molecules

Article
Nanoemulsion Formulations of Fungicide
Tebuconazole for Agricultural Applications
Vianney Díaz-Blancas 1 , Dora I. Medina 2 , Erika Padilla-Ortega 1, *, Raquel Bortolini-Zavala 1 ,
Melissa Olvera-Romero 1 and Gabriel Luna-Bárcenas 1, *
1 Cinvestav Queretaro, Libramiento Norponiente 2000, Fracc. Real de Juriquilla, Queretaro, Queretaro 76230,
Mexico; vian_versus@hotmail.com (V.D.-B.); rachel_p53@hotmail.com (R.B.-Z.);
melissa_ireth7pr@hotmail.com (M.O.-R.)
2 Tecnologico de Monterrey, School of Engineering and Sciences, Atizapan de Zaragoza,
Estado de Mexico 52926, Mexico; dora.medina@cantab.net
* Correspondence: erikapadilla@cinvestav.mx (E.P.-O.); gabriel.luna@cinvestav.mx (G.L.-B.);
Tel.: +52-442-211-9900 (E.P.-O. & G.L.-B.)

Academic Editor: Derek J. McPhee

Received: 26 July 2016; Accepted: 17 September 2016; Published: 26 September 2016

Abstract: Tebuconazole (TBZ) nanoemulsions (NEs) were formulated using a low energy method.
TBZ composition directly affected the drop size and surface tension of the NE. Water fraction and the
organic-to-surfactant-ratio (RO/S ) were evaluated in the range of 1–90 and 1–10 wt %, respectively.
The study was carried out with an organic phase (OP) consisting of an acetone/glycerol mixture
containing TBZ at a concentration of 5.4 wt % and Tween 80 (TW80) as a nonionic and Agnique
BL1754 (AG54) as a mixture of nonionic and anionic surfactants. The process involved a large dilution
of a bicontinuous microemulsion (ME) into an aqueous phase (AP). Pseudo-ternary phase diagrams of
the OP//TW80//AP and OP//AG54//AP systems at T = 25 ◦ C were determined to map ME regions;
these were in the range of 0.49–0.90, 0.01–0.23, and 0.07–0.49 of OP, AP, and surfactant, respectively.
Optical microscope images helped confirm ME formation and system viscosity was measured in the
range of 25–147 cP. NEs with drop sizes about 9 nm and 250 nm were achieved with TW80 and AG54,
respectively. An innovative low-energy method was used to develop nanopesticide TBZ formulations
based on nanoemulsion (NE) technology. The surface tension of the studied systems can be lowered
50% more than that of pure water. This study’s proposed low-energy NE formulations may prove
useful in sustainable agriculture.

Keywords: tebuconazole; nanoemulsion; organic-to-surfactant ratio

1. Introduction
The increased demand for crop raw materials needed to develop new applications, including
biofuels, is creating an increasingly competitive agriculture sector, according to a June 2013 report
on worldwide agriculture by the United Nations (UN) Food and Agriculture Organization and
the Organization for Economic Cooperation and Development [1]. Consequently, development
of more-efficient technologies for formulating pesticides is being sought worldwide [2,3].
Nanotechnology from the food and pharmacy fields are being used to develop a more efficient
agrochemical industry featuring nanopesticides that are less harmful to the environment than
conventional formulation [4]. However, there is controversy about the fate of nanopesticides and
nanofertilizers, including a lack of consensus about a definition of ‘nanopesticide’ that satisfies
international regulations.
A nanopesticide may be defined as a nanomaterial containing an active ingredient with pesticidal
action [5]. In this regard, nanoemulsions (NEs) [6,7] are of special interest for use as pesticide

Molecules 2016, 21, 1271; doi:10.3390/molecules21101271 www.mdpi.com/journal/molecules

Molecules 2016, 21, 1271 2 of 12

formulations,
Molecules 2016, 21,since
1271 many of them are marketed as emulsifiable concentrates or microemulsions 2 of 12
(MEs) [8–10] that contain their active ingredient at high concentrations.
NEs are dispersions of two liquid phases whose drop sizes are less than 0.5 microns; the drop
size of
ofthe
thediscrete
discretephase
phase guarantees
guarantees mechanical
mechanical stability
stability [11–13].
[11–13]. The advantages
The advantages of NEsofover NEs over
typical
typical emulsions are their smaller drop sizes, which give the system greater wettability,
emulsions are their smaller drop sizes, which give the system greater wettability, spreadability, and spreadability,
and superior
superior mechanical
mechanical stability.
stability. These
These characteristics
characteristics maymayhelp help reduce
reduce volatilization
volatilization andand hydrolysis
hydrolysis of
of
thethe active
active ingredient,
ingredient, thus
thus augmentingbioavailability.
augmenting bioavailability.TheseThesenew newproperties
properties allow
allow NE formulation
with lower concentrations of active ingredients and surfactants [6,14–17]. NEs generally are obtained
by diluting an ME; this process is called phase inversion composition (PIC) and is promoted by the
drastic
drastic change
changeininthethesystem’s
system’s concentration.
concentration. The emulsification
The emulsification method is designated
method a PICawhen
is designated there
PIC when
is inversion
there of the of
is inversion spontaneous
the spontaneous curvature of the surfactant
curvature film during
of the surfactant emulsification.
film during This low-energy,
emulsification. This low-
non-spontaneous
energy, non-spontaneousNE formulation requiresrequires
NE formulation mechanical or chemical
mechanical energy to
or chemical disperse
energy the discrete
to disperse the
phase as nanodroplets [18].
discrete phase as nanodroplets [18].
In addition
additiontotoactive
active ingredients,
ingredients, formulating
formulating pesticides
pesticides involves
involves solvents
solvents and adjuvants
and adjuvants whose
whose toxic characteristics
toxic characteristics may resultmay inresult in formulations
formulations more more toxic the
toxic than thanactive
the active ingredient
ingredient itself.itself.
The
The advantage
advantage of biodegradable
of biodegradable solvents
solvents is that
is that thethe toxicity
toxicity of ofthethe final
final productwould
product wouldnotnotbebeincreased.
increased.
Commonly used pesticides, including tebuconazole (TBZ), as seen in Figure 1, have this degree
of toxicity.
toxicity. TBZ is a fungicide that is on the list list of
of the
the most
most dangerous
dangerous pesticides
pesticides (Pesticide
(Pesticide Action
Network International, 2009).

