Journal of Experimental Botany, Vol. 57, No. 11, pp.

2501–2513, 2006
doi:10.1093/jxb/erl023 Advance Access publication 7 July, 2006

FOCUS PAPER

Characterization of the diffusion of non-electrolytes across

plant cuticles: properties of the lipophilic pathway

Anke Buchholz*
Syngenta Crop Protection AG, Research Biology, CH-4332 Stein, Switzerland

Received 25 November 2005; Accepted 10 April 2006

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Abstract
Systemic crop protection products are commonly
sprayed onto foliage, whereupon the active substances
must penetrate into the leaves in order to become
biologically active. Penetration of the plant cuticle is
the rate-limiting step. The diffusion of organic non-
electrolytes within cuticles is a purely physical pro-
cess that can be described and analysed in the same
way as is done for diffusion in synthetic polymer
membranes. Solute mobilities in cuticles vary consid-
erably between plant species. For a given species
they decrease with increasing solute size, and this
size selectivity holds for all of the plant species in-
vestigated so far. Wax extraction from leaf cuticles
increases the mobility of solutes tremendously, but
size selectivity is not affected. Furthermore, diffusion
within plant cuticles is extremely temperature depen-
dent. An analogous increase in solute mobility can
be achieved by using accelerators, which enhance
the fluidity of the polymer matrix and of the waxes.
The effects of temperature and plasticizers on the dif-
fusion of non-electrolytes in wax and the cutin matrix
have been used to characterize the nature of the lipo-
philic pathway. The ‘free volume’ theory can be used
to explain the influence of the size and shape of
the solute, and its dependence on temperature. The
physico-chemical nature of the diffusion pathway has
been shown, by thermodynamic analysis, to be ident-
ical for a wide range of solute lipophilicities. This ap-
proach also explains the mode of action and the
intrinsic activity of plasticizers.
Key words: Cuticular membrane, diffusion, foliar uptake, leaf
surface, penetration, solute mobility.
Introduction
Foliar uptake is a complex process determined by several
mutually affecting parameters. General characteristics—
required for the optimization of foliar uptake—can only be
obtained when interdependencies have been excluded. Ex-
tensive work, focusing on the diffusion of non-electrolytes
across plant cuticles, has been done, which this paper
attempts to review. The relevant characterization of cu-
ticle penetration for polar compounds is given separately
by Schönherr (2006). After a general introduction on
the function and character of plant cuticles, the applied
methodology is presented. Parameters affecting plant
cuticle penetration are displayed individually so that finally
the complex scenario of foliar uptake can be outlined in
a simplified way.
Function of plant cuticles
All aerial surfaces of the primary parts of terrestrial
higher plants are covered with a cuticle, the function of
which is to prevent uncontrolled water loss (Riederer
and Schreiber, 2001). The plant cuticle is not only exposed
to abiotic factors like sunlight, wind, rain, etc. but it
also interacts with microbes, fungi, and insects (Juniper,
1995; Kolattukudy et al., 1995; Kerstiens, 1996; Schreiber
et al., 2004). Furthermore, the plant cuticle is the initial
contact point between an agrochemical and the plant and,
where uptake into plant tissues is required for biological
activity, it is the main barrier to penetration.
Morphology, chemistry, and structure of
plant cuticles
Cuticles are not simple homogeneous structures like syn-
thetic polymer films. Various structural types occur in

* E-mail: anke.buchholz@syngenta.com
Abbreviations: CM, cuticular membrane; k*, solute mobility; MX, matrix membrane; UDOS, unilateral desorption of the outer surface; Vx, molar volume.

