Journal of Hazardous Materials 186 (2011) 1645–1651
Contents lists available at ScienceDirect
Journal of Hazardous Materials
journal homepage: www.elsevier.com/locate/jhazmat
Controlled release system for ametryn using polymer microspheres:
Preparation, characterization and release kinetics in water
Renato Grilloa,b, Anderson do Espirito Santo Pereirab,c, Nathalie Ferreira Silva de Meloa,b,
Raquel Martins Portoc, Leandro Oliveira Feitosac, Paulo Sergio Tonelloa, Newton L. Dias Filhod,
André Henrique Rosaa, Renata Limac, Leonardo Fernandes Fracetoa,b,∗
aDepartment of Environmental Engineering, UNESP – Univ Estadual Paulista, Avenida Três de Marc ¸o, n◦511, CEP 18087-180 Sorocaba, SP, Brazil
bDepartment of Biochemistry, Institute of Biology, UNICAMP, Cidade Universitária Zeferino Vaz, s/n, Campinas, SP, Brazil
cDepartment of Biotechnology, University of Sorocaba, Sorocaba, SP, Brazil
dDepartment of Physics and Chemistry, UNESP – Univ Estadual Paulista, Ilha Solteira, SP, Brazil
a r t i c l ei n f o
Received 29 June 2010
Received in revised form
25 November 2010
Accepted 10 December 2010
Available online 17 December 2010
a b s t r a c t
The purpose of this work was to develop a modified release system for the herbicide ametryn by encap-
sulating the active substance in biodegradable polymer microparticles produced using the polymers
poly(hydroxybutyrate) (PHB) or poly(hydroxybutyrate-valerate) (PHBV), in order to both improve the
herbicidal action and reduce environmental toxicity. PHB or PHBV microparticles containing ametryn
were prepared and the efficiencies of herbicide association and loading were evaluated, presenting simi-
lar values of approximately 40%. The microparticles were characterized by scanning electron microscopy
(SEM), which showed that the average sizes of the PHB and PHBV microparticles were 5.92±0.74?m
and 5.63±0.68?m, respectively. The ametryn release profile was modified when it was encapsulated
in the microparticles, with slower and more sustained release compared to the release profile of pure
ametryn. When ametryn was associated with the PHB and PHBV microparticles, the amount of herbicide
released in the same period of time was significantly reduced, declining to 75% and 87%, respectively.
For both types of microparticle (PHB and PHBV) the release of ametryn was by diffusion processes due
to anomalous transport (governed by diffusion and relaxation of the polymer chains), which did not fol-
low Fick’s laws of diffusion. The results presented in this paper are promising, in view of the successful
encapsulation of ametryn in PHB or PHBV polymer microparticles, and indications that this system may
help reduce the impacts caused by the herbicide, making it an environmentally safer alternative.
© 2010 Elsevier B.V. All rights reserved.
Herbicides are molecules used worldwide for weed control.
However, these compounds and their degradation products show
varying degrees of persistence and mobility in the environment,
tials, as well as effects on the endocrine systems of non-target
organisms, including humans . To mitigate the toxicity of these
compounds in the environment, new and improved controlled
release systems are emerging that aim to increase the effective-
ness of herbicides while minimizing their environmental impacts
and aiding sustainable development.
∗Corresponding author at: Department of Environmental Engineering, UNESP –
Univ Estadual Paulista, Avenida Três de Marc ¸o, n◦511, CEP 18087-180 Sorocaba, SP,
Brazil. Tel.: +55 15 3238 3415; fax: +55 15 3228 2842.
E-mail address: email@example.com (L.F. Fraceto).
Controlled release systems have also been applied extensively
in the food and pharmaceutical industries for the release of active
substances such as nutrients, drugs and aromas [2–7].
among the new technologies under study as potential alternatives
for the development of release systems in agribusiness. Polymer
micro- and nanostructured systems can act as transport media
for bioactive substances, and are able to alter the physicochem-
ical properties of the substances they incorporate. In the case of
herbicides, these systems can offer the following advantages: (a)
reduction of the amount of chemical substance required for weed
control; (b) diminished risk of environmental contamination; (c)
reduction of energy consumption, since fewer applications are
needed compared to conventional formulations; and (d) increased
safety of the individuals who apply the product in the field.
The literature offers a wide range of release systems applica-
ble to the bioactive compounds of interest in agriculture [3,7–11].