Figure 1.
Figure 1. Chemical
Chemical structure of TBZ.
structure of TBZ.

TBZ belongs to the chemical group of triazoles and is noted for its preventive and curative actions.
TBZ belongs to the chemical group of triazoles and is noted for its preventive and curative
Its disadvantage is low water solubility (32 mg/L); therefore, existing formulations are usually
actions. Its disadvantage is low water solubility (32 mg/L); therefore, existing formulations are usually
emulsifiable concentrates based on methylene chloride and acetone [19,20].
emulsifiable concentrates based on methylene chloride and acetone [19,20].
TBZ has a Groundwater Ubiquity Score (GUS) index of 2.3, which means it is considered as
TBZ has a Groundwater Ubiquity Score (GUS) index of 2.3, which means it is considered as having
having moderate potential for leaching into groundwater. TBZ is a persistent pesticide with an
moderate potential for leaching into groundwater. TBZ is a persistent pesticide with an adsorption
adsorption constant Koc (for organic carbon) of between 803 and 1251 mL/g [21]. TBZ’s systemic action
constant Koc (for organic carbon) of between 803 and 1251 mL/g [21]. TBZ’s systemic action allows it to
allows it to be absorbed through plant leaves and roots to inhibit the synthesis of ergosterol, a lipid
be absorbed through plant leaves and roots to inhibit the synthesis of ergosterol, a lipid component of
component of the cell membrane of fungi and micro-organisms. This inhibition prevents the
the cell membrane of fungi and micro-organisms. This inhibition prevents the formation of pathogens
formation of pathogens on the cell walls and slows the growth of germ tubes [20]. There have been
on the cell walls and slows the growth of germ tubes [20]. There have been other studies about
other studies about interesting formulations for pesticides [2,22–25].
interesting formulations for pesticides [2,22–25].
The objective of this work was to develop TBZ NEs by constructing a pseudo-ternary phase
The objective of this work was to develop TBZ NEs by constructing a pseudo-ternary phase
diagram and identifying TBZ NE concentration regions. This study proposes a low-energy TBZ NE
diagram and identifying TBZ NE concentration regions. This study proposes a low-energy TBZ
method that may help decrease the required dose of the fungicide and thus the residual active
NE method that may help decrease the required dose of the fungicide and thus the residual active
ingredient. The solvents used were less toxic than those currently used, which may help minimize
ingredient. The solvents used were less toxic than those currently used, which may help minimize the
the environmental impact.
environmental impact.
2. Results and Discussion

2.1. Pseudo-Ternary Phase Diagrams

An initial requirement for the PIC method to produce NEs is to obtain a bicontinuous ME whose
dilution will form an NE [26]. To find the appropriate compositions on the ternary system
Molecules 2016, 21, 1271 3 of 12

2. Results and Discussion

2.1. Pseudo-Ternary Phase Diagrams

An initial requirement for the PIC method to produce NEs is to obtain a bicontinuous ME
whose dilution will form an NE [26]. To find the appropriate compositions on the ternary system
(OP//SURFACTANT//AP)
Molecules 2016, 21, 1271 for phase equilibrium to be achieved and therefore, for an 3 of ME
12 to
exist, it is necessary to generate a phase diagram. The phase diagrams from the systems studied
(OP//SURFACTANT//AP)
(OP//TW80//AP and OP//AG54//AP at T = 25 ◦toC)beare
for phase equilibrium achieved
shownand therefore,
in Figure 2a,b,forrespectively.
an ME to exist, it 2a
Figure
is necessary to generate a phase diagram. The phase diagrams
shows that over a wide range of compositions, two phases were detected: one solid and one from the systems studied
(OP//TW80//AP
homogeneous liquid.and TheOP//AG54//AP
solid phase was at Tsediment
= 25 °C) are
TBZ, shown in Figure
and the liquid 2a,b,
phase respectively.
was a liquid Figure 2a
equilibrium
shows that over a wide range of compositions, two phases were detected: one solid and one
phase. The sedimentation effect may be due to the higher solubility parameter for the acetone-aqueous
homogeneous liquid. The solid phase was sediment TBZ, and the liquid phase was a liquid
phase (AP) pair compared with the acetone-TBZ pair. The ME phase equilibrium area predominated,
equilibrium phase. The sedimentation effect may be due to the higher solubility parameter for the
with composition
acetone-aqueous ranges
phaseof(AP)
0.49–0.9
pair from the organic
compared with thephase (OP), 0.01–0.23
acetone-TBZ pair. Thefrom the AP,
ME phase and 0.07–0.49
equilibrium
from area
TW80. predominated, with composition ranges of 0.49‒0.9 from the organic phase (OP), 0.01‒0.23 from
Figure ◦
the AP,2b andrepresents the system
0.07‒0.49 from TW80. corresponding to OP//AG54//AP at T = 25 C. The figure shows
the predominance
Figure 2b of two liquid
represents the phases (2P) in the W/O
system corresponding section. No TBZ
to OP//AG54//AP at Tsedimentation
= 25 °C. The figure wasshows
observed.
the predominance of two liquid phases (2P) in the W/O section. No TBZ sedimentation
As in the previous system, the phase diagram was related to the ratios of the components. In this was observed.
case, As
thein the previousforces
electrostatic system,
were theunable
phase diagram was equilibrium
to maintain related to thebetween
ratios of the components.
phases. All this In this the
caused
case, the electrostatic forces were unable to maintain equilibrium between
presence of areas in which the three liquid phases (3P) were denoted. The ME area corresponded phases. All this caused the to
presence of areas in which the three liquid phases (3P) were denoted. The ME area corresponded to
the compositions of the OP to AP: 0.49–0.90 and 0.01–0.23 and AG54: 0.07–0.49; the O/W section was
the compositions of the OP to AP: 0.49‒0.90 and 0.01‒0.23 and AG54: 0.07‒0.49; the O/W section was
selected. Although two phases occurred, the proportions were considered in order to compare the
selected. Although two phases occurred, the proportions were considered in order to compare the
methods of NE
methods of synthesis
NE synthesis by byvarious
varioussurfactants.
surfactants.