ª The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
For Permissions, please e-mail: journals.permissions@oxfordjournals.org
2502 Buchholz
gymnosperms and angiosperms, which were categorized
by Holloway (1982) into six morphological types. Inves-
tigators of the cuticular ultrastructure of different plant
species have used different terms to describe these layered
structures. Wattendorf and Holloway (1980) provided a
comparative overview of the different terminologies used.
The following generalization as given by Jeffree (1986)
(Fig. 1) distinguishes three main zones. The cuticle proper
with epicuticular and intracuticular waxes often has a
lamellate layer and typically extends to a thickness of
50–150 nm (Jeffree, 1996). The cuticular layer is bonded
to the periclinal walls of the epidermal cells by a pectin-
rich layer. This pectinaceous layer is equivalent to the
middle lamella, with which it is continuous. Enzymatic
hydrolysis of this layer enables the cuticular membranes
(Orgell, 1955), which comprise the cuticle proper and the
cuticular layer, to be isolated.
The epicuticular wax film is a distinct layer on the
surface of the cutin matrix (Jetter et al., 2000). Depending
on their chemical composition, epicuticular waxes may
form an amorphous film, granules, or crystalline structures
of various shapes (Baker, 1982; Jeffree, 1986). This fine-
surface structure might be modified with increasing age
(Bukovac et al., 1979), by environmental stress (Hoad
et al., 1992), and also by agrochemical spray solutions
(Whitehouse et al., 1982; Tamura et al., 2001). Epicuticu-
lar waxes have a considerable influence on the wettability
of plant surfaces, often causing poor retention or spread-
ing of spray droplets (Bukovac et al., 1979; Turunen and
Huttunen, 1990; Holloway, 1994), but they rarely affect
a compound’s rate of penetration into leaves (Baur, 1998).
The penetration barrier (Schönherr and Schmidt, 1979)
is made up of the cuticular membrane, which consists of
the cutin polymer matrix (Holloway, 1993) and associated
Fig. 1. Generalized structure of a plant cuticle (modified according to
Jeffree, 1986). EW, Epicuticular wax; CP, cuticle proper with lamellate
structure; CL, cuticular layer traversed by cellulose microfibrils; PL,
pectinaceous layer and middle lamella; CW, cell wall; P, plasmalemma.
waxes (Bianchi, 1995). However, the permeability of this
barrier is independent of the overall thickness of the cuti-
cle (Becker et al., 1986; Knoche et al., 2000). The intra-
cuticular waxes are predominantly located in the outer
layers of the cuticle where they form the transport-limiting
layer or skin (Schönherr and Riederer, 1988). This wax
layer acts as a barrier to both diffusion and solubility
(Shafer and Schönherr, 1985), and it has been established
that the solid and crystalline wax aggregates within this
layer determine the transport properties of the plant cuticle
(Riederer and Schreiber, 1995).
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Penetration across plant cuticles
The penetration of non-electrolytes across plant cuticles
requires three steps: sorption into the cuticular lipids, dif-
fusion across the cuticular membrane, and finally desorp-
tion into the apoplast of the epidermal cells (Kirkwood,
1999; Schönherr et al., 1999). Rates of penetration de-
pend upon solute mobility in the cuticle and on the driving
force (product of partition coefficient and concentration
gradient; see also below). Solute mobility can be meas-
ured using unilateral desorption from the outer surface
(UDOS). This method takes advantage of the fact that
cuticles are asymmetric membranes (see above) and distin-
guishes two functional layers: the limiting skin and the
sorption compartment (Fig. 2). In the cuticles studied, the
thin limiting skin comprised only about 10% of the total
Fig. 2. Functional layers (not to scale) of cuticular membranes as
differentiated in the UDOS (unilateral desorption of the outer surface) and
SOFU (simulation of foliar uptake) methods: limiting skin (LS) and
sorption compartment (SC). Occurrence and dimension of cuticular pegs,
and protrusion of the cuticular layer into anticlinal cell walls of epidermal
cells, are species dependent. The arrows denote the direction of diffusion
according to the concentration gradient. For one time, the sorption
compartment (UDOS) and, for the other time, the formulation residue
facing the limiting skin (SOFU) serve as donors. With UDOS rate
constants of desorption, k*, and with SOFU rate constants of penetration,
k, can be determined.

mass of the cuticular membrane (CM), and both sorption
capacity and solute mobility were very small. The sorption
compartment underneath the limiting skin amounted to
about 90% of the mass of the CM, its sorption capacity
was high, and solute mobility was much higher than in
the limiting skin (Schönherr and Baur, 1994). Model com-
pounds are applied to the morphological inner surface
of the CM and are subsequently sorbed in the sorption
compartment. When they are homogeneously distributed
within the sorption compartment they are unilaterally de-
sorbed from the morphological outer surface.
These measured solute mobilities are first-order rate
constants of desorption (k*), which are independent of
Lipophilic pathway 2503
where K is the partition coefficient and Dx is the membrane
thickness. The reader is referred to Bauer and Schönherr
(1992), Schönherr and Baur (1994), and Baur et al. (1996b)
for further details.
The benefit of this method is that the contributions
of mobility and solubility to the rates of foliar uptake
of pesticides can be determined separately, due to the fact
that the measured rate constants of desorption (k*) are
independent of the partition coefficient. When the donor
is applied to the outer surface of the CM and solutes are
desorbed from the morphological inner side (SOFU=
simulation of foliar uptake), rates of desorption also
follow first-order kinetics, but they depend on both solu-

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the partition coefficient and are directly proportional to
the diffusion coefficient in the waxy outer limiting skins
of cuticles.
D = k*3lls 3lsoco
where lls3lsoco is the diffusion path length in the limiting
skin, ls, and sorption compartment, soco. The translation
to diffusion coefficients D (m2 s
1) is only restricted
since the diffusion path length is not known precisely
(Schönherr and Baur, 1994). When only the permeability
P is determined, sorption processes are additionally in-
cluded and cannot be separated.
P = ðD3KÞ=Dx
bility and mobility (Baur et al., 1997b).
Plant species
ð1Þ
When solute mobility in cuticles is studied using the
UDOS method, astomatous CM must be used. Enzymatic
isolation does not affect the transport properties of cuti-
cles (Kirsch et al., 1997) but it is feasible only with a
limited number of species, i.e. those where a continuous
pectinaceous layer is present. These are most often peren-
nial plants. The range of solute mobilities (e.g. for bifenox)
measured for the CMs studied covered three orders of
magnitude (Fig. 3). These different mobilities are species-
ð2Þ specific characteristics and do not correlate with either

log k*
-4.5 -5 -5.5 -6 -6.5 -7 -7.5 -8 -8.5
Prunus persica
Tilia cordata
Populus canescens
Juglans regia
Pyrus pyrifolia
P. communis cv. Conference
Prunus armeniaca
Stephanotis floribunda
Malus baccata
Pyrus communis
Pyrus pyrifolia
M. domestica cv. Golden D.
M. domestica cv. Gloster
Citrus grandis
Prunus serotina
Ilex aquifolium
Prunus laurocerasus
Citrus aurantium
Strophanthus gratus
Ginkgo biloba
Melicoccus bijugatus
Hedera helix
Vanilla planifolia
Ilex paraguariensis
Decreasing solute mobility