These include materials composed of silica , clays such as ben-
tonites  and sepiolite , polymers such as alginate  and
0304-3894/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
R. Grillo et al. / Journal of Hazardous Materials 186 (2011) 1645–1651
lignin , and synthetic polymers such as polyhydroxyalkanoates
(PHAs) , including polyhydroxybutyrate (PHB) and its copoly-
mer hydroxyvalerate (PHBV), which are the most widely used PHA
polymers . The advantage of using polymers such as PHB and
produced by the fermentation of a variety of bacteria and degrade
during natural biological processes, making them important for
the production of release systems for bioactive materials [19,20].
decreases the speed of degradation in comparison to lactic (PLA)
and glycolic (PGA) homo- and copolymers .
structure presents an aromatic hexamer ring and which is widely
used in the pre- and post-emergence control of broadleaf weeds
and annual grasses . This type of compound acts by inhibit-
to the blockage of electronic transport. Plants that are sensitive to
sue necrosis . Prolonged exposure of humans to ametryn can
cause intoxication and contact dermatitis.
The aim of this work was to develop (involving preparation,
tem for the herbicide ametryn, using microparticles produced with
two different polymers, PHB and PHBV, since these polymers are
biodegradable and their degradation products are nontoxic to the
environment . The purpose of encapsulating ametryn in PHB
or PHBV microparticles was to produce a modified release system
that could enable the herbicide to be used more safely in agricul-
ture, minimizing its environmental impact. The importance of the
present work lies in the fact that the new formulations are not only
safer in environmental terms, but also safer for the individuals who
apply the product in the field, since smaller quantities of herbicide
imizes possible environmental contamination while at the same
time improving herbicidal activity.
alcohol (PVA) were purchased from Sigma Chemical Co. The
solvents employed for the chromatographic analysis were ace-
tonitrile, HPLC grade methanol (JT Baker) and Milli-Q water.
The solutions were filtered through 0.22?m nylon membranes
(Millipore®, Belford, USA).
2.2.1. Preparation of polymer microparticles containing ametryn
Microparticles consisting of the PHB or PHBV polymers were
prepared by forming an oil-in-water emulsion using the emul-
sion/solvent evaporation method [25,26]. 100mg of polymer (PHB
or PHBV) and 10mg of herbicide were dissolved in 10mL of chlo-
roform at 50◦C to produce the organic phase. The aqueous phase
(200mL) was prepared at 50◦C using 0.5% (m/v) of polyvinyl
alcohol. The organic phase was then transferred to the aqueous
phase (at 50◦C), under magnetic stirring (1000rpm, 15min) .
After this step, the chloroform was evaporated from the emul-
sion under reduced pressure, at 50◦C, using a rotary evaporation
system. The resulting microparticle suspension, containing a final
herbicide concentration of 50mg/L, was stored in an amber jar to
prevent photodegradation of the herbicide. Microparticles were
prepared without ametryn for use as controls, using the same
method described above.
2.2.2. Evaluation of encapsulation efficiency and herbicide
PHB or PHBV microparticles containing herbicide (10mg) were
dissolved in 50mL of methanol, and the percentage of ametryn
encapsulated in the microparticles was determined by the HPLC
ultrafiltration/centrifugation method [27,28].
The microparticle samples containing ametryn were cen-
trifuged in ultrafiltration filters composed of regenerated cellulose
matograph (HPLC, Varian®ProStar) coupled to a pump (PS 210),
410). The chromatograms were processed using Galaxy Worksta-
tion software. The concentration of ametryn was calculated using a
ponents of the colloidal suspension, and it was found that these
factors did not affect the quantification of ametryn.
The association rate of ametryn was determined from the dif-
ference between the concentration of the herbicide measured in
the filtrate and its total concentration (100%) in the microparticle
The chromatographic conditions employed for the quantifica-
tion were: mobile phase composed of acetonitrile/water (70:30,
v/v), at a flow rate of 1.4mLmin−1, and a Phenomenex Gem-
ini chromatographic column (C18 reversed phase, 5? 110A,
150mm×4.6mm). Ametryn was detected at a wavelength of
260nm, using an ultraviolet (UV) detector. The injection volume
was 100?L, and all samples injected were previously filtered
through a 0.22?M polyethersulfone membrane (Millipore®). Total
ametryn (100%) in the microparticle suspension was determined
after diluting the suspension in acetonitrile. Methanol was used
to dissolve the polymer, completely releasing the ametryn, which
was quantified using the calibration curve. Measurements were
performed in triplicate for each batch.
