it was chemically synthe‐ sized by Adams and Johnson [11]. Wilbaut and Klipool [12] isolated mimosine from *Leucaena leucocepphala* which they named "leucaenine" and verified that the three different substances are analogs. The chemical structure of mimosine was determined by Bickel [13] as β-N-(3 hydroxy-4pyridone)-α-amino propionic acid. The structure of mimosine is similar to dihy‐ droxyphenylalanine with a 3-hydroxy-4-pyridone ring instead of a 3,4-dihydroxy-phenyl ring

Mimosine exists in large quantities in leaves, pods and seeds of tropical legumes of the genus *Leucaena.* This compound is present in a much greater quantity in Leucaena than in *Mimosa* [14]. The Leucaena hybrid has lower mimosine content than the original *Leucaena leucocepha‐ la* [15]. In the non-hybridized Leucaena legume plant, mimosine accounted for 2-5% of fresh weight, and the level of concentration could increase to 10% in young leaves[16]. Small amounts of mimosine were in the nodules and the root exudates of Leucaena as well. The seed, stem, pod, and leaf tissue of different Leucaena species contained 1-12% mimosine, whereas the highest amount of mimosine was found in growing tips of Leucaena [14,16,17]. More recently, Xuan et al. [5] noted that all plant parts of Leucaena contained mimosine; however, the amount of mimosine in the young leaves and mature seeds was the highest, varying from

C named "leucenol". Its empirical

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301

very little success with synthesis of mimosine derivatives has been reported.

sive activities against plants and fungi are also demonstrated.

isolated an optically inactive crystal line solid, m.p.287o

**2. Discovery of mimosine**

**3. Content of mimosine**

(Fig.1).

**Figure 2.** Metabolic relations between cysteine, mimosine and DHP [3-hydroxy-4(1H)-pyridone] [41]

Mimosine has been known as an allelochemical, which is responsible for the strong allelopathic activity of Leucaena and *Mimosa* spp., by suppressing growth of plants and plant fungi [2,5]. Our previous work examined the possibility of exploiting higher plants selected from the plant ecosystems in Southeast Asia for paddy weed control, of which *Leucaena glauca* was among those which reduced paddy weeds up to 70% and increased rice yield by 20% [6]. We concluded that mimosine was responsible for significant weed reduction of *L. glauca* in paddy fields, and rich nutrients contained in plant materials of the legume also contributed to the increase of the rice yield.

Because mimosine is a compound that is responsible for many interesting biological activities as mentioned above, many works have been conducted to purify mimosine, typically from Leucaena because it contains higher mimosine content than other species of Mimosaceae. However, most methods published so far have been too complicated and costly to successfully yield mimosine in highly pure grade. Our group has already developed a simple method to purify mimosine at an industrial scale by the use of ion exchange resin. Although mimosine shows herbicidal and antifungal activity, the synthesis of mimosine-derived compounds is indispensable because of the need to find mimosine derivatives with stronger suppression against pests and fungi. Synthesized compounds exerting strong activity and require a simple process for synthesis will be selected for development of novel bioactive pesticides. However, very little success with synthesis of mimosine derivatives has been reported.

This paper describes the allelopathic interaction of mimosine as well as its efficacy in appli‐ cation for biological control of weeds and pests. The analytical and purified methods of this compound developed in our laboratory and the synthesis of its derivatives and their suppres‐ sive activities against plants and fungi are also demonstrated.

### **2. Discovery of mimosine**

Ac

300 Herbicides - Advances in Research

rice yield.

H2 C CH

HO

NH2

*O*-Acetyl-*L-*serine

OH

HO

O NH

CO2H

3,4-Dihydroxypyridine

N

H3C

3-Hydroxy-4(1H)-pyridone Pyruvate Ammonia

**Figure 2.** Metabolic relations between cysteine, mimosine and DHP [3-hydroxy-4(1H)-pyridone] [41]

O

Mimosine has been known as an allelochemical, which is responsible for the strong allelopathic activity of Leucaena and *Mimosa* spp., by suppressing growth of plants and plant fungi [2,5]. Our previous work examined the possibility of exploiting higher plants selected from the plant ecosystems in Southeast Asia for paddy weed control, of which *Leucaena glauca* was among those which reduced paddy weeds up to 70% and increased rice yield by 20% [6]. We concluded that mimosine was responsible for significant weed reduction of *L. glauca* in paddy fields, and rich nutrients contained in plant materials of the legume also contributed to the increase of the

