e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 4 ( 2 0 0 8 ) 257–262

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Stability of fenbendazole suspensions for veterinary use

Correlation between zeta potential and sedimentation

José L. Arias ∗ , Margarita López-Viota, Beatriz Clares, Ma Adolfina Ruiz

Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Granada, Spain

a r t i c l e i n f o a b s t r a c t

Article history: In this paper we have carried out a detailed investigation of the stability and redispersibil-
Received 14 March 2008 ity characteristics of fenbendazole aqueous suspensions, through a thermodynamic and
Accepted 24 April 2008 electrokinetic characterization, considering the effect of both pH and ionic strength. The
Published on line 3 May 2008 hydrophobic character of the drug, and the surface charge and electrical double-layer
thickness play an essential role in the stability of the system, hence the need for a full char-
Keywords: acterization of fenbendazole. It was found that the drug suspensions displays “delayed”
Fenbendazole or “hindered” sedimentation, determined by their hydrophobic character and their low
Influence of pH and electrolytes zeta potential (indicating a small electrokinetic charge on the particles). The electrostatic
Sedimentation repulsion between the particles is responsible for the low sedimentation volume and poor
Stability redispersibility of the drug. However, only low concentrations of AlCl3 induced a significant
Suspension effect on both the zeta potential and stability of the drug, leading to a “free-layered” sedi-
Zeta potential mentation and a very easy redispersion which could be of great interest in the design of an
Veterinary dosage forms oral pharmaceutical dosage form for veterinary.
© 2008 Elsevier B.V. All rights reserved.

1. Introduction Despite it is a widely used compound, little is known

The treatment of helmintic diseases consists in a pub-
lic health subject of great importance. Fenbendazole (FBZ)
[5-(phenylthio)-1H-benzimidazol-2-yl]carbamic acid methyl
ester; methyl-5-(Phenylthio) benzimidazole-2-carbamate; see
Fig. 1] is a broad spectrum benzimidazole antihelmintic indi-
cated worldwide in the treatment and prevention of several
endoparasitic diseases in veterinary (Iosifidou et al., 1997;
Praslička et al., 1997; Garossino et al., 2005; Ghazaei, 2007;
Willesen et al., 2007). This drug is thought to bind to tubulin
and thereby preventing its polymerization to form micro-
tubules, but it also inhibits fumarat reductase and glucose
transport. As a result, parasites may die of starvation. FBZ also
has an antifungal effect (McKellar, 1997).
about its surface properties, and nothing has been published
concerning its colloidal behaviour, which may be important
to understand its adhesion and aggregation characteristics.
Moreover, it must be considered that the thermodynamic
properties and, the electrokinetic potential and the electrical
double-layer thickness will play an essential role in the sta-
bility of the system. Hence, the need for the investigation of
the surface thermodynamics and the electrical state of the
particles if a full characterization of the system is sought.
In this paper, our aim is to perform a simultaneous analy-
sis of the hydrophobicity/hydrophilicity and the electrokinetic
properties, and the stability of FBZ. The effects of both pH (an
essential variable, given the extreme pH values of the gas-
trointestinal tract) and ionic content of the suspension will be

∗
Corresponding author at: Departamento de Farmacia y Tecnologı́a Farmacéutica, Facultad de Farmacia, Universidad de Granada, 18071
Granada, Spain. Tel.: +34 958 24 39 00; fax: +34 958 24 89 58.
E-mail address: jlarias@ugr.es (J.L. Arias).
0928-0987/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejps.2008.04.008
258 e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 4 ( 2 0 0 8 ) 257–262

These quantities can be related to the contact angle 

between the liquid and the solid, using the Young’s equation
(Adamson and Gast, 1997):
  
(1 + cos )LTOT = 2 SLW LLW + 2 S+ L− + 2 S− L+ (3)
Fig. 1 – Chemical structure of fenbendazole.

