Pest Management Science Pest Manag Sci 62:137–147 (2006)

DOI: 10.1002/ps.1139

Modelling the effects of alcohol ethoxylates

on diffusion of pesticides in the cuticular wax
of Chenopodium album leaves
Markus Burghardt,1 Adrian Friedmann,2 Lukas Schreiber3∗ and Markus Riederer1
1 Julius-von-Sachs-Institut für Biowissenschaften, Lehrstuhl für Botanik II— Ökophysiologie und Vegetationsökologie, Universität Würzburg,
Julius-von-Sachs-Platz 3, D-97082 Würzburg, Germany
2 Syngenta Bioperformance Research, Jealott’s Hill International Research Centre, Bracknell, Berks RG42 6ET, UK
3 Institut für Zelluläre und Molekulare Botanik (IZMB), Abteilung Ökophysiologie, Universität Bonn, Kirschallee 1, D-53115 Bonn, Germany

Abstract: Cuticular waxes represent the first and, in most cases, the limiting barrier for foliar uptake
of pesticides from solution. Sorption of pesticides in reconstituted cuticular wax (wax/water partition
coefficients) of Chenopodium album L. and in isolated cuticular membranes (cuticle/water partition
coefficients) of Prunus laurocerasus L. was determined. Diffusion coefficients of pesticides in reconstituted
cuticular wax of C. album leaves were size-dependent, increasing with increasing molar volume. In the
presence of alcohol ethoxylates, diffusion coefficients were enhanced by up to two orders of magnitude,
and size selectivity was significantly decreased. The accelerating effect and the decrease in size selectivity
were attributed to plasticisation of the cuticular wax by the alcohol ethoxylates increasing the fluidity
in the wax. A free volume model adopted from polymer science was successfully applied to predict
diffusion coefficients of pesticides on the basis of the transport properties of the wax (size selectivity and
crystallinity), the molar volume of the diffusing compound and the accelerator concentration in the wax.
 2005 Society of Chemical Industry

Keywords: adjuvants; alcohol ethoxylate; accelerator; cuticular wax; diffusion coefficient; partition coefficient

1 INTRODUCTION property have been termed accelerators.13 The action

After foliar application, many crop pesticides have
to penetrate the plant cuticle, which represents a
rate-limiting step on the way to their target sites.1
The barrier properties of the cuticle are established
by cuticular waxes embedded in the outer layer of
the cutin matrix (intracuticular waxes) and deposited
on the cuticle surface (epicuticular waxes).2 Wax
extraction by organic solvents increases cuticular
permeability by up to four orders of magnitude.3
Cuticular waxes are semicrystalline aggregates4,5
leading to much lower diffusion coefficients and lower
solubilities of pesticides compared with the amorphous
cutin polymer.6,7
Pesticides are usually formulated with, for example,
adjuvants to enhance the rates of foliar uptake.8
The formulation can influence foliar uptake of
pesticides in two major ways:9 (1) the driving force
of uptake represented by the concentration gradient
in the transport-limiting wax layer may be altered
by adjuvants owing to changes in partitioning of the
pesticide between the cuticle and the formulation
residue;10 – 12 (2) mobility of pesticides in the cuticular
wax barrier can be enhanced. Adjuvants having this
of accelerators is not related to surface activity,
since it was shown that other substances such as
aliphatic alcohols,13 tributyl phosphate14 or even
pesticides themselves such as chlorfenvinphos15 and
permethrin16 can have accelerating properties.
Transport across cuticles can be measured experi-
mentally using isolated cuticular membranes.17 Since
their isolation is limited to a small number of plant
species, an alternative approach using isolated and
reconstituted cuticular waxes was developed.6 Results
obtained by this method reflect transport properties
of isolated cuticular membranes and intact leaves7,18
and led to the following conclusions: (1) diffusion
coefficients of pesticides exhibited a pronounced size
selectivity;7,19,20 (2) accelerators caused a plastici-
sation of cuticular waxes, leading to an enhance-
ment of diffusion coefficients of the pesticides;16,21,22
(3) accelerator effects on diffusion of pesticides were
correlated with accelerator concentrations in the wax
barrier.15,22 – 24
This study was intended to develop and verify
a general model summarising the dependence of
diffusion coefficients on (1) the transport properties

∗ Correspondence to: Lukas Schreiber, IZMB, University of Bonn, Kirschallee 1, D-53115 Bonn, Germany