Because mimosine is a compound that is responsible for many interesting biological activities as mentioned above, many works have been conducted to purify mimosine, typically from Leucaena because it contains higher mimosine content than other species of Mimosaceae. However, most methods published so far have been too complicated and costly to successfully

H2S

SH

H2 C CH

O N

C CO2H NH3

HO

NH2

*L-*Cysteine

CO2H

H2

*L-*Mimosine

C CH CO2H NH2

> Mimosine was first isolated from the sap of *Mimosa pudica* by Renz [7] and was given the name "mimosine". Later, minosine was biologically characterized from *M. pudica* by Nienburg and Taubõck [8]. From the extraction of ground *Leucaena glauca* seeds, Mascré [9] successfully isolated an optically inactive crystal line solid, m.p.287o C named "leucenol". Its empirical formula was elucidated to be (C4H5O2N)χ, and further experimental research has shown that it contains an α-amino acid including a phenolic hydroxyl. Bickel and Wibaut [10] named leucaenol and concluded the formula of mimosine was C8H10N2O4; it was chemically synthe‐ sized by Adams and Johnson [11]. Wilbaut and Klipool [12] isolated mimosine from *Leucaena leucocepphala* which they named "leucaenine" and verified that the three different substances are analogs. The chemical structure of mimosine was determined by Bickel [13] as β-N-(3 hydroxy-4pyridone)-α-amino propionic acid. The structure of mimosine is similar to dihy‐ droxyphenylalanine with a 3-hydroxy-4-pyridone ring instead of a 3,4-dihydroxy-phenyl ring (Fig.1).

### **3. Content of mimosine**

Mimosine exists in large quantities in leaves, pods and seeds of tropical legumes of the genus *Leucaena.* This compound is present in a much greater quantity in Leucaena than in *Mimosa* [14]. The Leucaena hybrid has lower mimosine content than the original *Leucaena leucocepha‐ la* [15]. In the non-hybridized Leucaena legume plant, mimosine accounted for 2-5% of fresh weight, and the level of concentration could increase to 10% in young leaves[16]. Small amounts of mimosine were in the nodules and the root exudates of Leucaena as well. The seed, stem, pod, and leaf tissue of different Leucaena species contained 1-12% mimosine, whereas the highest amount of mimosine was found in growing tips of Leucaena [14,16,17]. More recently, Xuan et al. [5] noted that all plant parts of Leucaena contained mimosine; however, the amount of mimosine in the young leaves and mature seeds was the highest, varying from 2.4 to 2.7% of the fresh weight, whereas the lowest mimosine content was in the root xylems and xylems (0.11 to 0.18%, respectively). Our research team did not find mimosine content greater than 5% in any plant parts of Leucaena observed in previous reports [5].

mung bean (*Phaseolus aureus*) [25,26], lettuce [27,28]; hemp sesbania (*Sesbania exaltata*), ryegrass (*Lolium perenne* L), sicklepod (*Senna obtusifolia*), wheat (*Triticum aestivum*)[29], and rice (*Oryza sativa*)[28,30]. Similar to other phytotoxins, effects of mimosine against plant germination and growth are proportional to applied doses. Chou and Kuo [28] indicated that at 20 ppm, mimosine significantly suppressed growth of lettuce, rice and ryegrass; however, *Miscanthus floridulus* and *Pinus taiwanensis* were not inhibited by the mimosine at 200 ppm. Mimosine exhibited selective influence against the germination and growth of certain indicator plants including hair beggarticks (*Bidens pilosa* L), creeping grass (*Mimosa pudica* L), cabbage (*Brassica rapa*), Italian ryegrass (*Lolium multiflorum* L), and kidney bean (*Phaseoulus vulgaris* L) at 50-100 ppm. However, the effect of mimosine was the lowest against plants which are mimosine

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303

Mimosine also shows selective influence against certain bacteria and fungal growth. Some bacteria were inhibited, whereas growth of several bacteria was promoted by mimosine. Soedarjo and Borthakur [31] reported that growth of some root nodule bacteria was inhibited by mimosine. In contrast, some Leucaena-nodulating *Rhizobium* strains could utilize mimosine as a source of carbon and nitrogen. *Rhizobium* sp. strain TAL 1145 is such a strain that can catabolyze mimosine, which provides it a competitive advantage for nodulation of Leucaena [17]. Tawata et al. [32] revealed that *Escherichia coli* Crooks (1222) growth was inhibited by mimosine, but increased by DHP. *Aerobacter aerogenes* (1232) growth was increased by both mimosine and DHP. *Coryne bacterium psudodiphterium* (1471) growth was inhibited by DHP,