The three unknowns (SLW , S+ and S− ) can be obtained by

solving the resulting system of three equations if the contact
angles of three liquids of known LLW , L+ and L− , are measured.
investigated, in order to clarify the best formulation aspects
In our case, we used water (LLW = 21.8, L+ = L− = 25.5 mJ/m2 ),
of an oral dosage form that will facilitate the administration
formamide (LLW = 3.90, L+ = 2.28, L− = 39.6 mJ/m2 ) and
of exact dosages to animals.
diiodomethane (LLW = 5.08, L+ = L+ = 0 mJ/m2 ), all data
taken from van Oss (2006). The contact angles of the three
2. Materials and methods liquids were determined at 25.0 ± 0.5 ◦ C, using a Ramé-Hart
100–00 goniometer (USA), on pellets (radius: 1.3 cm) obtained
2.1. Materials by compressing the dry powders in a Spepac hydraulic press
(UK) set to 8 ton during 5 min.
The drug powder was obtained from Roig Farma-Fagron Finally, the surface electrical properties of FBZ suspensions
(Spain) and used as received. All other chemicals were of (≈0.1% (w/v)), were analyzed by electrophoresis measure-
analytical quality from Panreac (Spain) except for KOH and ments as a function of both pH (adjusted with either HCl
diiodomethane (Merck, Alemania) and formamide (Carlo Erba, or NaOH) and electrolyte concentration (NaCl, CaCl2 ·4H2 O
Italy). Water used in the experiments was deionized and fil- or AlCl3 ·6H2 O), using a Zetasizer 2000 (Malvern Instruments,
tered (Milli-Q Academic, Millipore, France). UK) electrophoresis device. Measurements were performed at
25.0 ± 0.5 ◦ C, after 24 h of contact at this temperature under
2.2. Methods mechanical stirring (50 rpm). The experimental uncertainty of
the measurements was below 5%. The theory of O’Brien and
2.2.1. Characterization methods White (1978) was used to convert the electrophoretic mobility
Size and shape of FBZ particles were deduced from SEM (ue ) into zeta potential () values.
pictures using a Zeiss DSM 950 (Germany) scanning elec-
tron microscope set at 80 kV accelerating voltage. Prior to 2.2.2. Stability of febendazole aqueous suspensions
observation, a dilute (≈0.1% (w/v)) drug suspension was Apart from the electrokinetic evaluation, another method was
sonicated for 5 min, and drops of the suspension were used to determine the stability of FBZ suspensions, consisting
placed on copper grids with formvar film. The grids were in the measurement of the sediment volume, Vs , after keeping
then dried at 40.0 ± 0.5 ◦ C in a convection oven. In order the suspensions in 100 mL cylinders (inner diameter: 2.4 cm)
to confirm these results, the mean particle diameters were placed in a thermostatted bath at 25.0 ± 0.5 ◦ C. The concen-
determined at 25.0 ± 0.5 ◦ C by quasi-electric light scattering tration of solids in the cylinders was 5% (w/v) in all cases. The
(QELS) using a Nanosizer (Coulter® N4MD, Coulter Electron- flocculation ratio, F, was the quantity chosen for the character-
ics, Inc., Hialeah, FL, USA). The selected angle was 90◦ and the ization of the stability. This is defined as 100 (Vs /V0 ), where V0
measurement was made after dilution of the aqueous suspen- is the initial volume of the suspension (Matthews and Rhodes,
sions. 1970). The redispersibility was ascertained by visual inspec-
A surface thermodynamic analysis of this antihelmintic tion of the suspensions after placing them for 2 min in an
drug was done by using the model developed by van Oss Branson 5200E4 ultrasonic bath (USA) operating at 40 kHz and
(2006) (see also Arias et al., 2007), according to which the with a sonic power of 100 W (Gallardo et al., 2005).
total surface free energy of any material i is the sum of two
contributions:
3. Results and discussion

iTOT = iLW + iAB = iLW +2 i+ i− (1)
3.1. Particle size and morphology
one of which, iLW ,
is the non-polar Lifshitz–van der Waals
FBZ particles were of irregular shape, in the colloidal size
component, and the second one, iAB or acid–base component,
range and moderately polydisperse. The average diameter
is related to the electron-donor (i− ) and electron-acceptor
(±S.D.) and polydispersity index were 235 ± 60 nm and 0.277,
(i+ ) characteristics of the material. Similarly, the interfacial
TOT respectively. Fig. 2 includes an example of scanning electron
solid/liquid free energy, SL , and its LW and AB compo-
LW and  AB , respectively) are related to the surface microscope (SEM) pictures of the samples prepared and the
nents (SL SL
size histogram (based on ≈200 particles counting).
free energies of both the solid (subscripts S) and the liquid
(subscripts L):
3.2. Surface thermodynamics
 
