**3. Results and discussion**

6 Herbicides – Properties, Synthesis and Control of Weeds

rate of ametryn was calculated from the difference between the concentration measured in the filtrate and the total concentration (100 %) in the microparticle suspension. The total concentration was measured after diluting the suspension with acetonitrile, which dissolved the polymer and ensured complete release of the herbicide. The measurements were performed in triplicate for each formulation. The encapsulation efficiency (EE, %) was

<sup>S</sup>

TOTAL <sup>W</sup> EE % x 100%

Where, *Ws* is the quantity of ametryn in the microparticles and *Wtotal* is the amount of

A scanning electron microscope (Model JSM-6700F, JEOL, Japan) was used to investigate the size distribution and surface morphology of the microparticles. Suspensions of microparticles containing the herbicide were filtered and the particles were then washed with 150 mL of distilled water. The solid residues were dried overnight over Na2SO4 in a desiccator. The samples were then attached to metallic supports (stubs) with double-sided tape, and metalized by deposition of a gold layer at a current of 25 mA for 150 s. Images (electron micrographs) of the samples were then generated using the microscope. Particle sizes were measured using the ImageJ 1.42 program, and the size distributions of the different microparticles were obtained using OriginPro 7.0. At least 1000 individual particles

The release profiles of ametryn, either free or associated with the microparticles, were investigated using a two-compartment experimental system. A cellulose membrane (Spectrapore, with a molecular exclusion pore size of 1000 Da) separated the donor compartment, containing 4 mL of solution (or suspension) of the herbicide, from the acceptor compartment, which contained 50 mL of deionized water maintained under gentle agitation at ambient temperature (Paavola et al., 1995). The pore size of the membrane only allowed passage of the free herbicide, while the herbicide associated with the microparticles was retained in the donor compartment until the equilibrium was shifted so as to release the ametryn present within the particles. The size of the microparticles prevented their passage through the pores of the membrane. These experiments were conducted under *dilution sink* conditions, whereby the volume of the dissolution medium was sufficiently large that the herbicide concentration never exceeded 10 % of the value of its saturation concentration

Samples were retrieved from the acceptor compartment as a function of time, and analyzed by HPLC at a detector wavelength of 260 nm. During the first hour, samples were collected every 15 min, during the second hour every 30 min, and subsequently at hourly intervals until the peak area stabilized. The peak area values were then converted into the percentage

of herbicide released as a function of time (De Araújo et al., 2004).

W (1)

expressed as the ratio:

(Aulton et al., 2002).

ametryn used in the formulation.

**2.2.4 Scanning electron microscopy (SEM)** 

of each sample were used for these measurements.

**2.2.5 Release of ametryn from the microparticles** 

The encapsulation efficiency values obtained for the different microparticles are listed in Table 2. Formulation A (100 % PHBV) showed the highest encapsulation efficiency (76.5 %). The efficiency decreased as the proportion of PHBV decreased, and formulation E (100 % PHB) provided the lowest encapsulation efficiency (26.2 %). The values obtained for formulations A and B were fairly high, relative to values that have been reported in the literature for other active principles (Bazzo et al., 2009; Grillo et al., 2010; Lobo et al., 2011; Sendil et al., 1999). Grillo and colleagues (2010) showed that the encapsulation efficiency of the herbicide atrazine in PHBV microparticles was in excess of 30 %. Lobo et al. (2011), using an experimental design optimization procedure, obtained an encapsulation efficiency of 24 % for atrazine in PHBV microparticles.


Table 2. Encapsulation efficiencies (EE, %) of the different microparticles.

The relationship between the percentage of PHBV and the encapsulation efficiency is illustrated in Figure 2. There was a polynomial relationship between the encapsulation efficiency and the PHBV concentration, which was positive for PHBV and negative for PHB. This can probably be explained by the structural differences between the microparticles, due to the different polymer ratios used in their preparation (Table 1).