There were 38 unknown microorganisms collected from the Leucaena population growing around Campus of University of the Ryukyus, Okinawa, Japan, including 12 from roots, 13 from top soil, and 8 from deep soil, and the remaining was from Leucaena stems; they were examined against mimosine and DHP. Among the unknown microorganisms, fungus D6-31 growth was inhibited by DHP, but increased by mimosine, whereas that of fungus D6-30 was inhibited by mimosine, but increased by DHP. The population of fungus D6-27 was dramati‐ cally increased by both mimosine and DHP, however, that of fungus D3-6 was inhibited by both mimosine and DHP. These four unknown fungi were selected for future research [32]. Other reports such as Murugesan and Radha [33] demonstrated that mimosine inhibited growth of bacteria and fungi, including *Alternaria* sp., *Cercospora canescens, Colletotrichum indemuthianum, Diplodia natalensis, Sclerotium rolfsii, Dreschlera oryzae*, and *Rhizoctonia solani*.

Anitha et al. [34] noted that mimosine was toxic against fungi rather than bacteria.

On the other hand, mimosine released from rhizomes and foliated leaves to soil caused inhibition of plants in the vicinity of Leucaena [5,20]. Soils amended with mimosine retarded growth of *Brassica rapa* [5]. Hong et al. [6] evaluated the potential of weed suppression of various plants collected from plant ecosystems in Southeast Asia. Several species showed the potential for weed suppression up to 70% and increased rice yield to 20%, including *Leucaena glauca*. Because of its weed suppression and rich nutrients as well as the wide adaptation of Leucaena in the tropics, the biomass of this plant is useful for weed control and serves as a

producers (*M. pudica* and *L. leucocepphala*) [5].

but increased by mimosine.

source of natural fertilizer.

The quantity of mimosine in Leucaena plants is species dependent. *Leucaena leucocephala* has a medium level of mimosine, whereas, *L. collinsii, L. diversifolia, L. escuienta, L. greggii,* and *L. pallida* have low mimosine content [18]. In addition, leaf size also showed different mimosine concentrations, with smaller-leaved species having lower mimosine content [18]. The afore‐ mentioned evidences suggest that mimosine concentration may be related to the genetic variation in Leucaena species. Among ecotypes, content of mimosine and DHP in the dry season were higher than in the rainy season [19,20]. The main difference may involve the antinutritional factors. The concentration of mimosine contained in Leucaena plants also fluctuates with the time of year and is proportionally related to growth rate. For example, better growth of Leucaena leads to higher mimosine content [19]. Moreover, some abiotic factors of envi‐ ronmental stress such as drought and moisture stress can dramatically increase mimosine levels in both new and old leaves [21].

### **4. Purification of mimosine**

Although mimosine levels are high in Leucaena, it is not easy to isolate pure mimosine. The determination of mimosine via extracting solvents and analytical instruments and spectro‐ photometrics was conducted [22,23]. However, it is complicated to separate mimosine from other amino acids in Leucaena, the cost of mimosine purchased from chemical companies is rather high. In our laboratory, mimosine can be easily purified at an industrial scale by the use of ion exchange resin. By this process, 6 kg of freshly harvested Leucaena leaves were immersed in 30 L of boiling water for 10 min. The water extract was cooled to room temperature and filtered with 300 mesh sieve. Ultrafiltration was carried out at 4 atm, 30o C and 700 rpm using a Filtron Miniset Omega equipped with the cassette system membrane. The filtrate was passed through a column packed with acid form Amberlite IRA (technical grade). The resin was washed with 1 L of 2N NH4OH. About 30 g of relatively pure mimosine was obtained after adjusting the pH to 4.5~5.0. We have examined various conditions for mimosine purification and observed that the type of ion exchange resin and adjustment of pH are crucial conditions to obtaining the maximum quantity and high purity of mimosine (5 g per 1 kg fresh Leucaena leaves, purity>95%).