Fig. 3. Solute mobilities, logk*, of the model compound bifenox (logKCW, 4.44; molar volume, 216 cm3 mol
1) measured in 24 plant species (CM) at
25 8C. Error bars denote 95% confidence intervals.
2504 Buchholz
cuticle thickness or the amount of wax embedded within,
and covering, the cuticular matrix. For example, Pyrus
communis, Malus domestica, Ilex aquifolium, and Ilex
paraguariensis exhibited a very similar amount of ex-
tractable wax (about 24%) but its contribution to the barrier
property of the CM varied by factors of 49 up to 4897
(Table 1). No clear relationship has been found to exist
between the particular composition of the soluble lipids
and the respective cuticular permeability (Haas and
Schönherr, 1979; Riederer and Schneider, 1990; Hauke
and Schreiber, 1998). Histochemical methods have also
been used to try to understand the relationship between
chemical composition and permeability (Leece, 1976;
homologous waxes (1-tetradecanol and 1-octadecanol) re-
vealed very complex mixing characteristics, and suggested
frequent phase immiscibility (Carreto et al., 2002). There-
fore, a mosaic of phase domains had to be expected even
at ambient temperatures.
This coexistence of crystalline and fluid or amorphous
phases was determined for the cuticular waxes extracted
from several different plant species (Reynhardt and
Riederer, 1991, 1994; Viougeas et al., 1995; Schreiber
et al., 1997), and was also demonstrated in situ (Merk
et al., 1998). The measurement of the diffusion character-
istics in reconstituted waxes is thought to be a reason-
able approach since the composition, and the proportion of

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Krüger et al., 1996).
Cuticular waxes and cutin matrix
Cuticular wax composition is very complex and varies
according to species, ontogenetic development, and envir-
onmental conditions (Bianchi, 1995; von Wettstein-
Knowles, 1995). The commonest components are n-alkanes
and primary alcohols, both of which range from C16 to C36
(Holloway, 1994). Long alkyl chain compounds exhibit
a solid-state polymorphism (liquid!hexagonally packed
solid phase!orthorhombically packed solid phase) across
a temperature scan (Watanabe, 1961). The physical state-
of the cuticular wax may contain several phases and
crystalline forms and is likely, therefore, to be the critical
feature that determines the transport properties of the cuti-
cle. Highly ordered crystalline regions are inaccessible to
permeation. The physical state of a mixture is a function
of its chemical composition and of physical variables like
temperature, pressure, etc. and is best described using a
phase diagram. Phase diagrams for such complex mixtures
as cuticular waxes are difficult to obtain and would not
be very informative due to their complexity. The ex-
amination of a simple binary mixture of two chemically
crystallites and amorphous phases is not altered during
recrystallization (Schreiber and Schönherr, 1993). A good
correlation between mobilities measured in reconstituted
cuticular waxes and in CMs was found, which confirmed
viscosity as an important property (Schreiber and Riederer,
1996; Kirsch et al., 1997). These investigations revealed
that the degree of crystallinity and the spatial arrangement
of crystals determined the permeating path through the
amorphous phase, which in turn determined the effective-
ness of the diffusion barrier (Schreiber et al., 1997).
Extracted waxes might, however, crystallize in a differ-
ent shape (spatial extension) compared with those con-
fined in a polymer matrix. Due to tortuosity (the path
taken around the crystalline obstacles) the length of the
diffusion path can be much greater than the thickness of
the barrier (Schreiber et al., 1996a; Baur et al., 1999).
The study of synthetic chemical polymer membranes
can also help to elucidate the mechanism of diffusion
across CMs, even though they are, in general, thicker and
less complex than plant cuticles. This enables a more de-
tailed view of the functional relationships to be obtained.
For instance, clay platelets are incorporated as inert fillers
into polymers to improve their mechanical and barrier
properties. To make the clay platelets compatible with
hydrophobic materials, such as polyolefins and waxes,

Table 1. Comparison of solute mobilities (k*; 695% CI) for bifenox within CM and MX of 13 plant species at 25 8C
Extractable wax fraction (mg cm
2; weight percentage) and differential factors of both solute mobilities are specified.
Plant species CM log k* 95% CI MX log k* 95% CI Factor Wax (mg cm
2) Wax (%)

Populus canescens –5.00 0.07 – – – 0.053 33.9

Pyrus pyrifolia –5.34 0.17 –3.29 0.05 112 0.055 25.5
P. communis cv. Conference –5.43 0.11 –3.74a – 49 0.061 23.7
Stephanotis floribunda –5.68 0.25 –3.73 0.02 89 0.026 10.2
M. domestica cv. Golden Delicious –6.20 0.08 –3.36 0.06 692 0.044 23.7
Ilex aquifolium –6.64 0.18 –4.14 0.12 316 0.162 24.6
Ginkgo biloba –6.97 0.11 –5.54 0.19 27 0.054 13.4
Melicoccus bijugatus –7.04 0.11 –4.81 0.06 170 0.049 8.8
Hedera helix –7.16 0.14 –3.75 0.05 2570 0.022 10.2
Strophanthus gratus –7.18 0.16 –5.34a – 69 0.060 19.5
Vanilla planifolia –7.67 0.11 –6.93 0.06 5 0.061 10.8
Ilex paraguariensis –7.89 0.11 –4.20 0.15 4897 0.114 22.3
Schefflera actinophylla –8.09a – –4.57a – 3311 0.084 29.8
a
Solute mobilities at 25 8C were calculated from Arrhenius graphs (Buchholz, 1998).