TOT
SL = LW
SL AB
+ SL = LW
SL +2 S+ S− +2 S− S+
  The contact angles of water, diiodomethane and formamide
−2 S+ L− − 2 S− L+ (2) measured on the FBZ pellets were 66 ± 1◦ , 29 ± 1◦ and
 e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 4 ( 2 0 0 8 ) 257–262
Fig. 2 – Scanning electron microscope picture of
fenbendazole (inset: size histogram of the particles). Bar
length: 250 nm.
47 ± 1◦ , respectively. The evaluation of the  S components
(LWS,  + S,  − S) constitute a set of physical quantities
that was used to analyze the nature of this drug, due to the
true physical information about the surface thermodynam-
ics given. The Lifshitz–van der Waals component (LWS) is
44.6 ± 0.4 mJ/m2 , the electron-acceptor component ( + S) is
0.05 ± 0.01 mJ/m2 , and the electron-donor component (S− ) is
13.9 ± 0.6 mJ/m2 . FBZ is thus essentially a monopolar electron-
donor material, in the sense given by van Oss: this material can
have acid–base interactions with phases of whatever polarity
( + ,  − , or both, different from zero) but the acid–base forces
do not contribute to its cohesion free energy (Arias et al., 2001).
These surface free energy components manifest them-
selves in the hydrophobicity/hydrophilicity characteristics of
the material. The evaluation of the free energy of interaction
GSLS (not considering the electrostatic component) between
the solid phases immersed in the liquid, can be used to
check whether a material can be considered hydrophobic or
hydrophilic (van Oss, 2006). This can be written, per unit area
259
Fig. 3 – Sedimentation ratio (sediment volume/suspension
volume, %) as a function of time for fenbendazole
suspensions at pH 3 (), 5 (䊉), 7 () and 9 ().
of interacting particles, as follows:
TOT
GSLS = −2SL (4)
This quantity was found to be negative for FBZ (GTOT SLS =
−33.6 ± 1.9 mJ/m2 ) and, therefore, the interfacial interactions
favour attraction of the particles to each other, and they are
considered hydrophobic.
3.3. Effect of pH on the stability
Due to the significant effect that pH typically has on the sur-
face charge of many solids of pharmaceutical interest (drugs,
polymers, etc.), we will first consider how the colloidal sta-
bility of FBZ depends on H+ concentration. Fig. 3 shows the
type of time evolution curves found. All suspensions devel-
oped a “delayed” or “hindered” sedimentation (Delgado et al.,
1990; Gallardo et al., 2005), characterized by a drop of the sed-
iment front from the upper level of the suspension, giving
rise to a marked boundary between the front and the clari-
fied supernatant. The short-time sedimented volume is high
(the aggregates extend to roughly the whole volume of the
suspension) and it decreases with time: only after ≈40 days
the particles settled giving about 18% sedimented volume. The
sedimentation rate was slightly affected by the pH: there is a
non-significant tendency to higher rates as the H+ concentra-
tion rises. In addition, redispersion was almost complete after
5 min of ultrasonic shaking.
In order to check to what extent can these results be quali-
tatively explained in the frame of the DLVO classical theory of
the stability of lyophobic colloids (Hunter, 2001), we have stud-
ied the electrophoretic mobility, ue , and the zeta potential, ,
of the particles as a function of pH in the same conditions as
those corresponding to the stability determinations. Note that
both ue and  are negative for the whole pH interval investi-
gated, and that it rises in absolute value as the pH increased
(Fig. 4). This behaviour can be explained on the basis of the
charge generation mechanism at the drug/solution interface:
we can expect that the chemical species responsible for the
260 e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 4 ( 2 0 0 8 ) 257–262
Fig. 4 – Electrophoretic mobility (a) and zeta potencial (b) of
fenbendazole particles as a function of pH in the presence
of 10−3 M NaCl.
generation of the negative surface charge on FBZ are weak
acid groups, presumably carboxylic. The increasingly negative
values and charge density may be explainable by the effect of
increasing OH− ion concentration in the solution, which tends
to favour a gain in protons. In contrast, a decrease in absolute
mobility as pH becomes more acidic may be explainable by
neutralization of the negative regions as a result of chemical
adsorption of increasing numbers of H+ ions (Vera et al., 1996;
Ruiz et al., 2004a; Gallardo et al., 2005; Arias et al., 2007).