### **5. Mimosine acts as an allelochemical**

Mimosine is considered as an allelochemical and is responsible for the allelopathic activity of the *Leucaena* genus and other species belonging to *Mimosa* spp. Leucaena is popular in intercropping with annual crops, using as a hedgerow, and alley cropping for yield promotion and weed control [24]. In bioassays, this compound exerted inhibition against seedlings of mung bean (*Phaseolus aureus*) [25,26], lettuce [27,28]; hemp sesbania (*Sesbania exaltata*), ryegrass (*Lolium perenne* L), sicklepod (*Senna obtusifolia*), wheat (*Triticum aestivum*)[29], and rice (*Oryza sativa*)[28,30]. Similar to other phytotoxins, effects of mimosine against plant germination and growth are proportional to applied doses. Chou and Kuo [28] indicated that at 20 ppm, mimosine significantly suppressed growth of lettuce, rice and ryegrass; however, *Miscanthus floridulus* and *Pinus taiwanensis* were not inhibited by the mimosine at 200 ppm. Mimosine exhibited selective influence against the germination and growth of certain indicator plants including hair beggarticks (*Bidens pilosa* L), creeping grass (*Mimosa pudica* L), cabbage (*Brassica rapa*), Italian ryegrass (*Lolium multiflorum* L), and kidney bean (*Phaseoulus vulgaris* L) at 50-100 ppm. However, the effect of mimosine was the lowest against plants which are mimosine producers (*M. pudica* and *L. leucocepphala*) [5].

2.4 to 2.7% of the fresh weight, whereas the lowest mimosine content was in the root xylems and xylems (0.11 to 0.18%, respectively). Our research team did not find mimosine content

The quantity of mimosine in Leucaena plants is species dependent. *Leucaena leucocephala* has a medium level of mimosine, whereas, *L. collinsii, L. diversifolia, L. escuienta, L. greggii,* and *L. pallida* have low mimosine content [18]. In addition, leaf size also showed different mimosine concentrations, with smaller-leaved species having lower mimosine content [18]. The afore‐ mentioned evidences suggest that mimosine concentration may be related to the genetic variation in Leucaena species. Among ecotypes, content of mimosine and DHP in the dry season were higher than in the rainy season [19,20]. The main difference may involve the antinutritional factors. The concentration of mimosine contained in Leucaena plants also fluctuates with the time of year and is proportionally related to growth rate. For example, better growth of Leucaena leads to higher mimosine content [19]. Moreover, some abiotic factors of envi‐ ronmental stress such as drought and moisture stress can dramatically increase mimosine

Although mimosine levels are high in Leucaena, it is not easy to isolate pure mimosine. The determination of mimosine via extracting solvents and analytical instruments and spectro‐ photometrics was conducted [22,23]. However, it is complicated to separate mimosine from other amino acids in Leucaena, the cost of mimosine purchased from chemical companies is rather high. In our laboratory, mimosine can be easily purified at an industrial scale by the use of ion exchange resin. By this process, 6 kg of freshly harvested Leucaena leaves were immersed in 30 L of boiling water for 10 min. The water extract was cooled to room temperature and

a Filtron Miniset Omega equipped with the cassette system membrane. The filtrate was passed through a column packed with acid form Amberlite IRA (technical grade). The resin was washed with 1 L of 2N NH4OH. About 30 g of relatively pure mimosine was obtained after adjusting the pH to 4.5~5.0. We have examined various conditions for mimosine purification and observed that the type of ion exchange resin and adjustment of pH are crucial conditions to obtaining the maximum quantity and high purity of mimosine (5 g per 1 kg fresh Leucaena

Mimosine is considered as an allelochemical and is responsible for the allelopathic activity of the *Leucaena* genus and other species belonging to *Mimosa* spp. Leucaena is popular in intercropping with annual crops, using as a hedgerow, and alley cropping for yield promotion and weed control [24]. In bioassays, this compound exerted inhibition against seedlings of

C and 700 rpm using

filtered with 300 mesh sieve. Ultrafiltration was carried out at 4 atm, 30o

greater than 5% in any plant parts of Leucaena observed in previous reports [5].

levels in both new and old leaves [21].

302 Herbicides - Advances in Research

**4. Purification of mimosine**

leaves, purity>95%).

**5. Mimosine acts as an allelochemical**

Mimosine also shows selective influence against certain bacteria and fungal growth. Some bacteria were inhibited, whereas growth of several bacteria was promoted by mimosine. Soedarjo and Borthakur [31] reported that growth of some root nodule bacteria was inhibited by mimosine. In contrast, some Leucaena-nodulating *Rhizobium* strains could utilize mimosine as a source of carbon and nitrogen. *Rhizobium* sp. strain TAL 1145 is such a strain that can catabolyze mimosine, which provides it a competitive advantage for nodulation of Leucaena [17]. Tawata et al. [32] revealed that *Escherichia coli* Crooks (1222) growth was inhibited by mimosine, but increased by DHP. *Aerobacter aerogenes* (1232) growth was increased by both mimosine and DHP. *Coryne bacterium psudodiphterium* (1471) growth was inhibited by DHP, but increased by mimosine.