their surfaces are treated with monolayers of specific
amines. The mechanism by which these organoclays im-
prove the barrier properties of the film relies on the high
aspect ratio (relationship between the width and height)
of the exfoliated clay platelets to impart a tortuous diffu-
sion path. The tortuosity factor can be as high as several-
hundred-fold for impermeable platelets with aspect ratios
of 100–500 and at modest mineral loadings of 5–10
vol% (Cussler et al., 1988).
The tortuosity of nanocomposite materials, however,
is rarely that high (typically 2- to 4-fold) and it has been
shown that the interphase surrounding the clay platelets
exerts a profound influence on gas permeation (Chaiko
and Leyva, 2005): The polymer systems, the polyolefins
together with the waxes, are semi-crystalline materials
consisting of crystalline and amorphous phases. If the hydro-
carbon chains are long enough, they will fold in on them-
selves to form crystallites (10–20 nm). The crystallites
are held together by amorphous chain segments that
contribute to the elastic strength of the material. If the
hydrocarbon chain length is shortened sufficiently, the am-
orphous chain segments become less able to bridge
the gap between the crystallites, and the material becomes
brittle. Gas diffusion can then take place at the interfaces
between the crystallites.
Similar interactions were also demonstrated with Hor-
deum wax. Longer chains bridged the amorphous zone
between two adjacent layers of crystalline material, thus
linking the two layers (Reynhardt and Riederer, 1994).
The relevance of tortuosity in cuticular waxes as an effec-
tive way of reducing the permeabilities of plant cuticles
is discussed by Schreiber (2006).
A significant amount of shrinkage might accompany
wax recrystallization due to the high degree of crystall-
inity and the large density difference between the amorph-
ous melt and the crystal phase. As the freezing points of
the individual components in the wax differ significantly
(Chaiko and Leyva, 2005), this shrinkage could lead to
extensive cracking. Nanocomposite materials mixed with
polyethylene, however, displayed reduced embrittlement
at high clay loadings, and the films were quite flexible
and free of gross structural defects. Within plant cuticles
the cutin polymer can be viewed as the analogous elas-
tomer ensuring flexibility and avoiding embrittlement.
The plant cuticle has to be flexible to follow deforma-
tions caused either by fluctuations in turgor pressure or
by wind stress. Petracek and Bukovac (1995) investigated
the rheological properties of tomato fruit cuticles and
identified the cuticle as a viscoelastic polymer, com-
prising both elastic (reversible) and plastic (irreversible)
components. Furthermore, water affected the mechanical
properties, and waxes reduced elasticity and susceptibility
to fracturing. The mechanical properties of cuticles are
not related to thickness but are likely to be altered by
chemical composition (Wiedemann and Neinhuis, 1998).
Lipophilic pathway 2505
Marga et al. (2001) specified that the chemical composition
determined both the biochemical properties as well as
the elastic or rigid behaviour.
Consequently, several structural principles described
for synthetic polymers can be applied to the plant cuticle.
The plant cuticle can be described as a matrix composed
of highly ordered crystalline and disordered amorphous
regions, within which the reduced dynamics in the crys-
talline regions of the wax barrier make them practically in-
accessible to permeants (Reynhardt and Riederer, 1991).
The cutin polymer not only serves as a matrix but also
contributes to the transport characteristics of the cuticle
(Santier and Chamel, 1998). Cutin monomers are cross-
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linked by ester, peroxide, and ether bridges (Kolattukudy,
1984), and the presence of large amounts of di- and
tri-hydroxyfatty acids may affect the degree of cross-
linking. It was demonstrated that the maturation of cutin
led to the addition of covalent bonds which resulted in
less polarity and reduced permeability (Schmidt et al.,
1981). The wax-free matrix membranes (MXs) of several
plant species showed a relatively wide range of solute
mobilities (Table 1). The applicability of the UDOS
method also provided evidence for the asymmetry of the
cutin matrix. Chloroform–methanol extraction of CM
to provide MX not only removes the impermeable wax
crystallites but also the amorphous waxes. Therefore, the
elevated solute mobilities in MX are due to the reduced
viscosity (absence of amorphous waxes) and the shortened
diffusion path (lack of crystalline waxes causing tortuosity).
Solute size effects
Size dependence of mobility was quantified by plotting
for each plant species logk* versus molar volume (Vx)
for the respective sets of model compounds. Characteristic
molar volumes were calculated according to McGowan
and Mellors (1986). These molar volumes represent the
volumes at absolute zero temperature and they are closely
correlated to van der Waals volumes (McGowan, 1969).
Solute mobilities in the CMs of numerous plant species