The encapsulation efficiency (EE, %) was expressed as the ratio
between the amount of herbicide encapsulated by the microparti-
cles and the total (100%) amount of herbicide, as described by Eq.
EE (%) =
where Wsis the weight of ametryn in the microspheres, and Wtotal
is the weight of ametryn used in the formulation.
The herbicide loading (HL, %) was determined by mixing 4mg of
sion for 4h, until the particles had fully degraded and the ametryn
final weight of the microparticles (Eq. (2)).
HL (%) =
and WP+S is the final weight of the microparticles (poly-
2.2.3. Scanning electron microscopy (SEM)
Scanning electron microscopy (SEM, JSM-6700F, JEOL, Japan)
was employed to evaluate the size distribution and surface mor-
phology of the PHB and PHBV microparticles, with and without
herbicide. The suspensions were filtered to collect the microparti-
water (150mL). The solids were dried overnight with Na2SO4in a
R. Grillo et al. / Journal of Hazardous Materials 186 (2011) 1645–1651
desiccator. The solid samples of microparticles were then attached
to metal holders (stubs) using double-sided tape, and coated with
a layer of gold for 150s, using a current of 25mA. After coating,
the stubs with the samples were placed in the scanning electron
microscope for analysis and imaging (electromicrography).
18.104.22.168. Sizedistribution. TheSEMmicrographswereusedtodeter-
mine the diameters and size distribution profiles of the PHB and
PHBV microparticles, with and without herbicide. Microparticle
size distributions employed OriginPro 7.0 software. At least 1000
spheres of each sample were used to determine the size distribu-
2.2.4. Study of ametryn release from the microparticles
ticles was determined based on in vitro release assays, using a
dual-compartment system to observe the release profiles of the
free herbicide alone and of the herbicide encapsulated in the
microparticles. In this system, which was maintained under slight
shaking at ambient temperature, a cellulose membrane (Spectra-
to separate the donor compartment (containing 4mL of herbicide
solution or microparticle suspension) from the acceptor compart-
ment (containing 50mL of deionized water) . The pore size
of the membrane in this system did not permit the passage of
microparticles, while the free herbicide could pass unimpeded
through the membrane.
The samples were collected in the acceptor compartment as a
function of time, and analyzed by HPLC at a wavelength of 260nm
(at 15min intervals during the first hour, 30min intervals during
the second hour, and at hourly intervals thereafter until the peak
area stabilized). The area values were converted into the amount
(%) of herbicide released as a function of time . All the mea-
surements were made in triplicate, and followed the dissolution
sink condition .
22.214.171.124. Release efficiency (RE). The release efficiency (RE, %), first
suggested by Khan and Rhodes , is a useful parameter that
can provide information on the release kinetics. This term can be
defined as the area under the release curve in a given time interval,
and allows different formulations containing the same active sub-
stance to be compared. The release efficiency was calculated based
on the values obtained from the area under the curve (AUC) of the
herbicide release profile (for the free herbicide or the herbicide in
the microparticle suspensions) at time intervals (t) ranging from
zero to 4.3 days. This calculated value was then divided by the area
above the theoretical curve of a rectangle, assuming a release of
100% between times zero and 4.3 days (AUC 100%) . The RE was
expressed in percentage terms, and can be defined by Equation 3.