There were 38 unknown microorganisms collected from the Leucaena population growing around Campus of University of the Ryukyus, Okinawa, Japan, including 12 from roots, 13 from top soil, and 8 from deep soil, and the remaining was from Leucaena stems; they were examined against mimosine and DHP. Among the unknown microorganisms, fungus D6-31 growth was inhibited by DHP, but increased by mimosine, whereas that of fungus D6-30 was inhibited by mimosine, but increased by DHP. The population of fungus D6-27 was dramati‐ cally increased by both mimosine and DHP, however, that of fungus D3-6 was inhibited by both mimosine and DHP. These four unknown fungi were selected for future research [32]. Other reports such as Murugesan and Radha [33] demonstrated that mimosine inhibited growth of bacteria and fungi, including *Alternaria* sp., *Cercospora canescens, Colletotrichum indemuthianum, Diplodia natalensis, Sclerotium rolfsii, Dreschlera oryzae*, and *Rhizoctonia solani*. Anitha et al. [34] noted that mimosine was toxic against fungi rather than bacteria.

On the other hand, mimosine released from rhizomes and foliated leaves to soil caused inhibition of plants in the vicinity of Leucaena [5,20]. Soils amended with mimosine retarded growth of *Brassica rapa* [5]. Hong et al. [6] evaluated the potential of weed suppression of various plants collected from plant ecosystems in Southeast Asia. Several species showed the potential for weed suppression up to 70% and increased rice yield to 20%, including *Leucaena glauca*. Because of its weed suppression and rich nutrients as well as the wide adaptation of Leucaena in the tropics, the biomass of this plant is useful for weed control and serves as a source of natural fertilizer.

### **6. Synthesis of mimosine and its derivatives**

Mimosine toxicity is ascribed to the presence of –OH and –O in the pyridine ring and known to suppress iron-containing enzymes and compete with tyrosine [35]. The characteristic activity of growth inhibitory properties of mimosine is a hydroxyl group α to the oxo function of the pyridone ring (Fig.1). The location of the amino acid side chain seems to be less critical and an isomer (Fig.1). The synthesis of two mimosine isomers with the position of the αhydroxy-oxo function in the pyridine ring of mimosine was at least as active *in vitro* and *in vivo* as the natural amino acid [36]. The constituent properties of the α-hydroxy-oxo group are involved in the biological activity of mimosine and other systems and may play a key factor in growth suppression [25,37-39]. The structure of the heterocyclic ring in mimosine is possible to modify the chelate properties of the molecule and their biological activity which could lead to the design of a mimosine analogue [36].

Even though mimosine shows a great potential as an allelochemical, it is difficult to apply this amino acid as a natural herbicide because it may be unstable in natural conditions. Mimosine can be easily degraded in soil by soil factors such as nutrients, minerals, pH and microorgan‐ isms. Therefore, synthesis of mimosine derivatives with stronger activity and greater stability is needed. Although many interesting experiments on mimosine have been conducted, very sporadic work on the synthesis of mimosine-derived compounds has been carried out and reported. This is the first synthesis of propionates as mimosine derivatives and was carried out in our laboratory [32].

### **6.1. Synthesis of propionates as mimosine derivatives**

Each of 2-hydroxypyridine (material A) and 4-hydroxypyridine (material B) were well blended with each 12 different acrylates (Fig. 3), at 90-110o C for 4-6 h to receive oily substances with deep yellow and brown color (Fig. 4). The reactive products were applied to TLC for purifi‐ cation. Solvents of TLC were benzene: methanol (1:1 or 1: 2, v/v). Yielded compounds were recovered by methanol and subsequently subjected to 1 H-NMR, 13C-NMR and IR to determine their chemical structures. The synthesized propionates are shown in Fig. 5. Herbicidal and antifungal activities of the propionates were examined against growth of *Brassica rapa* and two noxious fungi *Schlerotium dellfinii* and *Rhizoctonia solani* at 100 ppm, respectively.

### **6.2. Herbicidal activity**

Among synthesized propionates, two compounds including A2 and B2 [chloro-3-(2-oxohy‐ doropyridyl) and chloro-3-(4-oxohydoropyridyl) propionates] exhibited the strongest herbi‐ cidal activity against growth of *B. rapa* (50-70% of inhibition) (Fig. 6). On the other hand, lengths of radicle and hypocotyl were either promoted or inhibited by the propionates A3, A4, A11, B4, B5, B6, B11, and B12. The other compounds reduced growth of *B. rapa* by lower magnitudes (20-40%). The chloric group in the two propionates A2 and B2 may be responsible for the greater herbicidal activities than other compounds. However, none of these synthesized propionates could exert stronger herbicidal activities than mimosine, which showed a 80-90% inhibition.