have been measured using linear (Baur et al., 1996b) and
cyclic organic model compounds varying in molar volume
from 99 to 349 cm3 mol
1 (Bauer and Schönherr, 1992;
Schönherr and Baur, 1994; Buchholz et al., 1998; Baur
et al., 1999). Solute mobilities (k*) decreased exponent-
ially with increasing molar volumes (Vx) and, with all
plant species tested, good linear correlations between
logk* and Vx were found. This dependence could be
described with equations of the type
logk* = logk*0
b9Vx ð3Þ
The y-intercepts (k*0) represent the mobility of a hypo-
thetical molecule having zero molar volume and the
slopes of the graphs (b9) represent the size selectivity of
2506 Buchholz
7
-log k *
5 Hedera helix
-log k * = 5.22 + 0.009 . V x
Malus domestica
4 -log k * = 4.11 + 0.010 . Vx
Populus canescens
-log k * = 2.33 + 0.011 . Vx
3
50 100 150 200 250 300 350
Vx (cm3 . mol-1)
Fig. 4. The effect of molar volumes (Vx) of model compounds on their
mobility (k*) in cuticular membranes of three selected plant species.
Averages of 16–20 cuticular membranes and 95% confidence intervals
are shown.
the barrier. The differences in size selectivity (b9) were
not significant but the y-intercepts ranged from
2.33
(Populus) to
5.22 (Hedera) (Fig. 4). The latter are pure
membrane parameters, which are completely indepen-
dent of the properties of solutes (size and lipophilicity),
and they can be related to the free volume available for
diffusion in cuticles and cuticular waxes. (The real conver-
gence of this graph with the y-intercept is not known as
data describing the mobilities of non-electrolytes having
molar volumes significantly below 100 cm3 mol
1 are still
unavailable.)
No significant differences were observed in b9 among
all the species tested, with the average value of b9 being
0.0095 mol cm
3 at 25 8C. This equates to a reduction
in solute mobility of 700 times if Vx is increased from
100 to 400 cm3 mol
1. This size discrimination was found
to be the same when cuticular waxes were extracted and
comparable size selectivities were determined for poly-
mer MXs (Baur et al., 1999). A similar correlation
between diffusivity and solute size was also found for
reconstituted waxes (Schreiber and Schönherr, 1993;
Schreiber et al., 1996a; Schreiber, 2006).
Temperature
Size selectivity within plant cuticles is very temperature
sensitive. For example, in ivy cuticles, the difference in
solute mobilities between IAA (Vx=130 cm3 mol
1) and
tebuconazole (Vx=241 cm3 mol
1) amounts to factors of
24.5, 10.5, and 2.2 at 25 8C, 35 8C, and 55 8C, respectively
(Buchholz, 1998). Therefore, size selectivity diminishes at
elevated temperatures and intensifies at low temperatures.
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Fig. 5. Exponential hole-size distribution of a polymer matrix as
affected by temperature (modified according to Lieb and Stein, 1972).
400 (A) The distribution function provides the probability (P) of finding
a free-volume hole of a specific size (v*) at a given temperature (T). v* is
the minimum volume hole size into which a molecule can jump. The
diffusion coefficient is considered to be proportional to the probability
of finding a hole of volume v* or larger. (B) Increasing temperature
increases the overall free volume available for migration (shaded area).
The benefit of this relationship for the estimation of
the solute mobilities of new compounds at a given temper-
ature and plant species has been impressively demonstrated
(Buchholz et al., 1998). If the temperature sensitivity of
a plant species is known for only two compounds (i.e.
having two respective Arrhenius graphs) then a graph
of b9 versus temperature can be made. With this infor-
mation, the size selectivity (b9) for any desired temperature
can be derived and consequently the solute mobility for
any third compound (Vx) is predictable.
An explanation for this (temperature-dependent) size
discrimination can be derived from the free volume
theory (Coughlin et al., 1991; Duda and Zielinski, 1996).
For a successful diffusion step within a polymer the pen-
etrant needs a hole (a space between polymer chains) di-
rectly adjacent to its actual position. Enough energy to
make the jump is ensured by the motion of the solute
itself, via Brownian motion. Hence it follows that only
the movement of the adjacent polymer chains limits the
solute’s spatial motion. At a given temperature, a polymer
has a characteristic distribution of these temporary holes.
Elevating the temperature causes an increase in the motion
of the polymer chains which, in turn, produces a shift in
the hole-size distribution towards bigger holes. (Increased
energy enables the formation of larger gaps between adja-
cent polymer chains due to loosened bonds.) By contrast,
the temporal abundance of small holes is relatively reduced
at higher temperatures (Fig. 5).
An investigation of the response of solute mobility,
which is independent of time, revealed a large increase
with rising temperature (Baur and Schönherr, 1995;
Knoche and Bukovac, 2001; Schreiber, 2002). The tem-
perature coefficients Q10 ranged from 3 (IAA in Prunus