RE (%) =AUC(zero − 4.3)
126.96.36.199. Mathematical modeling of ametryn release. The release pro-
files of bioactive compounds in microparticle polymer systems can
be modeled mathematically in order to obtain information con-
cerning the release mechanism. The model described by Peppas is
able to predict release mechanisms based on processes governed
by Fick’s laws of diffusion, non-Fickian processes and Case II diffu-
sion [33,34]. Using this model, it is possible to calculate the release
sion exponent (n) characteristic of the release mechanism. A value
of n=0.5 is expected for Fickian diffusion, while values of n=1.0
and 0.5<n<1.0 are expected for Case II diffusion and non-Fickian
diffusion, respectively [33–39]. The model proposed by Korsmeyer
and Peppas  was applied to the release curves in order to char-
acterize the mechanism of release of the herbicide encapsulated in
the PHB and PHBV microparticles. These curves can explain how
the molecules are released from polymer matrix systems, enabling
determination of the values of k, n and the linear correlation coef-
3. Results and discussion
3.1. Characterization of the microparticles containing ametryn
shown). The influence of four variables, at two levels, was exam-
ined in order to obtain formulations with optimized association
efficiencies. It was observed that there was a greater dependence
of association efficiency on PVA concentration (negative) and the
mass of polymer (positive), with lesser influence of both stirring
speed and organic phase volume. The present work was performed
using the microparticle formulations containing ametryn that had
been previously optimized using the experimental design proce-
The PHB and PHBV polymer microparticles containing ametryn
were prepared, and the efficiency of association of the herbicide
was evaluated by the method proposed by Schaffazick . The
encapsulation efficiencies (EE, %) of the PHB:AMT and PHBV:AMT
microparticles were 34.3% and 38.2%, respectively. The herbicide
loadings were 13.2% and 11.1% for the PHB and PHBV micropar-
ticles, respectively. These values are similar to those reported in
the literature for formulations involving the association of other
Bazzo et al.  studied the use of PHB microparticles con-
taining chitosan with two bioactive compounds, and found similar
encapsulation efficiency values. Sendil et al.  found associa-
tion efficiency values of 30% for PHBV microparticles containing
tetracycline, while Grillo et al.  showed that the encapsula-
tion efficiency of the herbicide atrazine in PHBV microparticles
was higher than 30%. The low efficiency of encapsulation of bioac-
tive substances in some microparticles could be related to the use
of high concentrations of emulsifiers, as is the case for the PHB
and PHBV microparticles, where poly-vinyl alcohol (PVA) is used
in the preparation procedure. The emulsifier increases the solubil-
ity of the herbicide in the aqueous phase, and therefore reduces
the association of the compound [44–47]. This could be useful in
agricultural practices, since a controlled release system in which
only 30% of the herbicide is incorporated within a carrier enables
the fraction of the active compound that is not associated with the
microparticles to be immediately released at the application site,
where it acts rapidly, while the remaining fraction is progressively
released with time.
Scanning electron microscopy (SEM) was used to examine the
influence of the encapsulation of ametryn on physical and mor-
phological characteristics of the microparticles. Fig. 1 presents
micrographs obtained for the PHB and PHBV microparticles con-
Both PHB and PHBV microparticles were spherical, although
their surface aspects differed. The PHB microparticles presented a
ticles were rough with numerous surface pores. Morphological
knowledge of the surface characteristics can help shed light on
the mechanism of release of the herbicide associated with them.
In other words, the larger the number of pores, the greater the
probability of the solvent coming into contact with the interior
R. Grillo et al. / Journal of Hazardous Materials 186 (2011) 1645–1651
Fig. 1. Images of the polymer nanoparticles: (a) PHB, and (b) PHBV.
of the particle, aiding the release of the herbicide molecules .
Grillo et al.  showed that PHBV microparticles, prepared using
the same methodology as that used in the present study, exhib-
ited roughness and high porosity, and that these features were
important for the release mechanism of the herbicide atrazine.
the size distribution profiles of the different polymer microparti-
cles. Fig. 2 shows representative images used for determining the
PHBV microparticles without herbicide.
Fig. 2. SEM micrographs (10kV, bar=10?m) and microparticle size distribution: (a) PHB, and (b) PHBV.
R. Grillo et al. / Journal of Hazardous Materials 186 (2011) 1645–1651
Fig. 3. Size distribution of PHB and PHBV microparticles containing ametryn: (a) PHB/AMT, (b) PHBV/AMT.
From Fig. 2, the PHB and PHBV microparticles (without her-
bicide) presented size distributions in the ranges 1–10?m and
1–12?m, respectively. The average sizes of the PHB and PHBV
microparticles were 5.92±0.74?m and 5.63±0.68?m, respec-
tively, indicating that there were no significant differences in size
(using the unpaired t-test, p<0.05). These data demonstrate that
although the surfaces of the particles displayed distinct character-
istics, they possessed similar size distributions.
The same size distribution analysis was performed for the PHB
and PHBV microparticles containing ametryn (Fig. 3).
As can be seen in Fig. 3, both types of microparticle (PHB
and PHBV) showed an increase in size, with distributions in the
ranges 3–21?m and 6–35?m for the PHB:AMT and PHBV:AMT
in the literature for microparticles produced with the same poly-
mers but containing other bioactive substances [41,43]. The mean
sizes of the PHB and PHBV microparticles containing ametryn
were 10.6±0.43?m and 20.5±0.93?m, respectively. These dif-
ferences in microparticle size were due to the encapsulation of the
herbicide in the microparticles, as described in the literature for
other molecules [41,43]. Suave et al.  investigated the size and
morphology of microparticles of poly(3-hydroxybutyrate)/poly(e-
caprolactone) containing malathion, and also observed alteration
of the size of the microparticles when they were associated with
The surface morphology of the particles was not affected by
the presence of ametryn, with the PHB microparticles remaining
smooth and the PHBV microparticles remaining rough (data not
3.2. Release kinetics of ametryn from microparticles
The release assays provided the release profiles of ametryn,
either free or encapsulated in the PHB and PHBV microparticles,
as a function of time (Fig. 4). In this assay, the herbicide passes
through the pores of the membrane while the microparticles do
not, hence allowing observation of the effect of the association
of the herbicide on its speed of release from the polymer matrix
of the microspheres. During the assay, aliquots were collected at
preestablished times and the herbicide was quantified by HPLC.