The morphological characteristics of the microparticles, as well as the influence of the encapsulation of ametryn, were analyzed using the SEM procedure. Electron micrographs of the microparticles containing ametryn are illustrated in Figure 3. All types of microparticle were spherical, although the surface structures were different. Most of the PHB microparticles possessed smooth surfaces with few pores, while most of the PHBV microparticles were rough-surfaced with many cavities and pores, some of which were quite large, as can be clearly seen for formulation A (Figure 3, a1 and a2). Grillo et al. (2010) also found that PHBV microparticles, prepared using the same methodology as that

Preparation and Characterization

of Polymeric Microparticles Used for Controlled Release of Ametryn Herbicide 9

a 1) a 2)

b) c)

d) e)

Fig. 3. SEM images of the polymeric microparticles: a) Formulation A; b) Formulation B;

c) Formulation C; d) Formulation D; e) Formulation E.

described here, were rough-surfaced with pores, while PHB microparticles had smooth surfaces and fewer pores.

Fig. 2. Encapsulation efficiency according to PHBV content of the microparticles.

A higher encapsulation efficiency of ametryn was therefore related to a greater number of pores in the microparticles, probably due to greater contact (and/or affinity) of the herbicide with the microparticles during the formulation preparation procedure. Ametryn is likely to have greater affinity for the PHBV polymer, since both of these molecules possess alkyl branches, with interaction being further enhanced by the porosity of the PHBV microparticles.

The size distribution profiles (Figure 4) differed between microparticle formulations (it was not possible to measure the size distribution of the formulation D microparticles due to focusing problems). The average size of the microparticles (Table 3) increased as the PHBV concentration decreased and the PHB concentration increased, and was greatest for the PHB microparticles (formulation E). These size differences could be related to the incorporation of the herbicide as well as to associations between the molecules (as discussed above). At higher encapsulation rates, the amount of ametryn present within the microparticle increased, and the potential for reactions and interactions with the polymer therefore also increased. Ametryn is likely to have a higher affinity for PHBV, and as a result of this affinity (and/or reaction) the polymer contracts due to the formation of linkages between the polymer chains. As the proportion of PHBV decreases, the affinity of ametryn for the polymer mixture also diminishes (due to the lower affinity of ametryn for PHB), so that there is less shrinkage.

described here, were rough-surfaced with pores, while PHB microparticles had smooth

0,0 0,2 0,4 0,6 0,8 1,0

[PHBV], %

A higher encapsulation efficiency of ametryn was therefore related to a greater number of pores in the microparticles, probably due to greater contact (and/or affinity) of the herbicide with the microparticles during the formulation preparation procedure. Ametryn is likely to have greater affinity for the PHBV polymer, since both of these molecules possess alkyl branches, with interaction being further enhanced by the porosity of the PHBV

The size distribution profiles (Figure 4) differed between microparticle formulations (it was not possible to measure the size distribution of the formulation D microparticles due to focusing problems). The average size of the microparticles (Table 3) increased as the PHBV concentration decreased and the PHB concentration increased, and was greatest for the PHB microparticles (formulation E). These size differences could be related to the incorporation of the herbicide as well as to associations between the molecules (as discussed above). At higher encapsulation rates, the amount of ametryn present within the microparticle increased, and the potential for reactions and interactions with the polymer therefore also increased. Ametryn is likely to have a higher affinity for PHBV, and as a result of this affinity (and/or reaction) the polymer contracts due to the formation of linkages between the polymer chains. As the proportion of PHBV decreases, the affinity of ametryn for the polymer mixture also diminishes (due to the lower affinity of ametryn for PHB), so that

Fig. 2. Encapsulation efficiency according to PHBV content of the microparticles.

surfaces and fewer pores.

20

microparticles.

there is less shrinkage.

30

40

50

%EE

60

70

80

Fig. 3. SEM images of the polymeric microparticles: a) Formulation A; b) Formulation B; c) Formulation C; d) Formulation D; e) Formulation E.

Preparation and Characterization

solvent.

0

ambient temperature (n = 3).

20

40

60

Release percentage, %

80

100

D) and 40 % (formulation E) being released after 360 min.