armeniaca) to 14 (cholesterol in Hedera helix) at 20 8C
(Baur et al., 1997a). The temperature effect can be quan-
tified using the Arrhenius equation:
k* = k*0 3expð
ED =RTÞ ð4Þ

1
where k*0 is the pre-exponential factor, ED (kJ mol ) is
the activation energy of diffusion, R (J mol
1 K
1) the
gas constant, and T (K) the temperature. By plotting logk*
(s
1) versus 1/T (K
1) the activation energy of diffusion
(ED) can be calculated from the slope (b) of the graph:
ED =
13b3R32:3 ð5Þ
The Arrhenius graphs published were linear in the phy-
siologically relevant temperature range of 10–45 8C, show-
ing that ED was constant and that no distinct phase transitions
caused by sudden structural changes occurred. At temper-
atures above 50 8C, a slight curvature of Arrhenius graphs
towards lower ED occurred for some species. This was an
additional hint that cuticles could be classified as elasto-
mers, since this flattening quite often occurs with rubbery
polymers, as ED values reflect the onset of viscous flow
(Schlotter and Furlan, 1992). Depending on species and
solute size, ED ranged from 60 to 232 kJ mol
1 (Baur et al.,
1997a). This demonstrated that plant cuticles are very
effective diffusion barriers.
The structural and functional integrity of cuticles can
be examined by measuring mobility after temperature
cycling. The comparison of subsequent experiments after
annealing can identify hysteresis effects due to re-
arrangement of the wax constituents. Such experiments
were conducted at temperatures up to 70 8C (Baur et al.,
1997a). Results were plant-species specific and ranged
from superimposed graphs (Prunus laurocerasus) to
the straightening of previously curved graphs (Citrus
aurantium), and in one case showed 10-fold increased
mobilities at 25 8C (Hedera helix). These different results
were related to differences in wax composition causing
different melting behaviours and redistribution during
annealing.
The sonication of CMs increased solute mobility at
25 8C by a factor of 4 and reduced the ED from123 kJ mol
1
to 92 kJ mol
1. Phase transitions or structural defects
were not indicated by the Arrhenius graphs (Fig. 6). The
interpretation of these results remains speculative (e.g.
loosening of chemical bonds), although a further observa-
tion was that these effects diminished during storage
(several months), and mobilities reverted to those of untreated
CMs. This indicated a re-equilibration between the wax
crystallites (‘healing of defects’) over time, as previously de-
scribed for water transpiration (Geyer and Schönherr, 1990).
Based on the transition state theory (Glasstone et al.,
1941) data can be plotted according to the thermodynamic
relationship between the pre-exponential factor (k*0) of
the Arrhenius equation (proportional to entropy) and act-
Lipophilic pathway 2507
T (°C)
60 50 40 30 20
-3.5
Pyrus cv. ‚Gellerts B.‘ / Tebuconazole
-4.0
-4.5
log k*
-5.0
-5.5
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sonication
-6.0
check
-6.5
3.0 3.1 3.2 3.3 3.4
1/T.1000(K-1)
Fig. 6. Arrhenius graphs obtained for tebuconazole in CM of Pyrus
(cv. Gellerts Butterbirne). One sample of CMs (n=10) was treated
with sonication for 5 min in a water bath 1 d before experimental use,
whereas the other sample (n=11) remained untreated.
ivation energy (enthalpy) of diffusion (ED). A strict linear
correlation was obtained for all of the CMs from 14 plant
species and organic compounds having molar volumes
ranging from 130 to 349 cm3 mol
1 and cuticle/water
partition coefficients of 18–108 (Fig. 7). This provided
evidence that all of these compounds diffused—irrespec-
tive of their size and lipophilicity—along the same
lipophilic diffusion path. Each of the solutes must have
travelled through a microenvironment with identical phys-
icochemical properties (Buchholz and Schönherr, 2000).
There are indications that water and lipophilic solutes
can share the lipophilic path. This co-permeability was
also confirmed by the simultaneous measurement of
3
H-labelled water and 14C-labelled organic compounds
across plant cuticles (Niederl et al., 1998). Water can,
however, take two parallel paths of diffusion: the lipo-
philic path made of cutin and wax and the polar path com-
posed of humidity-sensitive pores traversing the cuticles
(Schreiber, 2002). Since wax extraction enhanced water
permeability by several orders of magnitude (Schönherr,
1982), whereas the effect of humidity on cuticular water
permeability was only 2–3-fold, it is reasonable to
argue that the largest fraction of water diffused across
the lipophilic paths in the cuticle (Schreiber et al., 2001;
see also Table 2). The additional path predominated at
temperatures higher than 30 8C and is likely to exhibit
a threshold in size and/or lipophilicity (Schreiber, 2002).
Arrhenius graphs obtained by repeat measurements
on the same CM showed that, although the data points
remained on the general line for CMs, they were shifted
2508 Buchholz
35
Model Compounds:
IAA
NAA
30 Bifenox
Tebuconazole
Cholesterol
25
log k*o
20
15
log k *o = -3.61 -1.28 ED 10-3 / R
10
r² = 0.99
5
5 10 15
ED 10-3 / R
Fig. 7. Correlation between the Arrhenius slopes (ED/R) and y-intercepts of Arrhenius graphs k*0. Data for individual CMs from 14 plant species were
plotted. The solute mobilities were calculated for heating experiments with a temperature range of 25–50/55 8C for five different model compounds
(modified from Buchholz and Schönherr, 2000, with kind permission of Springer Science and Business Media).
towards lower entropy and enthalpy values (Fig. 8). A
consecutive third run rarely caused any further change in
this free energy relationship. Extraction of the cuticular
waxes from the CMs greatly reduced the activation en-
ergies and increased the solute mobilities. In the thermo-
dynamic plot the linear dependency was maintained, which
means that the free energy was identical in both membrane
types, CM and MX. Additionally, the graphs obtained
for MX exhibited a parallel displacement towards higher
entropy. This displacement was interpreted as a temperature-
independent tortuosity factor directly related to entropy
(Buchholz and Schönherr, 2000). Consecutive heating
cycles caused a shift along the line of a given membrane
type towards lower entropy and enthalpy. This means that
the individual chemical components might have been
redistributed during the annealing phase but this did not
affect the physicochemical properties of the diffusion path.
Plasticizer effects
Foliar uptake can be affected by formulation and adjuv-
ants (Falk, 1994; Baur, 1998). Formulants are any added
material in a pesticide formulation other than the biol-
ogically active ingredient(s), whereas adjuvants are those
formulants designed to enhance the activity or other
properties of an agrichemical product (Holland, 1996).
Surfactants that enhance foliar uptake by increasing
the mobility of a given compound are called accelerating
adjuvants (Schönherr, 1993b). This accelerator effect has
been demonstrated for various surfactants which were also
plasticizers (Schönherr, 1993a; Schönherr and Baur, 1996;
Citrus aurantium
Citrus grandis
Hedera helix
Ilex aquifolium
Ilex paraguariensis
Malus cv. Golden Delicious
Populus canescens
Prunus laurocerasus
Pyrus cv. Conference
Pyrus cv. Gellerts Butterbirne
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Pyrus cv. Williams Christ
Pyrus pyrifolia
Schefflera actinophylla
Strophanthus gratus
20 25 30 35
Baur et al., 1997b; Schönherr et al., 2001; Shi et al.,
2005), and also for active ingredients (Baur et al., 1996a).
This increased diffusivity is dependent on concentra-
tion and is interpreted as an unspecific plasticizing effect
of the surfactant molecule on the ultrastructure of the wax
(Schreiber et al., 1996b). Rapid induction and complete
reversibility of the effects indicate that the physical wax
structure is not irreversibly altered (e.g. solubilization of
wax or destruction of crystalline wax domains) by the
surfactant-induced acceleration (Schreiber, 1995, 2006).
The plasticizing effect of accelerators on cuticular
waxes in situ (CM) and on the cutin matrix (MX) was
investigated by applying the entropy–enthalpy relationship
(Buchholz and Schönherr, 2000). The plasticizer tributyl
phosphate (TBP) increased solute mobilities and reduced
ED significantly for both CM and MX. This indicated
that sorption of TBP increased segmental mobility of
polyethylene chains in both cutin and wax. This effect
was almost completely reversible, and for both plasticized
membrane types (CM, MX) the thermodynamic relation-
ship existed whereby all data points lay on the same line.
These data were used to characterize the lipophilic path-
way across plant cuticles in terms of the free-volume
theory, giving evidence that both high temperature and
accelerators act similarly on the barrier properties of a CM.
It is known that the efficacy of an accelerator is
dependent on both the type of plasticizer and on its concen-
tration. Arrhenius diagrams indicate that lower activation
energies are produced with higher loadings of plasticizers
(Schönherr et al., 2001). The different intrinsic efficacies
of two plasticizers have been elucidated using this method.