The results were expressed in terms of the percentage release.
Fig. 4 illustrates the release profiles of ametryn encapsulated in
PHB and PHBV microparticles as a function of time (up to approx-
imately 4.5 days), at ambient temperature. It should be noted that
in the tests using microparticles containing herbicide, a fraction
of non-encapsulated herbicide was present in the donor com-
partment (only about 40% of the herbicide was incorporated into
the particles). This means that the effect of the encapsulation of
this herbicide in the microspheres was not as marked as that
described by several previous authors [11,49,50], whose studies
ous medium (possibly also containing some co-solubilizer). In our
release tests, we aimed to evaluate the release profile of the col-
loidal suspension prepared without processing by filtration and
drying of the spheres, which would undoubtedly have resulted in
much longer release times comparable with the previous reports
Analysis of the release kinetics curves indicated that free
ametryn was released much more rapidly than when it was
encapsulated in the microparticles, with nearly 100% release after
1.2 days. In contrast, when associated with the PHB and PHBV
of time declined significantly, to 75% and 87%, respectively. This
modification of the release behavior of pesticides after association
with microstructured polymer systems has been widely reported
for other bioactive compounds [11,42,43,48–51].
Fig. 4. Results of in vitro release assays, comparing the kinetic profiles of pure AMT
and AMT encapsulated in PHB and PHBV microparticles at ambient temperature
R. Grillo et al. / Journal of Hazardous Materials 186 (2011) 1645–1651
Release constants (k), correlation coefficient (r) and diffusion exponent (n) obtained
by adjusting the curves of the release kinetics of the herbicides encapsulated in the
Parameters PHB:AMT PHBV:AMT
Release constant (k)
To better quantify the differences in the release profiles, the
release efficiencies (RE %) were calculated from the areas under
the herbicide release profile curves, for a given time interval (t),
using the method described in the literature . The RE values
tively, indicating that ametryn was released faster from the PHBV
The difference observed between the release profiles of encap-
by the structural characteristics of the microparticles. The associa-
tion of PHB with hydroxyvalerate (HV) causes a plastification effect
in the microparticles, increasing the polymer’s free volume and
reducing its crystallinity. The advantage of polymer plastification
due to lower resistance to diffusion inside the microparticle. Simi-
lar studies conducted by Gangrade and Price  on progesterone
encapsulation in PHB and PHBV (9–24% of HV) also concluded that
the higher the HV content the lower the polymer’s crystallinity,
due to the break in interchain regularity, rendering the particle
more porous and increasing the speed of release of the active prin-
ciple. Moreover, PHB has a lower speed of degradation by in vivo
hydrolysis than PLA and PHBV, which degrade more easily .
Another factor that could influence the release profile is the
presence of crystals in solution, which might occur if the herbicide
was not fully dissolved during the preparation of the microparti-
cles, and which could influence the release kinetics. However, the
The herbicide release profile curves were analyzed to obtain
involves several mechanisms, including desorption from the sur-
and dissolution and erosion of the polymer matrix or wall [27,53].
Fig. 5. Results of the analyses of PHB and PHBV microparticles containing ametryn,
using the mathematical model of Peppas.
Several mathematical models are used extensively to analyze
the characteristics of release of active substances from polymer
systems . The results obtained from the in vitro release assay
(Fig. 4) were analyzed by the model described by Peppas [33–39].
Table 1 and Fig. 5 present the values obtained for k, n and the cor-
relation coefficient, for ametryn release from the PHB and PHBV
The Peppas model, which is based on a semi-empirical equation
, is frequently employed in the absence of information about
a system’s release mechanism. In the present study, the values
found for the release constant k were 0.64min−1and 1.00min−1,
respectively, for the PHB:AMT and PHBV:AMT systems, indicating
cles. Values of the release coefficient (n) were 0.83 and 0.90 for the
involved diffusion processes due to anomalous transport, not gov-
erned by Fick’s laws of diffusion, but by diffusion and relaxation of
the polymer chains.