(a) (b)
Figure 2. Pseudo-ternary phase diagrams of the systems at T = 25 °C: (a) OP//TW80//AP and (b)
Figure 2. Pseudo-ternary phase diagrams of the systems at T = 25 ◦ C: (a) OP//TW80//AP and
OP//AG54//AP.
(b) OP//AG54//AP.
The difference between the ME areas in the systems studied was due mainly to the nature of the
The difference
surfactant. between
In both the the
systems, MEinterfacial
areas in the systems studied
equilibrium was due mainly
was a consequence of the to the nature of
component
proportions In
the surfactant. andboth
the intermolecular
systems, the forces. The TW80
interfacial is a nonionic
equilibrium wassurfactant that mainly
a consequence favors
of the Van
component
der Waals forces and hydrogen bonds. The AG54 surfactant is a mixture of
proportions and the intermolecular forces. The TW80 is a nonionic surfactant that mainly favors nonionic and anionic
amphiphilic
Van der components,
Waals forces whereinbonds.
and hydrogen hydrogen
Thebonds
AG54occur along is
surfactant with the interactions
a mixture of attraction
of nonionic and anionic
and repulsion. As a result of the existing ionic forces, the AG54 system did not present the
amphiphilic components, wherein hydrogen bonds occur along with the interactions of attraction and
phenomenon of TBZ sedimentation. However, the presence of electrostatic interactions required
repulsion. As a result of the existing ionic forces, the AG54 system did not present the phenomenon of
more energy input to maintain the balance between phases and avoid their separation as a result of
TBZ sedimentation.
the repulsion amongHowever, the presence
molecules. of electrostatic
The stability interactions
of the final products (NE)required more energy
was determined using input
the to
maintain the balance
polydispersity between
index (PDI) asphases and avoid
the parameter. Thistheir separation
parameter as a the
determines result of thesize
particles’ repulsion among
variability
molecules.
throughThe
thestability of the final
dimensionless mediaproducts
from the(NE) was determined
amplitude using the
of size distribution intopolydispersity
the Gaussian bell; index
it is(PDI)
as thea parameter. This parameter
function of solution determines
properties, the particles’ size
system thermodynamics, variability through
and preparation methods. the dimensionless
Higher values
media from the amplitude of size distribution into the Gaussian bell; it is a function ofcould
are indicative of the heterogeneity of the particles’ diameters, and if this increases over time, it solution
be a consequence
properties, of the aggregates’and
system thermodynamics, formation [27]. The
preparation PDI can Higher
methods. be obtained from
values aredynamic
indicativelight-of the
scattering measurements (DLS); however, when this parameter is very high the sample is very
heterogeneity of the particles’ diameters, and if this increases over time, it could be a consequence of
polydisperse and may not be suitable for DLS measurements. Moreover, the sample contains large
the aggregates’ formation [27]. The PDI can be obtained from dynamic light-scattering measurements
particles, aggregates, and dust, which is an indication of an unstable sample through time. The PDI
value selected was 0.7; if the PDI of the sample was larger than this value, the sample was discarded.
Molecules 2016, 21, 1271 4 of 12

(DLS); however, when this parameter is very high the sample is very polydisperse and may not be
suitable for DLS measurements. Moreover, the sample contains large particles, aggregates, and dust,
which is an indication of an unstable sample through time. The PDI value selected was 0.7; if the PDI
of the sample was larger than this value, the sample was discarded.

2.2. Microemulsions and Nanoemulsions

Molecules 2016, 21, 1271 4 of 12

Once the composition range of the ME area was identified, the samples were prepared as shown
2.2. Microemulsions and Nanoemulsions
in Table 1. To confirm the formation of the bicontinuous ME, the samples were characterized by
Once the composition range of the ME area was identified, the samples were prepared as shown
optical microscopy to identify whether the system was isotropic or anisotropic. Anisotropic samples
in Table 1. To confirm the formation of the bicontinuous ME, the samples were characterized by
are denoted by the birefringence of the sample (liquid crystal conformation) or by differences in the
optical microscopy to identify whether the system was isotropic or anisotropic. Anisotropic samples
refractive index of the system [28].
are denoted by the birefringence of the sample (liquid crystal conformation) or by differences in the
refractive index of the system [28].
Table 1. Composition of different nanoemulsions.
Table 1. Composition of different nanoemulsions.
OP//TW80//AP OP//AG54//AP RO/S a OP (wt %) S (wt %) AP (wt %)
OP//TW80//AP OP//AG54//AP RO/S a OP (wt %) S (wt %) AP (wt %)
T1 A1 1.0 5.0 5.0 90.0
T1 A1 1.0 5.0 5.0 90.0
T2 A2 1.0 49.0 49.0 2.0
T3 T2 A3A2 1.0
1.5 49.0
30.0 49.0
20.0 2.0 50.0
T4 T3 A4A3 1.5
1.5 30.0
58.0 20.0
38.7 50.0 3.3
T5 T4 A5A4 1.5
3.0 58.0
60.0 38.7
20.0 3.3 20.0
T6 T5 A6A5 3.0
3.0 60.0
74.0 20.0
24.7 20.0 1.3
T7 T6 A7A6 6.0
3.0 50.0
74.0 24.78.3 1.3 41.7
T8 T7 A8A7 6.0
6.0 60.0
50.0 10.0
8.3 41.730.0
T9 T8 A9A8 6.0
6.0 70.0
60.0 11.7
10.0 30.018.3
T10 T9 A10A9 10.0
6.0 80.0
70.0 11.78.0 18.312.0
T11 A11 10.0 90.0 9.0 1.0
T10 A10 10.0 80.0 8.0 12.0
a
T11 A11RO/S : organic-to-surfactant-ratio.
10.0 90.0 9.0 1.0
a RO/S: organic-to-surfactant-ratio.
Formulations from various samples were selected by the parameter of translucence, since
Formulations
this indicates from various
the apparent samples
formation ofwere selected
an ME. by the
Figure 3 parameter
shows images of translucence, since this
corresponding to the
indicates the apparent formation of an ME. Figure 3 shows images
OP//TW80//AP system at 5× magnification. The table shows that samples T3, T8, T10, and T11 corresponding to the
OP//TW80//AP
exhibited homogeneity system in at
the5×refraction
magnification. Thepolarized
of their table shows thatthat
light, samples
is, noT3,disparity
T8, T10, in
andtheT11
modes
exhibited homogeneity in the refraction of their polarized light, that is, no disparity in the modes by
by which they refracted light. In addition, samples T2 and T4 showed a shift in brightness, that is,
which they refracted light. In addition, samples T2 and T4 showed a shift in brightness, that is, a
a contrasting color within the same area. However, T4 also showed birefringence, which is characteristic
contrasting color within the same area. However, T4 also showed birefringence, which is characteristic
of theofformation of aofliquid
the formation crystal.
a liquid crystal.These
Theseresults indicatethat
results indicate thatsamples
samples T2 T2
andand T4 were
T4 were anisotropic
anisotropic
and that samples
and that T3,T3,
samples T8,T8,
T10,
T10,and
andT11
T11had
had one refractiveindex
one refractive index andand therefore
therefore an isotropic
an isotropic character.
character.
This suggests the the
This suggests formation
formation of of
thethe
ME.
ME.TheThesamples werestored
samples were storedforforone
one month
month to evaluate
to evaluate stability.
stability.