These results can also qualitatively explain the stability
and redispersibility data above described. At acid pH, the zeta
potential of the particles will be low, and formation of volumi-
nous aggregates can occur rapidly because of van der Waals
attraction, hence the large volume of aggregates in Fig. 3.
These open flocculi will settle slowly, and a long time will thus
be needed for producing relatively compact sediments. Fur-
thermore, due to the open structure of the aggregates, they
will enclose comparatively large volumes of the supporting
solution. Hence, the average particle–particle distances will
be rather large, and because of the weak van der Waals attrac-
tions, the particles can get separated even after a mild shaking,
so the suspensions will be easily redispersible. This behaviour
is also observed for basic pH values, despite the high elec-
trokinetic potential of the particles. Therefore, this slight trend
towards faster sedimentation rates for acid pH values can be
Fig. 5 – Sedimentation ratio F (sediment
volume/suspension volume, %) as a function of time for
fenbendazole suspensions without electrolyte () and at
different NaCl (a) or CaCl2 (b) molar concentrations: 10−2
(䊉), 5 × 10−4 () and 10−5 ().
partially explained if we take into account the hydrophobic
character of this drug (see Section 3.2.), which determines that
the interfacial interactions favours the existence of attractions
between the particles that induce the formation of volumi-
nous aggregates, even at basic pHs (high  values), where
electrostatic repulsions are expected.
3.4. Effects of electrolyte type and concentration
Fig. 5 shows the sedimentation rate curves of the drug sus-
pensions at pH 8.2 (the natural pH of FBZ suspensions) when
two common electrolytes, NaCl and CaCl2 , were added in con-
centrations ranging from 10−5 to 10−2 M. As observed in the
study of the effect of the pH (Fig. 3), all suspensions developed
a “delayed” or “hindered” sedimentation. The increase in the
electrolyte concentration induced a slight increment in the
sedimentation rate. Moreover, the comparison between both
electrolytes showed kinetics of aggregation slightly slower
in NaCl compared to CaCl2 . However, as previously pointed
out, the absence of significant differences between both elec-
trolytes and their concentrations can be partially explained by
considering the hydrophobic character of FBZ which induces
 e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 4 ( 2 0 0 8 ) 257–262
Fig. 6 – Sedimentation ratio F (sediment
volume/suspension volume, %) as a function of time for
fenbendazole suspensions without electrolyte () and at
different AlCl3 molar concentrations: 10−2 (䊉), 5 × 10−4 ()
and 10−5 ().
the formation of aggregates despite the tendency of the drug
to settle as individual particles, even at low electrolyte con-
centrations (high  values, as discussed below). Furthermore,
whatever both the electrolyte and its concentration, the sed-
imentation rate was slightly faster than the sedimentation
developed by the FBZ suspensions prepared without ionic
strength (high  values at pH 8.2, see Fig. 4). Redispersion was
easily achieved when the drug suspensions contained elec-
trolyte (≈5 min), without significant differences between NaCl
and CaCl2 and their concentrations.
With respect to the effect of AlCl3 on the sedimentation of
the drug suspensions, as can be seen in Fig. 6, only at high elec-
trolyte concentrations the suspensions developed a “delayed”
or “hindered”. Low ionic strengths in the suspension induced
a “free-layered” sedimentation described by many authors
(Gallardo et al., 2005), where the sediment height increases
over time (as predicted by Stokes’ law) up to a maximum value
after an interval long enough for all the particles to sediment
out (except perhaps the smallest fraction, prevented from set-
tling by a Brownian effect).
We now investigate to what extent these aggregation deter-
minations are explained by electric double-layer properties.
Fig. 7 a shows that the mobility was negative throughout the
range of concentrations tested for NaCl and CaCl2 , and that the
absolute value tended to decrease as concentration of either
electrolyte increased. This trend was more evident for CaCl2
than for NaCl due to a stronger specific adsorption in the Stern
layer (Ruiz et al., 2004a; Gallardo et al., 2005). Decreased mobil-
ity was manifested as a decrease in the zeta potential that
resulted from the effect of double-layer compression (Fig. 7b).