In this study, the herbicide ametryn was incorporated into
microparticles composed of two polymers, PHB and PHBV, with
encapsulation efficiencies of ∼40%. SEM analysis revealed that the
microparticles were spherical but had different surface proper-
ties (smooth or rough with pores). The formulations of PHB and
PHBV microparticles without herbicide showed similar size distri-
butions. However, encapsulation of the herbicide in PHB and PHBV
microparticles increased the size distributions of the microparti-
cles of both polymers. The release profile of ametryn was modified
by encapsulation in the microparticles, which provided a slower
and more sustained release compared to the release kinetics of the
free herbicide. This is a desirable feature in the use of herbicides,
since it diminishes their impacts on ecosystems, human health and
the environment. Mathematical modeling revealed that the ame-
tryn release mechanism was the same for both types of polymer
microparticle. The new carrier system has the potential to reduce
harmful effects of the herbicide, as well as provide a safer handling
This research was supported by the Brazilian agencies FAPESP,
CNPq (National Council for Scientific and Technological Develop-
ment) and FUNDUNESP. RG and NFSM are also grateful to CNPq for
granting them scholarships. AHR and LFF are recipients of fellow-
ships from CNPq.
agrotóxicos em cana-de ac ¸úcar na bacia do rio Corumbataí e o risco de poluic ¸ão
hídrica, Quim. Nova 30 (2007) 1119–1127.
 N.F. Melo, R. Grillo, A.H. Rosa, L.F. Fraceto, Study of the interaction between
Pharm. Biomed. Anal. 47 (2008) 865–869.
 R. Grillo, N.F.S. De Melo, D.R. De Araújo, E. De Paula, A.H. Rosa, L.F. Fraceto,
Polymeric alginate nanoparticles containing the local anesthetic bupivacaine,
J. Drug Target. 18 (2010) 688–699.
 R. Grillo, N.F.S. Melo, C.M. Moraes, R. Lima, C.M. Menezes, E.I. Ferreira, A.H.
Rosa, L.F. Fraceto, Study of the interaction between hydroxymethylnitrofu-
razone and 2-hydroxypropyl-beta-cyclodextrin, J. Pharm. Biomed. Anal. 47
 C.M. Moraes, E. De Paula, A.H. Rosa, L.F. Fraceto, Physicochemical stability of
poly(lactide-co-glycolide) nanocapsules containing the local anesthetic Bupi-
vacaine, J. Braz. Chem. Soc. 21 (2010) 995–1000.
 R. Duncan, L.W. Seymour, Controlled Release Technologies: A Survey of
R. Grillo et al. / Journal of Hazardous Materials 186 (2011) 1645–1651 Download full-text
 Z. El Bahri, J.L. Taverdet, Elaboration and characterisation of microparticles by
pesticide model, Powder Technol. 172 (2007) 30–40.
 K. Hirech, S. Payan, G. Carnelle, L. Brujes, J. Legrand, Microencapsulation of an
 Z. El Bahri, J.L. Taverdet, Optimization of an herbicide release from ethylcellu-
lose microspheres, Polym. Bull. 54 (2005) 353–363.
 B. Singh, D.K. Sharma, A. Gupta, In vitro release dynamics of thiram fungicide
from starch and poly(methacrylic acid)-based hydrogels, J. Hazard. Mater. 154
 B. Singh, D.K. Sharma, R. Kumar, A. Gupta, Development of a new controlled
pesticide delivery system based on neem leaf powder, J. Hazard. Mater. 177
 T.K. Barik, B. Sahu, V. Swain, Nanosilica—from medicine to pest control, Para-
sitol. Res. 103 (2008) 253–258.
 M. Fernández-Pérez, F. Flores-Céspedes, E. González-Pradas, M. Villafranca-
Sánchez, S. Pérez-García, F.J. Garrido-Herrera, Use of activated bentonites in
controlled-release formulations of atrazine, J. Agric. Food Chem. 52 (2004)
 C. Maqueda, J. Villaverde, F. Sopena, T. Undabeytia, E. Morillo, Novel system for
reducing leaching of the herbicide metribuzin using clay-gel-based formula-
tions, J. Agric. Food Chem. 56 (2008) 11941–11946.
 M. Fernádez-Pérez, Controlled release systems to prevent the agro-
environmental pollution derived from pesticide use, J. Environ. Sci. Health B
42 (2007) 857–862.