Figure
Figure 3. Optical
3. Optical microscopeimages,
microscope images, with
with crossed
crossed polarizers
polarizersatat5×5×
magnification, of the
magnification, of ME
the ME
corresponding to the OP//TW80//AP system at T = 25 °C. ◦
corresponding to the OP//TW80//AP system at T = 25 C.
After one month, samples T2, T3, T4, and T10 remained translucent, indicating good stability,
After onesamples
whereas month,T11samples
and T8T2, T3, T4,a and
showed T10
white remained
aspect, typicaltranslucent, indicating
of the formation good stability,
of macroscopic
whereas samples
emulsion. T11 4and
Figure T8 showed
shows a white aspect,
images obtained typicalsamples
from selected of the formation of macroscopic
from the OP//AG54//AP emulsion.
system
and illustrates that samples A2, A4, A5, A6, A9, A10, and A11 showed anisotropy. The presence of
the birefringence phenomenon denoted formation of liquid crystals. Furthermore, samples A3 and
A8 showed isotropy. Another selected sample of this system was observed to have translucent
Molecules 2016, 21, 1271 5 of 12

Figure 4 shows images obtained from selected samples from the OP//AG54//AP system and
illustrates that samples A2, A4, A5, A6, A9, A10, and A11 showed anisotropy. The presence of
the birefringence phenomenon denoted formation of liquid crystals. Furthermore, samples A3 and
Molecules 2016, 21, 1271 5 of 12
A8 showed isotropy. Another selected sample of this system was observed to have translucent
Molecules 2016, 21, 1271 5 of 12
formulations
formulationsafterafter
oneone
month
monthofofstorage,
storage,meaning thatititcould
meaning that couldbebe assumed
assumed to correspond
to correspond to anto
ME.an ME.
Sample A11 A11
Sample waswas
formulations the the
after exception,
one month as it
ofas
exception, showed
storage,
it macroscopic
meaning
showed emulsions
that it could
macroscopic (a(a
be assumed
emulsions white solution).to an ME.
tosolution).
white correspond
Sample A11 was the exception, as it showed macroscopic emulsions (a white solution).

Figure 4. Optical
Figure microscope
4. Optical microscopeimages,
images, with
with crossed polarizersatat5×5×
crossed polarizers magnification,
magnification, of ME
of the the ME
Figure 4.
corresponding Optical
to the
corresponding microscope
OP//AG54//AP
to the images, with
system
OP//AG54//AP system crossed
atat
T =T25
= 25 ◦
polarizers
°C. C. at 5× magnification, of the ME
corresponding to the OP//AG54//AP system at T = 25 °C.
To evaluate the behavior of the systems based on the percentage of the AP, their viscosity was
To evaluate thethe
To evaluate behavior of the
thesystems based on
onthethepercentage
percentage of the AP, their viscosity was
determined. Figure 5behavior
plots theof
curvessystems
for eachbased
sample in both systems.of the AP, their viscosity was
determined. Figure 5 plots the curves for each sample in both systems.
determined. Figure 5 plots the curves for each sample in both systems.

Figure 5. Viscosity of ME as a function of AP % in the OP//TW80//AP and OP//AG54//AP systems at

Figure 5. Viscosity of ME as a function of AP % in the OP//TW80//AP and OP//AG54//AP systems at
T =5.25Viscosity
°C.
Figure of ME as a function of AP % in the OP//TW80//AP and OP//AG54//AP systems
T = 25◦ °C.
at T = 25 C.
The figure illustrates that at concentrations ranging from 4 to 50 wt %, both studied systems
haveThe figure tendency.
a similar illustrates When
that at the
concentrations ranging
AP concentration from 4 tofrom
increased 50 wt %,30
4 to bothwt studied systems
%, the viscosity
The
have figure
a similar
decreased illustrates
tendency.that
approximately Whenat concentrations
theMoreover,
four-fold. ranging
AP concentration
when APfrom
theincreased 4from
to 50
concentration wt
4 to %,wt
30
increasedboth
%, studied
the
from to 50systems
30viscositywt
havedecreased
a similar
%, approximately
tendency.
the viscosity four-fold.
When
was augmented, theandMoreover,
can when
AP itconcentration the AP
be noted concentration
increased
that fromincreased
the system 4 to TW80
with 30 wtfrom %,30the
enhancedto 50viscosity
wt
the
%, the approximately
viscosity
decreased viscosity
more than wasthe
augmented,
AG54 system.
four-fold. andInitcontrast,
can be when
Moreover, noted thethat AP
concentrations the ranging
system from
with 1TW80
concentration 4 wtenhanced
toincreased
% in samples
fromthe 30 to
viscosity
with AG54 more than
surfactant the AG54 system.
increased the In contrast,
viscosity up concentrations
to five-fold. ranging
Thus, the
50 wt %, the viscosity was augmented, and it can be noted that the system with TW80 enhanced from
minimum 1 to 4 wt %
viscosity in samples
of 27.6 cP the
with
(TW80)AG54 and surfactant
24.3 cP increased
(AG54) the viscositytoup
corresponded 30towtfive-fold.
% of theThus,
AP theboth
in minimum
systems,viscosity
and a of 27.6 cP
maximum
viscosity more than the AG54 system. In contrast, concentrations ranging from 1 to 4 wt % in samples
(TW80)
viscosityand 24.3cP
of 147 cPand
(AG54) corresponded
123 cP was obtainedtowith30 wt % of
1 wt % theandAP 2 wtin%both systems,
of the and a and
AP of TW80 maximumAG54,
with AG54 surfactant increased the viscosity up to five-fold. Thus, the minimum viscosity of 27.6 cP
viscosity of 147
respectively. The cPminimum
and 123 cP was obtained
viscosity of both with
systems1 wtwas
% and 2 wt % of
for isotropic the AP of TW80
formulations and
(T8 and AG54,
A8), and
respectively. The minimum viscosity of both systems was for isotropic formulations (T8 and A8), and
Molecules 2016, 21, 1271 6 of 12