**6.3. Antifungal activity**

**Figure 3.** Chemical structures of materials for propionate synthesis

The fungal activity varied among the mimosine derivatives. The compounds A1, A2, A11, B6, and B8 were the most inhibitive against both *R. solani* and *S. dellfinii* (50-70% inhibition) (Fig. 7), whereas there were 5 propionates B3, B4, B5, B11, and B12 that stimulated growth of the two fungi up to 20%. Growth of *R. solani* and *S. dellfinii* were either stimulated or suppressed

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**Figure 3.** Chemical structures of materials for propionate synthesis

#### **6.3. Antifungal activity**

**6. Synthesis of mimosine and its derivatives**

to the design of a mimosine analogue [36].

**6.1. Synthesis of propionates as mimosine derivatives**

with each 12 different acrylates (Fig. 3), at 90-110o

recovered by methanol and subsequently subjected to 1

out in our laboratory [32].

304 Herbicides - Advances in Research

**6.2. Herbicidal activity**

inhibition.

Mimosine toxicity is ascribed to the presence of –OH and –O in the pyridine ring and known to suppress iron-containing enzymes and compete with tyrosine [35]. The characteristic activity of growth inhibitory properties of mimosine is a hydroxyl group α to the oxo function of the pyridone ring (Fig.1). The location of the amino acid side chain seems to be less critical and an isomer (Fig.1). The synthesis of two mimosine isomers with the position of the αhydroxy-oxo function in the pyridine ring of mimosine was at least as active *in vitro* and *in vivo* as the natural amino acid [36]. The constituent properties of the α-hydroxy-oxo group are involved in the biological activity of mimosine and other systems and may play a key factor in growth suppression [25,37-39]. The structure of the heterocyclic ring in mimosine is possible to modify the chelate properties of the molecule and their biological activity which could lead

Even though mimosine shows a great potential as an allelochemical, it is difficult to apply this amino acid as a natural herbicide because it may be unstable in natural conditions. Mimosine can be easily degraded in soil by soil factors such as nutrients, minerals, pH and microorgan‐ isms. Therefore, synthesis of mimosine derivatives with stronger activity and greater stability is needed. Although many interesting experiments on mimosine have been conducted, very sporadic work on the synthesis of mimosine-derived compounds has been carried out and reported. This is the first synthesis of propionates as mimosine derivatives and was carried

Each of 2-hydroxypyridine (material A) and 4-hydroxypyridine (material B) were well blended

deep yellow and brown color (Fig. 4). The reactive products were applied to TLC for purifi‐ cation. Solvents of TLC were benzene: methanol (1:1 or 1: 2, v/v). Yielded compounds were

their chemical structures. The synthesized propionates are shown in Fig. 5. Herbicidal and antifungal activities of the propionates were examined against growth of *Brassica rapa* and two

Among synthesized propionates, two compounds including A2 and B2 [chloro-3-(2-oxohy‐ doropyridyl) and chloro-3-(4-oxohydoropyridyl) propionates] exhibited the strongest herbi‐ cidal activity against growth of *B. rapa* (50-70% of inhibition) (Fig. 6). On the other hand, lengths of radicle and hypocotyl were either promoted or inhibited by the propionates A3, A4, A11, B4, B5, B6, B11, and B12. The other compounds reduced growth of *B. rapa* by lower magnitudes (20-40%). The chloric group in the two propionates A2 and B2 may be responsible for the greater herbicidal activities than other compounds. However, none of these synthesized propionates could exert stronger herbicidal activities than mimosine, which showed a 80-90%

noxious fungi *Schlerotium dellfinii* and *Rhizoctonia solani* at 100 ppm, respectively.

C for 4-6 h to receive oily substances with

H-NMR, 13C-NMR and IR to determine

The fungal activity varied among the mimosine derivatives. The compounds A1, A2, A11, B6, and B8 were the most inhibitive against both *R. solani* and *S. dellfinii* (50-70% inhibition) (Fig. 7), whereas there were 5 propionates B3, B4, B5, B11, and B12 that stimulated growth of the two fungi up to 20%. Growth of *R. solani* and *S. dellfinii* were either stimulated or suppressed

**7. Analytical determination of mimosine**

**Figure 5.** Chemical structures of synthesized propionates

and 4.8 min, respectively.