 F.J. Garrido-Herrera, I. Daza-Fernández, E. González-Pradas, M. Fernández-
Pérez, Lignin-based formulations to prevent pesticides pollution, J. Hazard.
Mater. 168 (2009) 220–225.
 H. Salehizadeh, M.C.M. Van Loosdrecht, Production of polyhydroxyalkanoates
by mixed culture: recent trends and biotechnological importance, Biotechnol.
Adv. 22 (2004) 261–279.
 W. Amass, B. Tighe, A review of biodegradable polymers: use, current devel-
opments in the synthesis and characterization of biodegradable polyesters,
blends of biodegradable polymers and recent advances in biodegradable stud-
ies, Polym. Int. 47 (1998) 89–144.
 C.W. Pouton, S. Akhtarb, Biosynthetic polyhydroxyalkanoates and their poten-
tial in drug delivery, Adv. Drug Delivery Rev. 18 (1996) 133–162.
 K.C. Reis, J. Pereira, A.C. Smith, C.W.P. Carvalho, N. Wellner, I. Yakimets, Char-
acterization of polyhydroxybutyrate-hydroxyvalerate (PHB-HV)/maize starch
blend films, J. Food Eng. 89 (2008) 361–369.
 K. Sudesh, T. Fukui, T. Iwata, Y. Doi, Factors affecting the freeze-fracture mor-
phology of in vivo polyhydroxyalkanoate granules, Can. J. Microbiol. 46 (2000)
 A.H. Tennant, P. Peng, A.D. Kligerman, Genotoxicity studies of three triazine
herbicides: in vivo studies using the alkaline single cell gel (SCG) assay, Mutat.
Res. 493 (2001) 1–10.
 L. Prade, R. Huber, B. Bieseler, Structures of herbicides in complex with their
detoxifying enzyme glutathione S-transferase—explanations for the selectivity
of the enzyme in plants, Structure 11 (1998) 1445–1452.
 O.N. Vo˘ ınova, G.S. Kalacheva, I.D. Grodnitskaia, T.G. Volova, Microbial poly-
mers as a degradable carrier for pesticide delivery, Prikl. Biokhim. Mikrobiol.
45 (2009) 427–431.
 B. Conti, I. Genta, T. Modena, F. Pavanetto, Investigation on process parameters
involved in polylactide-co-glycolide microspheres preparation, Drug Dev. Ind.
Pharm. 21 (1995) 615–622.
 M.I.Z. Lionzo, M.I. Ré, S.S. Guterres, A.R. Pohlmann, Microparticles prepared
with poly(hydroxybutyrate-co-hydroxyvalerate) and poly((-caprolactone)
blends to control the release of a drug model, J. Microencapsul. 24 (2007)
 S.R. Schaffazick, S.S. Guterres, L.L. Freitas, A.R. Pohlmann, Physicochemical
characterization and stability of the polymeric nanoparticle systems for drug
administration, Quim. Nova 5 (2003) 726–737.
 A.C. Kilic, Y. Capan, I. Vural, R.N. Gursoy, T. Dalkara, A. Cuine, A.A. Hincal, Prepa-
ration and characterization of PLGA nanospheres for the targeted delivery of
NR2B-specific antisense oligonucleotides to the NMDA receptors in the brain,
J. Microencapsul. 22 (2005) 633–641.
 A. Paavola, J. Yliruusi, Y. Kajimoto, E. Kalso, T. Wahlström, P. Rosenberg, Con-
trolled release of lidocaine from injectable gels and efficacy in rat sciatic nerve
block, Pharm. Res. 12 (1995) 1997–2002.
 D.R. De Araújo, C.M.S. Cereda, G.B. Brunetto, L.M.A. Pinto, M.H.A. Santana, E. De
Paula, Encapsulation of mepivacaine prolongs the analgesia provided by sciatic
nerve blockade in mice, Can. J. Anesth. 51 (2004) 566–572.
 M. Aulton, Dissolution and Solubility, Pharmaceutics: The Science of Dosage
from Design, 2nd ed., Churchill Livingstone, Edinburgh, 2002, 15–32 (Chapter
 K.A. Khan, C.T. Rhodes, The concept of dissolution efficiency, J. Pharm. Pharma-
col. 27 (1975) 48–49.
 P.L. Ritger, N.A. Peppas, A simple equation for description of solute release.