(TW80) and 24.3 cP (AG54) corresponded to 30 wt % of the AP in both systems, and a maximum
viscosity
Molecules of 147
2016, cP and 123 cP was obtained with 1 wt % and 2 wt % of the AP of TW80 and AG54,
21, 1271 6 of 12
respectively. The minimum viscosity of both systems was for isotropic formulations (T8 and A8),
this
and minimum
this minimum confirmed the existence
confirmed of a transition
the existence zonezone
of a transition (between
(between1 and 2 phases,
1 and as shown
2 phases, in the
as shown in
pseudo-ternary
the pseudo-ternary phase diagrams).
phase diagrams).
The
The results
results ofof the
the characterization
characterization ofof the
the bicontinuous
bicontinuous MEs MEs from
from the
the two
two studied
studied systems
systems were
were
used
used toto select
select the
the composition
composition of of the
the NEs
NEs developed.
developed. Figure
Figure 6a
6a shows
shows thethe drop
drop size
size as
as aa function
function ofof
the
the AP % for the system
system OP//TW80//AP.
OP//TW80//AP. When WhenthetheAP AP%%was
wasincreased
increasedfromfrom11toto50
50wtwt%, %,the
the drop
drop
size remained 9 ±±11nm;
size remained nm;the
thesystem
systemshowed
showedno nodependence
dependenceon onthe
theconcentration
concentrationofofthetheAP.
AP.However,
However,
the
the influence
influence of the organic-to-surfactant-ratio
organic-to-surfactant-ratio (R(RO/S
O/S) )parameter
parametercould
couldnotnotbeberuled
ruled out,
out, but
but Figure
Figure 6b
6b
shows
shows that
that the
the drop size did not vary significantly when the RO/S O/S ranged
rangedbetween
between11and and10.10.

(a) (b)
Figure
Figure 6.
6. Drop
Drop size for the
size for the OP//TW80//AP
OP//TW80//AP system
systematatTT= =2525°C
◦ Casasa afunction
functionofof(a)
(a)AP
AP%%and
and (b)
(b) R
RO/S.
O/S .

Drop size primarily was related to the structure of the generated micelles and therefore the type
Drop size primarily was related to the structure of the generated micelles and therefore the
of interactions between these components. The critical micelle concentration (CMC) of the TW80
type of interactions between these components. The critical micelle concentration (CMC) of the
system was obtained experimentally as 0.094 mmol/L, and the formulations developed in this study
TW80 system was obtained experimentally as 0.094 mmol/L, and the formulations developed in this
had greater surfactant concentrations than the CMC (minimum concentration = 0.3816 mmol/L).
study had greater surfactant concentrations than the CMC (minimum concentration = 0.3816 mmol/L).
Thus, the previously described results confirmed the spontaneous formation of micelles in the system,
Thus, the previously described results confirmed the spontaneous formation of micelles in the system,
ideally spherical, as these generally are achieved at higher concentrations than the CMC of the
ideally spherical, as these generally are achieved at higher concentrations than the CMC of the
surfactant. Furthermore, the values found for drop size were similar to those of the TW80 micelles
surfactant. Furthermore, the values found for drop size were similar to those of the TW80 micelles
without TBZ in the same conditions (10 nm) (results not shown).
without TBZ in the same conditions (10 nm) (results not shown).
Figure 7a shows the OP//AG54//AP system at T = 25 °C and their drop size as a function of the
Figure 7a shows the OP//AG54//AP system at T = 25 ◦ C and their drop size as a function of
water concentration in the ME. The strong influence of the AP was observed at percentages below 20
the water concentration in the ME. The strong influence of the AP was observed at percentages below
wt %. An increase in the AP from 1.3 to 3.3 wt % increased the drop size from 150 to 200 nm. However,
20 wt %. An increase in the AP from 1.3 to 3.3 wt % increased the drop size from 150 to 200 nm.
an increase of from 3.3 to 12 wt % more than doubled drop size (from 200 to 488 nm). In addition,
However, an increase of from 3.3 to 12 wt % more than doubled drop size (from 200 to 488 nm).
greater AP values (from 20 to 50 wt %) did not have a significant effect, with the drop size remaining
In addition, greater AP values (from 20 to 50 wt %) did not have a significant effect, with the drop
at approximately 250 nm. Furthermore, as Figure 7b shows, the same RO/S system values resulted in
size remaining at approximately 250 nm. Furthermore, as Figure 7b shows, the same RO/S system
different drop sizes. For example, when the RO/S was 1.5, drop sizes were 285 nm and 201 nm at 50 wt
values resulted in different drop sizes. For example, when the RO/S was 1.5, drop sizes were 285 nm
% and 3.33 wt % AP, respectively, and when the RO/S was 10, drop sizes were 236 nm and 488 nm at
and 201 nm at 50 wt % and 3.33 wt % AP, respectively, and when the RO/S was 10, drop sizes were
1 wt % and 12 wt % AP, respectively. This implies that increased RO/S and AP % in the system affect
236 nm and 488 nm at 1 wt % and 12 wt % AP, respectively. This implies that increased RO/S and
the overall growth of drop size. Moreover, when the RO/S value was larger, increased AP % affected
AP % in the system affect the overall growth of drop size. Moreover, when the RO/S value was larger,
drop size more, since it was related to the micellar aggregates formed in the NE and their interaction
increased AP % affected drop size more, since it was related to the micellar aggregates formed in the
in the system. Specific systems were developed with AG54 concentration values of more than 500
NE and their interaction in the system. Specific systems were developed with AG54 concentration
mg/L of surfactant, exceeding the CMC obtained in the laboratory for AG54 (165.9 mg/L). Therefore,
values of more than 500 mg/L of surfactant, exceeding the CMC obtained in the laboratory for AG54
spontaneous formation of micelles was considered to exist in the NE.
(165.9 mg/L). Therefore, spontaneous formation of micelles was considered to exist in the NE.
Molecules 2016, 21, 1271 7 of 12
Molecules 2016, 21, 1271 7 of 12

(a) (b)
Figure 7.
Figure 7. Drop
Dropsize
sizefor
forthe
theOP//AG54//AP
OP//AG54//APsystem at T =at25T°C
system as a◦ C
= 25 function of: (a) AP
as a function of: %(a)and
AP(b)
% Rand
O/S.