Paper and thin layer chromatography were used to identify mimosine [28]; however, mimosine content could not be quantified. Gas-liquid chromatography, liquid chromatography, and reversed-phase ion-pair high-performance liquid-chromatography were also applied for mimosine determination. However, these methods require elaborate preparation of samples, but with no appreciable improvement in the range of sensitivity [23]. Other methods were the coupling of mimosine with *p*-nitroaniline [22] or mimosine with N-1(naphthyl)ethylenedia‐ mine (NEDA) forming a pink-colored azodye with an absorbance of 540 nm [23], and the use of indirect spectrophotometricity which is based on its reaction with diazotized sulfanilamide (DZSAM). These methods were reported to increase the sensitive estimation of mimosine. A useful HPLC system to determine mimosine and DHP contents that influenced *Rhizobium* isolates was reported by Soedarjo et al. [40]. They applied a C18 HPLC column, UV detection at 280 nm, a solvent system of 0.2% orthophosphoric acid to detect mimosine and DHP at 2.7

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Our laboratory also developed a simple method using HPLC to determine mimosine and DHP. This method is nottime consuming, uses simple reagents and procedures, and has a high level of accuracy. Of which, the HPLC system includes an 880-PU pump and column (Fine pak Sil C18, Nihonbunko company). The mobile phase employed was a mixture solution of 10 mM potassium-dihydrogen phosphate, 10 mM phosphoric acid, acetonitrile (45:45:10), and finally, 0.1% sodium 1-octanesulfonate was added to the mixture as the surface active agent. The flow rate was 1.5 mL per min. Mimosine and DHP were detected at a wave length of 280 nm. The fresh samples from Leucaena (leaves, stems, or roots) were boiled for 10 min, cooled at room temperature, centrifuged, filtered and injected into HPLC at 2-5 µL. The peaks of mimosine

**Figure 4.** Synthesis route of propionates

by A3 and A4. The other propionates exerted fungal activity by 10-40%. The compounds chloro-3-(2-oxohydoropyridyl) and chloro-3-(4-oxohydoropyridyl) propionates (A2 and B2) showed good antifungal activity, whereas the chloro-3-(4-oxohydoropyridyl) propionate exhibited weak suppression of *R. solani* (about 10% inhibition). The two compounds, A2 and B2, were the most potential among synthesized propionates for obtaining herbicidal and antifungal activities. Mimosine did not show any effects against *S. dellfinii*, but inhibited growth of *R. solani* by about 30%. The antifungal strength of these synthesized propionates was greater than that of mimosine, with the exception of compounds A4, A8, B4, B5, B6, B11, and B12 (Fig. 7).

Several compounds among the synthesized propionates from this research showed good herbicidal and antifungal activities. In general, antifungal activity of these propionates was greater than their herbicidal activity. The most promising compounds were chloro-3-(2 oxohydoropyridyl) and chloro-3-(4-oxohydoropyridyl) propionates.

by A3 and A4. The other propionates exerted fungal activity by 10-40%. The compounds chloro-3-(2-oxohydoropyridyl) and chloro-3-(4-oxohydoropyridyl) propionates (A2 and B2) showed good antifungal activity, whereas the chloro-3-(4-oxohydoropyridyl) propionate exhibited weak suppression of *R. solani* (about 10% inhibition). The two compounds, A2 and B2, were the most potential among synthesized propionates for obtaining herbicidal and antifungal activities. Mimosine did not show any effects against *S. dellfinii*, but inhibited growth of *R. solani* by about 30%. The antifungal strength of these synthesized propionates was greater than that of mimosine, with the exception of compounds A4, A8, B4, B5, B6, B11,

Several compounds among the synthesized propionates from this research showed good herbicidal and antifungal activities. In general, antifungal activity of these propionates was greater than their herbicidal activity. The most promising compounds were chloro-3-(2-

oxohydoropyridyl) and chloro-3-(4-oxohydoropyridyl) propionates.

and B12 (Fig. 7).

**Figure 4.** Synthesis route of propionates

306 Herbicides - Advances in Research


**Figure 5.** Chemical structures of synthesized propionates

### **7. Analytical determination of mimosine**

Paper and thin layer chromatography were used to identify mimosine [28]; however, mimosine content could not be quantified. Gas-liquid chromatography, liquid chromatography, and reversed-phase ion-pair high-performance liquid-chromatography were also applied for mimosine determination. However, these methods require elaborate preparation of samples, but with no appreciable improvement in the range of sensitivity [23]. Other methods were the coupling of mimosine with *p*-nitroaniline [22] or mimosine with N-1(naphthyl)ethylenedia‐ mine (NEDA) forming a pink-colored azodye with an absorbance of 540 nm [23], and the use of indirect spectrophotometricity which is based on its reaction with diazotized sulfanilamide (DZSAM). These methods were reported to increase the sensitive estimation of mimosine. A useful HPLC system to determine mimosine and DHP contents that influenced *Rhizobium* isolates was reported by Soedarjo et al. [40]. They applied a C18 HPLC column, UV detection at 280 nm, a solvent system of 0.2% orthophosphoric acid to detect mimosine and DHP at 2.7 and 4.8 min, respectively.