II. Fickian and anomalous release from swellable devices, J. Control. Release 5
 P.L. Ritger, N.A. Peppas, A simple equation for description of solute release I.
spheres, cylinders or discs, J. Control. Release 5 (1987) 23–36.
delivery from swellable systems with partial physical restrictions or imperme-
able coatings, Int. J. Pharm. 112 (1994) 47–54.
 P. Colombo, R. Bettini, G. Massimo, P.L. Catellani, P. Santi, N.A. Peppas, Drug
diffusion front movement is important in drug release control from swellable
matrix tablets, J. Pharm. Sci. 84 (1995) 991–997.
 G. Colombo, R. Padera, R. Langer, D.S. Kohane, Prolonged duration anesthesia
J. Biomed. Mater. Res. A 75 (2005) 458–464.
 C. Ferrero, A. Mu˜ noz-Ruiz, M.R. Jiménez Castellanos, Fronts movement as a
useful tool for hydrophilic matrix release mechanism elucidation, Int. J. Pharm.
202 (2000) 21–28.
 C. Costa, J.M.S. Lobo, Modeling and comparison of dissolution profiles, Eur. J.
Pharm. Sci. 13 (2001) 123–133.
 R.W. Korsmeyer, N.A. Peppas, Macromolecular and modeling aspects of
swelling-controlled systems, in: T.J. Roseman, S.Z. Mansdorf (Eds.), Controlled
Release Delivery Systems, Marcel Dekker Inc., New York, USA, 1991.
 G.C.Bazzo, E.Lemos-Senna, A.T.N.
chitosan/ketoprofen or piroxicam composite microparticles: preparation
and controlled drug release evaluation, Carbohydr. Polym. 77 (2009) 839–
 D. Sendil, I. Gursel, D.L. Wise, V. Hasircia, Antibiotic release from biodegradable
PHBV microparticles, J. Control. Release 59 (1999) 207–217.
 R.Grillo,N.F.Melo,R.Lima,R.Lourenc ¸o,A.H.Rosa,L.F.Fraceto,Characterization
of atrazine-loaded biodegradable poly(hydroxybutyrate-co-hydroxyvalerate)
microspheres, J. Polym. Environ. 18 (2010) 26–32.
 M. Cea, P. Cartes, G. Palma, M.L. Mora, Atrazine efficiency in an andisol as
affected by clays and nanoclays in ethylcellulose controlled release formula-
tions, R. C. Suelo Nutr. Veg. 10 (2010) 62–77.
 F. Sope˜ na, A. Cabrera, C. Maqueda, E. Morillo, Ethylcellulose formulations for
controlled release of the herbicide alachlor in a sandy soil, J. Agric. Food Chem.
55 (2007) 8200–8205.
 F. Sope˜ na, C. Maqueda, E. Morillo, Norflurazon mobility, dissipation, activity,
55 (2007) 3561–3567.
 R. Fernandez-Urrusu˜ no, J.M. Gines, E. Morillo, Development of controlled
release formulations of alachlor in ethylcellulose, J. Microencapsul. 17 (2000)
 J. Suave, E.C. Dall’Agnol, A.P.T. Pezzin, M.M. Meier, D.A.K. Silva, Biodegrad-
able microspheres of poly(3-hydroxybutyrate)/poly(e-caprolactone) loaded
release testing, J. Appl. Polym. Sci. 117 (2010) 3419–3427.
 C. Maqueda, J. Villaverde, F. Sope˜ na, T. Undabevtia, E. Morillo, Effects of soil
characteristics on metribuzin dissipation using clay-gel-based formulations, J.
Agric. Food Chem. 57 (2009) 3273–3278.
 X. Liu, W.S. Heng, L.Q. Paul, L.W. Chan, Novel polymeric microspheres con-
taining norcantharidin for chemoembolization, J Control. Release 116 (2006)
 Y. Wang, X. Wang, K. Wei, N. Zhao, S. Zhang, J. Chen, Fabrication, charac-
terization and long-term in vitro release of hydrophilic drug using PHBV/HA
composite microspheres, J. Mater. Lett. 61 (2007) 1071–1076.
 N. Gangrade, J.C. Price, Poly(hydroxybutyrate-hydroxyvalerate) microspheres
containing progesterone: preparation, morphology and release properties, J.
Microencapsul. 8 (1991) 185–202.
 M. Polakovic, T. Gorner, R. Gref, E. Dellacherie, Lidocaine loaded biodegrad-
able nanospheres II. Modelling of drug release, J. Control. Release 60 (1999)