(b) RO/S .
The NE’s drop size is a function of the quantity and nature of the components, the type of
surfactant (nature and molecular weight) and the obtaining method; however, there are also
The NE’s drop size is a function of the quantity and nature of the components, the type
phenomena that promote destabilization [29–31].
of surfactant (nature and molecular weight) and the obtaining method; however, there are also
According to the displayed results, the system with TW80 presents drops sizes much lower than
phenomena that promote destabilization [29–31].
the AG54. This result can be attributed to the fact that TW80 is mainly a hydrophilic nonionic
According to the displayed results, the system with TW80 presents drops sizes much lower
surfactant (HLB = 15); therefore, the types of interactions that may occur with it involve mainly the
than the AG54. This result can be attributed to the fact that TW80 is mainly a hydrophilic nonionic
London forces and hydrogen bonds that occur between polar and nonpolar regions of the chemical
surfactant (HLB = 15); therefore, the types of interactions that may occur with it involve mainly the
groups present in the components, and mainly in the organic phase. In contrast, AG54 is a mixture
London forces and hydrogen bonds that occur between polar and nonpolar regions of the chemical
of nonionic and anionic surfactants, so, in addition to contributing to interactions similar to those in
groups present in the components, and mainly in the organic phase. In contrast, AG54 is a mixture
TW80, it contributes strongly to electrostatic forces. The results allow the inference that the presence
of nonionic and anionic surfactants, so, in addition to contributing to interactions similar to those in
of electrostatic charges affects the OP/AP interface, destabilizing the hydration layer around the
TW80, it contributes strongly to electrostatic forces. The results allow the inference that the presence
particles and increasing the strength of colloidal repulsion and coalescence. Additionally, this may
of electrostatic charges affects the OP/AP interface, destabilizing the hydration layer around the
favor the Ostwald ripening phenomenon, thus generating the increases in drop size [32]. Even more,
particles and increasing the strength of colloidal repulsion and coalescence. Additionally, this may
when the micellar structure of AG54 formed, repulsive forces among the micellar structures that had
favor the Ostwald ripening phenomenon, thus generating the increases in drop size [32]. Even more,
already formed.
when the micellar structure of AG54 formed, repulsive forces among the micellar structures that had
In addition to drop size, another important feature of the NEs was whether their surface tension
already formed.
decreased compared to the surface tension of the water, which would indicate good wettability of the
In addition to drop size, another important feature of the NEs was whether their surface tension
system in future potential applications. Figure 8 shows results obtained with the AG54 and TW80
decreased compared to the surface tension of the water, which would indicate good wettability of the
systems. The semi-continuous line represents the CMC values obtained for TW80 and AG54.
system in future potential applications. Figure 8 shows results obtained with the AG54 and TW80
The TBZ composition directly affected the drop size and surface tension of the NE, both of which
systems. The semi-continuous line represents the CMC values obtained for TW80 and AG54.
were related to the wettability and availability of the active ingredient.
The TBZ composition directly affected the drop size and surface tension of the NE, both of which
All samples from the TW80 system exhibited very similar surface-tension values from 42 to 45
were related to the wettability and availability of the active ingredient.
mN/m, regardless of the RO/S. In addition, these values were up to 50% lower than the surface tension
All samples from the TW80 system exhibited very similar surface-tension values from 42 to
of water under the conditions of analysis (γ = 72.8 mN/m) and very similar to the corresponding
45 mN/m, regardless of the RO/S . In addition, these values were up to 50% lower than the
surface-tension values when the solution had only surfactant in the CMC (γ = 47 mN/m). For example,
surface tension of water under the conditions of analysis (γ = 72.8 mN/m) and very similar
a minimum surface-tension value might be expected due to the formation of micelles in the sample.
to the corresponding surface-tension values when the solution had only surfactant in the CMC
This corroborates that, as shown by the drop size, spontaneous micelle formation and stability both
(γ = 47 mN/m). For example, a minimum surface-tension value might be expected due to the
existed under these experimental conditions.
formation of micelles in the sample. This corroborates that, as shown by the drop size, spontaneous
The surface tension of the AG54 surfactant system samples showed surface-tension values close
micelle formation and stability both existed under these experimental conditions.
to 31 mN/m, without any significant effect from increasing the surfactant concentration and therefore
The surface tension of the AG54 surfactant system samples showed surface-tension values close
decreasing the RO/S. This surface tension value (31 mN/m) was the value of the surface tension at the
to 31 mN/m, without any significant effect from increasing the surfactant concentration and therefore
concentration CMC (CMC = 165.9 mg/L), indicating that this system contained micelle formation in
decreasing the RO/S . This surface tension value (31 mN/m) was the value of the surface tension at the
the solution. Unlike the TW80 systems, the AG54 systems had lower surface tensions. Considering
concentration CMC (CMC = 165.9 mg/L), indicating that this system contained micelle formation in
surface tension as a manifestation of the cohesive force among a liquid’s molecules, it can be deduced
the solution. Unlike the TW80 systems, the AG54 systems had lower surface tensions. Considering
that AG54 formulations have better wettability.
surface tension as a manifestation of the cohesive force among a liquid’s molecules, it can be deduced
that AG54 formulations have better wettability.
Molecules 2016, 21, 1271 8 of 12
Molecules 2016, 21, 1271 8 of 12
Molecules 2016, 21, 1271 8 of 12