Our laboratory also developed a simple method using HPLC to determine mimosine and DHP. This method is nottime consuming, uses simple reagents and procedures, and has a high level of accuracy. Of which, the HPLC system includes an 880-PU pump and column (Fine pak Sil C18, Nihonbunko company). The mobile phase employed was a mixture solution of 10 mM potassium-dihydrogen phosphate, 10 mM phosphoric acid, acetonitrile (45:45:10), and finally, 0.1% sodium 1-octanesulfonate was added to the mixture as the surface active agent. The flow rate was 1.5 mL per min. Mimosine and DHP were detected at a wave length of 280 nm. The fresh samples from Leucaena (leaves, stems, or roots) were boiled for 10 min, cooled at room temperature, centrifuged, filtered and injected into HPLC at 2-5 µL. The peaks of mimosine

**Figure 6.** Herbicidal activity of mimosine and its propionate derivatives against *Brassica rapa* (100 ppm)

and DHP appear at 2.5 min and 7.5 min retention time, respectively. However, these retention times varied among columns and HPLC conditions.

continued to yield novel derivatives of mimosine which can obtain stronger inhibition on plant

Herbicidal Activity of Mimosine and Its Derivatives

http://dx.doi.org/10.5772/55845

309

1 Graduate School for International Development and Cooperation, Hiroshima University,

growth than their parent, mimosine.

Tran Dang Xuan1\*, Shinkichi Tawata2\* and Tran Dang Khanh3

**Figure 7.** Antifungal activity of mimosine and its propionate derivatives (100 ppm)

3 Agricultural Genetics Institute, Hanoi, Vietnam

2 Faculty of Agriculture, University of the Ryukyus, Okinawa, Japan

**Author details**

Higashi Hiroshima, Japan

### **8. Conclusion**

Mimosine is a major secondary metabolite in Leucaena and Mimosaceae plants and is responsible for the biological activities of these plants. Its allelopathic interaction includes both inhibition of plants, fungi, and bacteria, and stimulation of several strains of bacteria. The biomass of Leucaena in the tropics is a potential source for reduction of weed emergence in paddy fields and simultaneous utilization as green manure. Although we have synthesized several propionates which exerted potent antifungal activity, further synthesis should be

**Figure 7.** Antifungal activity of mimosine and its propionate derivatives (100 ppm)

continued to yield novel derivatives of mimosine which can obtain stronger inhibition on plant growth than their parent, mimosine.

### **Author details**

and DHP appear at 2.5 min and 7.5 min retention time, respectively. However, these retention

**Figure 6.** Herbicidal activity of mimosine and its propionate derivatives against *Brassica rapa* (100 ppm)

Mimosine is a major secondary metabolite in Leucaena and Mimosaceae plants and is responsible for the biological activities of these plants. Its allelopathic interaction includes both inhibition of plants, fungi, and bacteria, and stimulation of several strains of bacteria. The biomass of Leucaena in the tropics is a potential source for reduction of weed emergence in paddy fields and simultaneous utilization as green manure. Although we have synthesized several propionates which exerted potent antifungal activity, further synthesis should be

times varied among columns and HPLC conditions.

**8. Conclusion**

308 Herbicides - Advances in Research

Tran Dang Xuan1\*, Shinkichi Tawata2\* and Tran Dang Khanh3

1 Graduate School for International Development and Cooperation, Hiroshima University, Higashi Hiroshima, Japan

2 Faculty of Agriculture, University of the Ryukyus, Okinawa, Japan

3 Agricultural Genetics Institute, Hanoi, Vietnam

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## *Edited by Andrew J. Price and Jessica A. Kelton*

Herbicides represent one of the most widely used groups of pesticides worldwide for control of weed species in agricultural and non-crop settings. Due to the extensive use of herbicides and their value in weed management, herbicide research remains crucial for ensuring continued, effective use of herbicides while minimizing detrimental effects to ecosystems. Presently, a wide range of research continues to focus on improved herbicide use, environmental impact of herbicides, and even medicinal application of herbicide chemistries. In Herbicides - Advances in Research, authors cover multiple topics concerning current, valuable herbicide research.

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Herbicides - Advances in Research

Herbicides

Advances in Research

*Edited by Andrew J. Price and Jessica A. Kelton*