The counterions accumulate closer to the particle surface,
such that the double-layer shrinks as concentration increases.
This leads to a lower electrical potential in the shear plane (or
slip surface) that limits the value of the zeta potential and,
therefore, favours the formation of aggregates that settle down
slightly slower (tendency also observed in NaCl solutions as
compared to CaCl2 ).
261
Fig. 7 – Electrophoretic mobility (a) and zeta potencial (b) of
fenbendazole particles as a function of the concentration of
NaCl (), CaCl2 (䊉) and AlCl3 () at pH 8.2.
Given the important effect of highly charged counterions
on the magnitude (and sign) of the surface charge of parti-
cles, we also performed electrokinetic determinations of FBZ
when the medium contained AlCl3 . Fig. 7a shows that mobil-
ity was positive within the range of concentrations 5 × 10−5 to
10−4 M. These results can be explained by adsorption of Al3+
ions on the surface of the drug particles with a consequent
increase in cationic charge and decrease in electrophoretic
mobility. In accordance with the model of adsorption we pro-
pose, this suggests that trivalent cations are more effective
than divalent cations, which in turn are more effective than
monovalent cations, in reducing ue and . This behaviour is
a logical reflection of the Schulze–Hardy rule, according to
which higher counterion valence is associated with greater
decrease in electrophoretic mobility or other electrokinetic
effect. The reasons for this response may lie in the interaction
between Cl− co-ions and the FBZ surface. The hypothetical
specific or chemical adsorption of Cl− ions on the particle
surface may account for the essential aspects of this phe-
nomenon. At low NaCl or CaCl2 concentrations, counterions
are unable to coat the entire surface, and Cl− ions might there-
fore be able to reach the particle surface. This in turn may
lead to a further increase in net negative charge and hence
mobility. When concentration increases, the greater number
262 e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 4 ( 2 0 0 8 ) 257–262
of cations may become more effective in impeding contact
between Cl− ions and the particles; this effect can thus pre-
vent co-ion adsorption (or at least lower the adsorption rate).
This effect is compensated for by double-layer compression
caused by the increasing counterion concentration. We may
assume that Ca2+ cations, thanks to their greater charge, are
more effective in this coating effect than Na+ cations. This
would explain why the low mobility detected in the presence
of Na+ cations (indicating a steady increase in the number
of Cl− ions adsorbed) is not found in CaCl2 solutions. Triva-
lent cation salts have a considerable effect on ue and . Fig. 7
illustrates the increase in ue and  with increasing concen-
tration of AlCl3 with a shift from negative to positive values.
The fact that the Al3+ ion is able to bring about this change
is a clear indication that AlCl3 interacts strongly with the
surface (Ruiz et al., 2004a). Moreover, the very low  values
generated at the weak ionic strengths of AlCl3 can explain the
“free-layered” sedimentation described above, because of the
generation of voluminous macroaggregates (favoured by the
hydrophobic character of the drug particles) and that only at
high electrolyte concentrations (probably due to high  val-
ues), the suspensions developed a “delayed” or “hindered”.
Redispersion of these voluminous macroaggregates was eas-
ily achieved (<1 min) in comparison to higher concentrations,
other electrolytes (NaCl and CaCl2 ) and acid or basic pHs,
because of the high volume of the solution included inside
(Ruiz et al., 2004b).
4. Conclusions
The stability of fenbendazole suspensions is mainly controlled
by its hydrophobic character and, the zeta potential of the par-
ticles and the thickness of their ionic double-layers. Despite
the former parameter strongly determine the sedimentation
curves, the modification of pH and the addition of varying con-
centrations of simple electrolytes also affects the stability of
the suspensions. The use of low concentrations of AlCl3 allows
obtaining fenbendazole suspensions with adequate proper-
ties (suitable stability and redispersability) for the oral route
in veterinary.
Acknowledgements
Financial support from Projects MAT2005-07746-C02-02 (MEC)
and FQM-410 (Junta de Andalucı́a) is acknowledged.
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