 Ametryn Formulation A Formulation B Formulation C Formulaçtion D Formulation E

of Polymeric Microparticles Used for Controlled Release of Ametryn Herbicide 11

almost total release after 360 min. Association with the microparticles resulted in retarded release, with around 70 % (formulations A and B), 30 % (formulation C), 20 % (formulation

The release of other bioactive compounds from systems composed of microstructured polymers has been described in the literature, but usually for only one type of polymer (Grillo et al., 2010; Maqueda et al., 2009; Sendil et al., 1999; Singh et al., 2010; Wang et al., 2007). However, interpretation of release profiles relies to a large extent on knowledge of the composition and structural characteristics of the microparticles concerned, and in this respect studies that use more than one type of microparticle are advantageous. In the present work, the release of ametryn increased in line with the content of PHBV for formulations A-D, indicating that increased porosity aided the exit of ametryn molecules due to increased contact with the solvent. However formulation E was an exception to the rule, since it was composed of PHB alone and showed the fastest release of ametryn. There are two possible explanations for this observation. Firstly, the encapsulation efficiency of this formulation was lower than those achieved using the other formulations, which could have resulted in higher concentrations of ametryn crystals in the solution, and consequently higher release rates. Secondly, it is possible that lengthy refrigerated storage of this sample could have resulted in solubilization of the herbicide, due to increased contact time with the

0 50 100 150 200 250 300 350 400 450

Time (minutes) Fig. 5. Results of the release experiments, comparing the kinetic profiles of free ametryn and ametryn associated with the different microparticles (PHB, PHBV and PHBV+PHB), at

Analysis of release curves can provide important information concerning the mechanisms involved in the release of compounds from microparticles (Polakovic et al., 1999). Possible mechanisms include desorption from the surface of the polymeric matrix, diffusion through the pores or wall of the matrix, disintegration of the microparticle with subsequent release

A=76,5 B=54,7

E=26,2

C=40,5

D =29,3

Fig. 4. Size distributions of the polymeric microparticles: a) Formulation A; b) Formulation B; c) Formulation C; d) Formulation E.


\* Not determined.

Table 3. Average sizes (± SD) of the different microparticles.

The release profiles of free ametryn (as the reference) and ametryn encapsulated in the microparticles are illustrated in Figure 5, as a function of time (up to approximately 360 min). In these experiments the herbicide could traverse the pores of the membrane, while the microparticles were retained, so that it was possible to measure the influence of the association of ametryn with the polymeric matrix of the microparticles on its release rate. The release kinetics of free ametryn was faster than that of the encapsulated herbicide, with

Percentage (%)

Diameter (m) 10 20 30 40 50 60 0 2 4 6 8 10 12

c d Fig. 4. Size distributions of the polymeric microparticles: a) Formulation A; b) Formulation

> **Formulation PHBV (%) PHB (%) Average size (µm) A** 100 0 24.14 ± 1.606 **B** 75 25 31.45 ± 2.797 **C** 50 50 33.5 ± 3.22 **D** 25 75 \*

**E** 0 100 110.2 ± 3.881

The release profiles of free ametryn (as the reference) and ametryn encapsulated in the microparticles are illustrated in Figure 5, as a function of time (up to approximately 360 min). In these experiments the herbicide could traverse the pores of the membrane, while the microparticles were retained, so that it was possible to measure the influence of the association of ametryn with the polymeric matrix of the microparticles on its release rate. The release kinetics of free ametryn was faster than that of the encapsulated herbicide, with

Percentage (%)

a b

Diameter (m)

40 60 80 100 120 140 160 180

Diameter (m)

Formulation B

Formulation E

10 20 30 40 50

20 25 30 35 40 45 50 55 60

Table 3. Average sizes (± SD) of the different microparticles.

Diameter (m)

B; c) Formulation C; d) Formulation E.

Formulation A

Formulation C

0

\* Not determined.

Percentage (%)

5

10

Percentage (%)

15

20

almost total release after 360 min. Association with the microparticles resulted in retarded release, with around 70 % (formulations A and B), 30 % (formulation C), 20 % (formulation D) and 40 % (formulation E) being released after 360 min.

The release of other bioactive compounds from systems composed of microstructured polymers has been described in the literature, but usually for only one type of polymer (Grillo et al., 2010; Maqueda et al., 2009; Sendil et al., 1999; Singh et al., 2010; Wang et al., 2007). However, interpretation of release profiles relies to a large extent on knowledge of the composition and structural characteristics of the microparticles concerned, and in this respect studies that use more than one type of microparticle are advantageous. In the present work, the release of ametryn increased in line with the content of PHBV for formulations A-D, indicating that increased porosity aided the exit of ametryn molecules due to increased contact with the solvent. However formulation E was an exception to the rule, since it was composed of PHB alone and showed the fastest release of ametryn. There are two possible explanations for this observation. Firstly, the encapsulation efficiency of this formulation was lower than those achieved using the other formulations, which could have resulted in higher concentrations of ametryn crystals in the solution, and consequently higher release rates. Secondly, it is possible that lengthy refrigerated storage of this sample could have resulted in solubilization of the herbicide, due to increased contact time with the solvent.