Figure 8.
Figure 8. Surface tension
Surface of the
tension of NE
theasNE
a function of AP %of
as a function forAP
the %
OP//TW80//AP and OP//AG54//AP
for the OP//TW80//AP and
Figure
systems 8.
at Surface
T = 25 tension
°C. of the NE ◦
as a function of AP % for the OP//TW80//AP and OP//AG54//AP
OP//AG54//AP systems at T = 25 C.
systems at T = 25 °C.
Finally, the experiments were monitored during the more than six hours required for the
Finally,
Finally,the experiments
thethe
experiments were monitored
wereNE.monitored during
during the more than six
six hours required for the
development of corresponding The purpose wasthe to more
evaluatethandrop hours
size required
over time infor the
those
development of the corresponding
developmentcharacterized
of the corresponding NE. The
NE.smallpurpose
The drop
purpose was
was to evaluate
to evaluate drop size over time in those
formulations by relatively sizes, storage stabilitydrop
(withsize over time
no phase in those
separation),
formulations
formulations characterized
characterized by
by relatively
relativelysmall
smalldrop
dropsizes,
sizes, storage
storage stability
stability (with
(with no
no phase
phase separation),
separation),
and non-recrystallization of TBZ. As Figure 9 shows, drop sizes over time for selected formulations
and
and non-recrystallization ofofTBZ. As Figure 9 shows, drop sizes over time for for
selected formulations of
of thenon-recrystallization
OP//TW80//AP system TBZ. at T =As25Figure
◦°C
9 shows,
corresponded drop sizes
to systems over
thattime
showedselected
anisotropyformulations
(T2) and
the OP//TW80//AP
of the crystal
OP//TW80//AP system at T = 25 C corresponded to systems that showed anisotropy (T2) and
liquid formationsystem
(T4). at T = 25 °C corresponded to systems that showed anisotropy (T2) and
liquid
liquidcrystal
crystalformation
formation(T4).
(T4).

Figure 9. Drop size as a function of time for selected NEs for the OP//TW80//AP system at T = 25 °C.
Figure9.9.Drop
Figure Dropsize
sizeasasa function
a function
ofof time
time forfor selected
selected NEs
NEs forfor
thethe OP//TW80//APsystem
OP//TW80//AP systematatTT==25
25◦°C.
C.
According to Figure 9, drop size measurements remained unchanged, confirming the stability
of NE According to Figure
over the period 9, drop
of time size measurements remained unchanged, confirming the stability
studied.
According to Figure 9, drop size measurements remained unchanged, confirming the stability of
of NE over
The the the
selected period
sample of time studied.
NE over period of timeofstudied.
the OP//AG54//AP system corresponded to the formulation with 2 wt %
of theThe
AP selected
and liquid sample of
crystal the OP//AG54//AP system corresponded to the formulation with 2 wt %
The selected sample of theformation
OP//AG54//APin its ME (Figure
system 10). A drop
corresponded sizeformulation
to the of about 225
withnm was
2 wt %
of the AP
maintained; and liquid
however, crystal formation
each attempt hadin in its
a its ME
deviation (Figure 10). A
in measurements drop size of about 225 nm was
of the AP and liquid crystal formation ME (Figure 10). A dropof approximately
size of about 225±nm 30 was
nm,
maintained; however,
which can behowever,
attributedeacheach
to the attempt had
nature had
of the a deviation
surfactant,in in measurements
since it generates ofof approximately
interactions, ± 30
attraction, nm,
maintained; attempt a deviation measurements approximately ± 30 and
nm,
which canamong
repulsion be attributed
the polarto the
partnature of the
and the surfactant,
loads, which since
may it
begenerates interactions,inattraction, and
which can be attributed to the nature of the surfactant, since itconstantly
generates present theattraction,
interactions, solution,
repulsion among the polar part and the loads, which may be constantly
provoking significant mobility among molecules. Although a similar average drop size was presentpresent in the solution,
provoking
over significant
a period mobility
of six hours, theamong
AG54 molecules.
showed a Although
considerablea similar average
instability in drop size wasthe
formulating present
NE,
over a period of six hours, the AG54 showed a considerable instability in formulating the NE,
Molecules 2016, 21, 1271 9 of 12

and repulsion among the polar part and the loads, which may be constantly present in the solution,
provoking significant
Molecules 2016, 21, 1271 mobility among molecules. Although a similar average drop size was present 9 of 12
over a period of six hours, the AG54 showed a considerable instability in formulating the NE, compared
with the systems
compared with the obtained
systems byobtained
TW80, whose molecules
by TW80, whosedid not involve
molecules did net
notcharges.
involve This study found
net charges. This
that the selected ME showed the formation of liquid crystals for both systems. The selected
study found that the selected ME showed the formation of liquid crystals for both systems. The ME appears
to favor TBZ
selected stability to
ME appears in favor
the system, preventing
TBZ stability its system,
in the sedimentation and thus
preventing improving the
its sedimentation kinetic
and thus
equilibrium
improving the of the NE.equilibrium of the NE.
kinetic

Figure 10.
Figure 10. Drop
Drop size
sizeas
asaafunction
functionofoftime
timefor
forselected
selected NEs
NEs forfor
thethe OP//AG54//AP system
OP//AG54//AP system at
atTT== 25
25 ◦°C.
C.

3. Materials
3. Materialsand
andMethods
Methods

3.1. Chemicals
TBZ fungicide (whose
(whose structure
structureisisshown
shownininFigure
Figure1),1),with
witha apurity
purityofof
9898wtwt
%%andandsolubility in
solubility
water
in of 0.1
water of g/L and in
0.1 g/L acetone
and of > 1000
in acetone g/L [33,34],
of >1000 was supplied
g/L [33,34], by Velsimex
was supplied (Mexico City,
by Velsimex Mexico).
(Mexico City,
Glycerol and the nonionic surfactant Tween 80 (Polyoxyethylenesorbitan monooleate,
Mexico). Glycerol and the nonionic surfactant Tween 80 (Polyoxyethylenesorbitan monooleate, MW: 1310
g/mol)1310
MW: [35] g/mol)
were purchased
[35] werefrom Sigma Aldrich.
purchased Acetone
from Sigma was purchased
Aldrich. Acetone from JT Baker Company,
was purchased from JT
and commercially
Baker Company, available Agnique BL1754—which
and commercially is a mixture
available Agnique of nonionicisand
BL1754—which anionic surfactants—
a mixture of nonionic
was anionic
and acquiredsurfactants—was
from BASF (a mixture of approximately
acquired from BASF (a30%–60%
mixture calcium dodecylbenzenesulfonate
of approximately 30%–60% calcium and
20%–40% nonylphenol, ethoxylates)
dodecylbenzenesulfonate and 20%–40% [36].nonylphenol, ethoxylates) [36].
experimentsused
All experiments useddeionized
deionizedwaterwater supplied
supplied by by Meyer
Meyer Chemical
Chemical Company.
Company. All products
All products were
were used as received.
used as received.
These two
These twosolvents
solvents have
have a very
a very low degree
low degree of toxicity
of toxicity in comparison
in comparison with the with the ones
ones used used
nowadays
nowadays in industry, such as toluene
in industry, such as toluene or xylene [37,38].or xylene [37,38].