20
Strophanthus gratus / IAA
15
log k*o
10
MX 1st run
MX 2nd run
5 When foliar uptake of lipophilic solutes is considered,
CM 1st run
CM 2nd run
CM 3rd run
0 solute mobilities in the limiting skin and driving forces
0 5 10 15 20
ED 10-3 / R
Fig. 8. Correlation between Arrhenius slopes (ED/R) and y-intercepts
of Arrhenius graphs k*0. Data for individual cuticular membranes (CM)
and polymer matrix membranes (MX) from Strophanthus gratus leaves
are plotted. The double arrow indicates the parallel displacement of
both lines with identical slope. This displacement amounted to 1.7
(log scale). Solute mobilities were calculated for two or three succes-
sive heating experiments with identical membranes at temperature
ranges of 25–55 8C (CM) and 15–30 8C (MX). Between two tem-
perature scans the membranes were kept at ambient temperature for 24 h.
This also demonstrated that the penetrating compound
and the surfactant must be present simultaneously in the
limiting skin in order to enhance the compound’s mobility.
Perkins et al. (2005) demonstrated the plasticizing ef-
fect on both reconstituted waxes and on the native leaf sur-
face. Localized thermal analysis measurements revealed
considerable variation in the wax melting point (Tm) over
the leaf surface, indicating lateral heterogeneity in wax
composition. The authors proposed areas of amorphous
wax formations of at least 1 lm2. After droplet application
of Synperonic A7 (ethoxylated alcohol) they observed a
depression in the wax melting point, mainly at the deposit
annulus, whereas the surfactant Synperonic A20 (with a
more homogenous deposit structure) had no observable
effect on the Tm of the wax.
According to the model presented to describe the foliar
uptake of non-electrolytes (Schönherr et al., 1999), these
two surfactants could be classified as active (accelerator;
plasticizing) or passive (donor; dissolving) adjuvants
(Cronfeld et al., 2001). Passive adjuvants, which remain
on the surface of the cuticles, can serve as solvents, and
they affect driving forces via their effects on Kwxfr
(partitioning of the active substance or adjuvants between
the formulation residue and the cuticular waxes) and Cfr
(concentration of the active substance in the formulation
Lipophilic pathway 2509
residue). If these passive adjuvants are hygroscopic, their
effect on driving forces depends greatly on humidity.
Active adjuvants are solvents as well, but in addition they
penetrate into cuticles and increase solute mobility. These
effects depend on the type of accelerator, temperature,
size of solute, and plant species. Accelerators decrease the
viscosity of amorphous waxes which results in reduced
size selectivity. Accelerators also reduce differences in
solute mobility among plant species (see above).
Foliar uptake
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one has to be aware of the three consecutive steps: sorption,
diffusion, desorption (see above). Generally, the rates of
cuticular penetration (J) can be modelled in terms of
(Schönherr and Baur, 1994; Schönherr et al., 1999):
J = M=ðAtÞ = k*3lls 3ðKwxfr Cfr
Kmxw Cw Þ ð6Þ
The amount (M) of active substance penetrating per area
(A and time (t) is the rate of penetration (J). The term in
brackets denotes the driving force across the CM.
The sorption process is especially affected by numerous
factors which change during droplet drying (or rewetting)
and can rarely be analysed individually. Figure 9 attempts
to illustrate the most relevant factors. Physicochemical
properties of the active substance (group 1) affect the
‘external’ term of the driving force: KwxfrCfr. For example,
low water solubility causes a low concentration of dis-
solved molecules (Cfr), and will result in minor sorption
in cuticular wax (Kwxfr), although partitioning could
be high. But a spray solution comprises, beside the active
substance, adjuvants and other formulants, like emulsifiers,
buffer, etc. (group 2) which affect, for example, solvation
and partitioning (Baur et al., 1997c). Further, environ-
mental factors like temperature and humidity govern the
velocity of these dynamic processes. Consequently, it has
to be realized that, in order to maximize rates of pen-
etration, the product of Kwxfr and Cfr has to be increased,
whereas both quantities are mutually dependent on each other.
The individual parameters affecting solute mobility
(group 3) were extensively described above. After travel-
ling through the cuticle the ‘internal’ term of the driving
force, KmxwCw becomes relevant. Here, the partitioning
in the aqueous phase of the apoplast and potential access
to the symplast (after traversing the biomembrane) de-
termine the translocation into plant tissues. Rapid distri-
bution will result in a low concentration underneath the
spray droplet and enable large driving forces.
Finally, all those parameters describing the lipophilic
pathway of non-electrolytes across plant cuticles—and
demarcating it from the aqueous pathway (Schönherr,
2006)—are summarized in Table 2.
2510 Buchholz
Conclusions 2006) allowed the quantification and interpretation of
size selectivity and tortuosity, as well as entropy/enthalpy
It is the big advantage of the UDOS method that, with
relationships. Therefore, the properties of the lipophilic
this desorption measurement and the determination of
pathway are now completely characterized. Only the
the solute mobility k*, a pure descriptor of the diffusion
interpretation of the plant species-specific y-intercept of
process in CMs can be obtained. Only the feasibility of
a hypothetical molecule having zero molar volume, k*0,
determining sorption-independent mobilities in CMs
and its reference to tortuosity, remain to be explained
(or diffusion coefficients in extracted wax; Schreiber,
in more detail.

