**Preparation and Characterization of Polymeric Microparticles Used for Controlled Release of Ametryn Herbicide**

Fabiana A. Lobo1, Carina L. de Aguirre2, Patrícia M.S. Souza2, Renato Grillo2,3, Nathalie F.S. de Melo2,3, André H. Rosa2 and Leonardo F. Fraceto2 *1UFOP - Universidade Federal de Ouro Preto 2UNESP – State University of São Paulo, 3Department of Environmental Engineering, Campus Sorocaba, SP, Brazil* 

#### **1. Introduction**

There is increasing pressure to improve agricultural productivity, due to rapid population growth, increased consumption and global demand for high quality products. As a result, agricultural chemicals have become essential for the control of weeds, pests and diseases in a wide range of crops. Ametryn (2-ethylamino-4-isopropylamino-6-methylthio-s-2,4,6 triazine) is a selective herbicide belonging to the s-triazine family, whose activity is the result of inhibition of photosynthesis by blocking of electron transport. The ametryn molecule (Figure 1) contains a symmetrical hexameric aromatic ring in its chemical structure, consisting of three carbon atoms and three nitrogen atoms in alternate positions. The herbicide is classified as a methylthiotriazine, due to the presence of the SCH3 group (Tennant et al., 2001).

Fig. 1. Structural formula of ametryn.

Ametryn is used for the control of graminaceous and broad-leaved weeds in plantations of annual crops (Tennant et al., 2001). Once in the soil the herbicide may be taken up by plants, absorbed by the soil and plant residues, biodegraded, or undergo chemical transformations that increase its volatilization and photocatalytic decomposition. Studies have shown that prolonged human exposure to triazine herbicides can lead to serious health problems including contact dermatitis, intoxication, hormonal dysfunction and cancers (Friedmann et

Preparation and Characterization

**2.2.1 Determination of ametryn** 

**2.2 Methodology** 

membranes.

of Polymeric Microparticles Used for Controlled Release of Ametryn Herbicide 5

The HPLC analyses were performed using a Varian ProStar instrument fitted with a PS 210 pump, a UV-VIS detector (PS 325), a Metatherm oven and an automatic injector (PS 410). The chromatograms were acquired and processed using Galaxy Workstation software. The eluent used was acetonitrile/water (70:30, v/v), at a flow rate of 1.4 mL min–1, and separation was achieved using a Phenomenex Gemini C18 reversed phase column (5 μm, 110 Å, 150 mm x 4.60 mm i.d.). Ametryn was detected at a wavelength of 260 nm. The injection volume was 100 μL, and all samples were previously filtered through 0.22 µm nylon

Microparticles were prepared with the PHB and PHBV polymers, used either individually or as a mixture, by formation of oil in water emulsions using the emulsification-solvent evaporation technique (Coimbra et al., 2008; Conti et al., 1995; Lionzo et al., 2007; Lobo et al., 2011). 200 mg of polymer (PHB, PHBV or a mixture of the two polymers, as described in Table 1) and 10 mg of herbicide were dissolved in 10 mL of chloroform to form the organic phase. The aqueous phase (200 mL) was prepared using 0.5 % (w/v) polyvinyl alcohol, at 50 oC. The organic phase was transferred to the aqueous phase (at 50 oC) with magnetic stirring (1000 rpm for 15 min). The chloroform was then evaporated from the emulsion. The suspension of microparticles formed was stored in an amber flask (to avoid any photodegradation of the herbicide). The final concentration of ametryn was 50 mg L-1.

**(mg) %** 

Table 1. Proportions of polymers used to prepare the different formulations.

**A** 200 *100* 0 *0*  **B** 150 *75* 50 *25* 

**C** 100 *50* 100 *50* 

**D** 50 *25* 150 *75*  **E** 0 *0* 200 *100* 

Portions (10 mg) of the different microparticles containing herbicide were dissolved in 50 mL of acetonitrile, and the association rate of the herbicide with the microparticles was determined by the technique described previously, which involves ultrafiltration/ centrifugation and analysis using HPLC (Kilic et al., 2005; Schaffazick et al., 2003). The samples were centrifuged in regenerated cellulose ultrafiltration filters that had a molecular size-exclusion pore size of 30 KDa (Microcon, Millipore), and the filtrate was analyzed using HPLC. The ametryn concentration was obtained from an analytical curve. The association

**PHB (mg) %**

**2.2.2 Preparation of the polymeric microparticles containing ametryn** 

**Formulation PHBV** 

**2.2.3 Measurements of encapsulation efficiency** 

al., 2002). It is therefore desirable to develop techniques whereby the physico-chemical properties of these chemicals can be altered and their usage made safer. The goal is to enable the use of soil management strategies that can produce foods at the current high levels of demand, without significant human or environmental risk.

Micro- and nanostructured polymeric materials can be used as transport systems for active chemicals. Advantages of these materials include good physical, chemical and biological stability, simple and reproducible preparation procedures, and applicability to a wide range of chemicals. In use, the active principle is released slowly and continuously, enabling the use of smaller quantities with greater efficiency, which reduces the risk of adverse environmental impacts (Sinha et al., 2004; Sopena et al., 2009).

Controlled release systems have been extensively used in the food and pharmaceutical industries for active substances including nutrients, drugs and aromas (El Bahri & Taverdet, 2007; Grillo et al., 2008; Mello et al., 2008; Moraes et al., 2010), and there has been a recent increase in their application in medicine (Natarajan et al., 2011; Parajo et al., 2010; Vicente et al., 2010).

Amongst the new controlled-release system technologies under development, the use of polymeric micro- and nanoparticles is of special interest in agribusiness. Several studies have investigated controlled-release systems for bioactive compounds in agricultural applications (Ahmadi & Ahmadi, 2007; Bin Hussein et al., 2010; El Bahri & Taverdet, 2005, 2007; Grillo et al., 2010; Hirech et al., 2003; Li et al., 2010; Lobo et al., 2011; Silva et al., 2010; Singh et al., 2008, 2010). Materials that have been used include silica, bentonite and sepiolite clays, and polymeric substances such as alginate, lignin and synthetic polymers. The latter include the poly(hydroxyalkanoates) (PHAs) (Salehizadeh & Loosdrecht, 2004), of which poly(3-hydroxybutyrate) (PHB) and its hydroxyvalerate copolymer (PHBV) have been most widely used (Amass & Tighe, 1998). The advantages of using polymers such as PHB and PHBV are that they are fully biodegradable, inexpensive and easily prepared by bacterial fermentation (Pouton & Akhtarb 1996; Reis et al., 2008). These polymers are isotactic and highly crystalline (55-80 %), so that their degradation rates are relatively slow compared to those of lactate (PLA) and glycolate (PGA) copolymers (Sudesh et al., 2000).

The objective of this work was to develop a novel release system for ametryn, employing microparticles prepared using two different polymers, PHB and PHBV (either individually or as mixtures). It was envisaged that the encapsulation of the herbicide in these microparticles would improve its chemical stability and enable the use of smaller quantities of the chemical, hence reducing the risk of environmental contamination.