3.2. Pseudo-Ternary
Pseudo-Ternary Phase
Phase Diagrams
Diagrams
The phase diagrams were determined for the pseudo-ternary system: the the OP,
OP, AP,
AP, and surfactant
phase (Tween
(Tween 80,
80, TW80
TW80 oror Agnique
Agnique BL1754,
BL1754, AG54)
AG54) and
and constructed
constructed through
through the
the observed
observed phase
behavior and predominant
predominant areas.
areas. The OP was prepared with 5.4 wt % of TBZ as an active active ingredient,
ingredient,
54.9 wt % of glycerol as a co-solvent,
glycerol a co-solvent, and 39.7 wt
wt % of acetone as a solvent; these components were
% of acetone as a solvent; these components
mixed and stirred at room temperature for 24 h before use to ensure homogeneity
homogeneity of the mixture.
mixture.
Initially,
Initially, experiments
experiments were carried
carried out weighing
weighing the OP and surfactant (analytical balance APX200;
Denver Instrument,
Instrument, Bohemia,
Bohemia, NY,
NY,USA)
USA) in
in falcon
falcon tubes
tubes with
with various
various Ro/s to map
Ro/s to map the
the entire
entire diagram.
diagram.
Then, AP was added
added and
and the
the three-component
three-component sample was mixed by stirring with a vortex vortex (G560
(G560
GVortex-2 Genie; Scientific Industries, Inc., Bohemia, NY, USA) at T = 25 °C in a water bath (TE-D10;
Techne Tempette, Staffordshire, UK) for 24 h. Each point of the diagram was done in triplicate. Areas
of ME predominance were recorded for further analysis. Criteria for assigning ME regions were
based on the observance of the cloud point (the incipient point of phase separation), that is, the visual
Molecules 2016, 21, 1271 10 of 12

GVortex-2 Genie; Scientific Industries, Inc., Bohemia, NY, USA) at T = 25 ◦ C in a water bath (TE-D10;
Techne Tempette, Staffordshire, UK) for 24 h. Each point of the diagram was done in triplicate. Areas of
ME predominance were recorded for further analysis. Criteria for assigning ME regions were based on
the observance of the cloud point (the incipient point of phase separation), that is, the visual transition
from a transparent, homogeneous mixture to an opaque or translucent one. To confirm the number of
phases, all samples were observed using optical microscopy.

3.3. Nanoemulsion Preparation

NE formulations were developed by the PIC method, as previously reported by Wang et al. [23].
Using the information from the pseudo-ternary diagrams, compositions of ME were mapped as shown
in Table 1. First, the OP and surfactant were mixed with magnetic stirring, 400 rpm, at T = 40 ◦ C for
30 min (stirring and hot plate, C-MAG HS4; IKA, Wilmington, NC, USA). Then the AP was added at
a flow rate of 3 mL/min, heating was turned off, and stirring continued for 40 min. In the final step,
samples were stored in closed containers at T = 25 ◦ C. This ME precursor was diluted with water at
a ratio of 1:100 and stirred for 1 min on a vortex mixer at room temperature.

3.4. Characterization of MEs and NEs

MEs were characterized by determining their viscosity with an Ostwald type viscometer (model
NDJ-8S, Changzhou, Jiangsu, China), and images were obtained using an optical microscope (Olympus
BX60, Tokyo, Japan) with crossed polarizers (Olympus U-AN360 and U-DICR, Tokyo, Japan) at
5× magnification [39,40]. Further characterization of NEs included drop size and surface tension (γ)
at T = 25 ◦ C. Measurements were performed in triplicate. The average drop size was determined using
dynamic light-scattering equipment (Zeta Sizer® , Malvern, UK). Measurements were performed using
the parameters of viscosity in a dispersion medium, water, at T = 25 ◦ C, and the refractive index of the
OP = 1.433.
Light-scattering measurements also helped verify that there was no precipitation of TBZ.
Surface-tension measurements were determined with a tensiometer (model 703, Sigma, Lichfield,
UK) using the corrected Nuoy Ring method.

4. Conclusions
This study achieved NE formulation of TBZ as an active ingredient with drop sizes of about 9 nm
for the OP//TW80//AP system, with oil-phase concentrations in the precursor ME of up to 90 wt %.
Stability values over time showed that with an RO/S of 1 and 1.5, the drop size stayed constant for
six hours.
The AG54 systems maintained storage stability with a drop size of between 148 nm and 488 nm,
which did not vary with the individual parameters studied. However, the RO/S and AP % together
influenced the system. The evolution of drop size over six hours showed instability. Selected MEs
formed liquid crystals, according to optical microscope images.
The phase diagrams showed that the balance between phases strongly depended on the nature of
the surfactant. The zone O/W was selected to study ME development.
Factors such as the nature of the surfactant’s molecules governed interactions during the formation
of micelles, which affected drop size. A larger drop size resulted in less cohesion of the anionic
surfactant’s molecules to the solution.

Acknowledgments: The authors thank partial financial support from CONACYT grant number 181678.
The authors are grateful to CINVESTAV and Tecnologico de Monterrey.
Author Contributions: V.D.-B., D.I.M., E.P.O., R.B.-Z., M.O.-R., and G.L.-B. wrote the paper, designed and carried
out experiments, performed characterization, and analyzed data and results. Additionally, the corresponding
authors led the work.
Conflicts of Interest: The authors declare no conflict of interest.
Molecules 2016, 21, 1271 11 of 12

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Sample Availability: Samples of the compounds one (each NE) are available from the authors.

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