Table 2. Comparison of the lipophilic and the aqueous pathways across plant cuticles
Lipophilic pathway Aqueous pathway

Plant Epicuticular waxes (Retention on leaf surface)

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No effect on permeability Limit access to aqueous pores
Intracuticular waxes Amorphous waxes determine viscosity Minor effect on penetration
Crystallites cause tortuosity and
reduction in free volume
Cutin matrix Cross-linkage affects mobility Quality and spatial arrangement of polar
constituents determine abundance of aqueous pores
Substance Molecular size Pronounced size selectivity Hydrate shells enforce weak size selectivity
(no plasticizer)
Lipophilicity Affects driving force (sorption in waxes) –
No effect on mobility
Point of deliquescence – Low POD reduces risk of crystallized
formulation residues
Concentration Effect on driving force Effect on driving force
Microclimate Temperature Effect on fluidity, viscosity of waxes Minor effect on penetration
and matrix
Humidity Effect on driving force (solubility) Effect on driving force (solubility) and
swelling of cuticle
Passive additives Surfactants (Retention; mass flow into open stomata)
Effect on driving force (solvation, partitioning) Facilitation of access to aqueous pores
Minor effect on penetration
Humectants Effect on driving force (partitioning, solvation, Effect on driving force (solubility,
concentration dilution) concentration dilution)
Prevention of crystallization extends time Prevention of crystallization facilitates
for penetration access to aqueous pores and extends time
for penetration
Active additives Plasticizer Penetrate into cuticle and decrease viscosity Negligible effect on penetration
(accelerator)
Concentration dependent efficacy
Plasticizer’s and substance’s mobility need
a mutual fit

Fig. 9. Schematic drawing of a spray droplet and the underlying plant epidermis. Factors affecting cuticular penetration via the ‘lipophilic pathway’ are
grouped into (1) physicochemical properties of the active substance, (2) conditions determined by the environment and the spray solution, and (3)
parameters affecting solute mobility, k*. J, Rate of cuticular penetration; lls, length of the diffusion path in the limiting skin (6¼ thickness); Kwxfr, partition
coefficient wax/formulation residue; Kmxw, partition coefficient polymer matrix/water; Cfr, concentration of active substance in formulation residue;
Cw, concentration of active substance in water (apoplast) (according to the model described by Schönherr et al., 1999).

This knowledge on factors affecting solute mobility
(group 3 in Fig. 9) and the physicochemical properties of
a given non-polar active substance (group 1 in Fig. 9) are
the starting points for tailored crop protection products.
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