Fig. 5. Results of the release experiments, comparing the kinetic profiles of free ametryn and ametryn associated with the different microparticles (PHB, PHBV and PHBV+PHB), at ambient temperature (n = 3).

Analysis of release curves can provide important information concerning the mechanisms involved in the release of compounds from microparticles (Polakovic et al., 1999). Possible mechanisms include desorption from the surface of the polymeric matrix, diffusion through the pores or wall of the matrix, disintegration of the microparticle with subsequent release

Preparation and Characterization

according to circumstances.


agents such as ametryn in modern agriculture.

Vol.19, No.1, pp. 187-194.

The authors thank FAPESP, CNPq and Fundunesp for financial support.

**4. Conclusions** 

**5. Acknowledgments** 

**6. References** 




ln( MM Total)




Formulation A Formulation B Formulation C Formulation D Formulation E

0.0

of Polymeric Microparticles Used for Controlled Release of Ametryn Herbicide 13

according to formulation, as expected considering the different structural characteristics of the microparticles, so that the release mechanisms were not identical. Nonetheless, the values obtained for all formulations were in the range 0.43 < n < 0.85, indicating that in all cases the release occurred as a result of anomalous transport, involving diffusion and relaxation of the polymeric chains. This information concerning the release mechanism is of vital importance in order to be able to adjust and optimize the release of the active principle

2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0

ln t (minutes) Fig. 6. Results obtained using the Korsmeyer-Peppas model applied to formulations A-E.

Ametryn herbicide was efficiently encapsulated in microparticles composed of PHB, PHBV and mixtures of the two polymers. The highest encapsulation efficiencies were achieved when higher proportions of PHBV were used. SEM analysis showed that the microparticles were spherical, although with different surface features (either smooth or rough with pores). The release profile of ametryn was modified when it was encapsulated, with slower and more sustained release compared to the free herbicide. This finding suggests that the use of encapsulated ametryn could help to mitigate adverse impacts on ecosystems and human health. This is particularly important given the increasingly widespread and intensive use of

Ahmadi, A. & Ahmadi, A. (2007). Preparation and characterization of chemical structure

composition of polyurethane's microcapsules pesticides. *Asian Journal of Chemistry*,

of the active principle, and dissolution and erosion of the matrix or the polymeric wall (Polakovic et al., 1999; Schaffazick et al., 2003).

A number of mathematical models have been extensively used to analyze the characteristics of the release of substances from polymeric systems (Costa & Lobo 2001). Here, the results of the release experiments (Figure 5) were analyzed using the zero order, first order, Higuchi and Korsmeyer-Peppas models (Table 4). For the formulations investigated, the Korsmeyer-Peppas model provided the best explanation of the ametryn release mechanism, according to the correlation coefficient obtained. The curves obtained for each formulation using this model are illustrated in Figure 6.


Table 4. Results of the application of four mathematical models to the release curves of ametryn associated with different microparticles.

The Korsmeyer-Peppas model is based on a semi-empirical equation (Korsmeyer & Peppas, 1991; Korsmeyer et al., 1983) that is widely used when the release mechanism is unknown. When the release exponent (n) is equal to 0.43 the mechanism involved is diffusion. When the value of the exponent is greater than 0.43 but smaller than 0.85, the release occurs due to anomalous transport that does not obey Fick's Law. Values less than 0.43 are indicative of porous systems in which transport occurs by a combination of diffusion through the polymeric matrix and diffusion through the pores. The values obtained (Table 4) differed according to formulation, as expected considering the different structural characteristics of the microparticles, so that the release mechanisms were not identical. Nonetheless, the values obtained for all formulations were in the range 0.43 < n < 0.85, indicating that in all cases the release occurred as a result of anomalous transport, involving diffusion and relaxation of the polymeric chains. This information concerning the release mechanism is of vital importance in order to be able to adjust and optimize the release of the active principle according to circumstances.

Fig. 6. Results obtained using the Korsmeyer-Peppas model applied to formulations A-E.