E-mail: lukas.schreiber@uni-bonn.de
Contract/grant sponsor: Novartis Crop Protection AG (Basel)
Contract/grant sponsor: DFG
(Received 29 May 2005; revised version received 12 August 2005; accepted 19 August 2005)
Published online 24 November 2005
 2005 Society of Chemical Industry. Pest Manag Sci 1526–498X/2005/$30.00 137
M Burghardt et al.

of the cuticular wax (size selectivity and crystallinity),
(2) the molar volume of the diffusing compound
and (3) the accelerator concentration in the wax.
Diffusion coefficients in reconstituted cuticular wax of
Chenopodium album L. (fat hen) leaves were measured
with model compounds of different size in the absence
and presence of monodisperse alcohol ethoxylates.
One polydisperse alcohol ethoxylate was included
in order to check whether its effects on diffusion
coefficients of pesticides were in agreement with the
behaviour of monodisperse alcohol ethoxylates.
2 MATERIALS AND METHODS
2.1 Plant material
In this experimental approach, C. album plants were
chosen as an example of a typical weed. Cuticular
wax was extracted at the three-leaf growth stage from
leaves of C. album plants cultivated in the Botanical
Garden of the University of Würzburg, Germany.
Leaves were dipped in chloroform (purity 99%; Riedel
de Haën, Seelze, Germany) for 5 s. Since the isolation
of C. album cuticles (CM) was not possible, cuticular
membranes were obtained from the adaxial leaf side
of Prunus laurocerasus L. (cherry laurel). Enzymatic
isolation was carried out as described previously.25
Wax-free polymer matrix membranes (MX) were
obtained by exhaustive extraction of the cuticular
membranes for 16 h with chloroform in a Soxhlet
apparatus.
2.2 Preparation of wax samples
Isolated cuticular wax was reconstituted on aluminium
discs (radius 4 mm) as described by Schreiber and
Schönherr.6 The amounts of reconstituted wax were
determined by weighing the discs before and after
wax deposition (±1 µg; S3D microbalance, Sartorius,
Göttingen, Germany). Wax amounts per disc were
in the range 150–250 µg, which corresponded to a
thickness of the wax layer in the range 1.7–2.8 µm.
2.3 Pesticides
14
C-labelled pesticides of different sizes were selected
as model compounds (Table 1). Benzoic acid was
included because of its relatively low molar vol-
ume. The radiochemical purity of all compounds
was better than 97%. The compounds were dis-
solved in deionised water containing sodium azide
(1 mmol litre−1 ; Merck-Schuchardt, Hohenbrunn,
Germany) to prevent growth of micro-organisms. The
ionisable compounds 2,4-D and benzoic acid were
dissolved in citric acid buffer adjusted to pH 3 with
potassium hydroxide.
2.4 Monodisperse and polydisperse alcohol
ethoxylates
Monodisperse alcohol ethoxylates (AEs) with alkyl
chain lengths C12 , C14 and C16 and with degrees
of ethoxylation ranging from E2 to E8 (Fluka,
Neu-Ulm, Germany) were selected (Table 2). Their
purity was better than 97%. Critical micelle con-
centrations (CMCs) were estimated according to
a prediction equation.23 Genapol C-050 (Hoechst,
Frankfurt/Main, Germany) was chosen as a poly-
disperse AE; its CMC determined by tensiom-
etry was 0.0297 g kg−1 , corresponding to 6.5 ×
10−5 mol kg−1 .22 AEs were dissolved in deionised
water containing sodium azide (1 mmol litre−1 ).
2.5 Determination of partition coefficients
Wax/water partition coefficients (Kww ) of the radiola-
belled compounds were determined as described by
Schreiber and Schönherr.27 In brief, reconstituted wax
samples were incubated in vials containing a solution
Table 2. Abbreviations and critical micelle concentrations (CMCs) of
the monodisperse alcohol ethoxylates (AEs)
CMC×
10−5
Abbrevia- (mol
AE tion kg−1 )
Triethylene glycol monododecyl ether C12 E3 3.5
Pentaethylene glycol monododecyl ether C12 E5 5.2
Hexaethylene glycol monododecyl ether C12 E6 6.5
Heptaethylene glycol monododecyl ether C12 E7 7.9
Octaethylene glycol monododecyl ether C12 E8 9.8
Triethylene glycol monotetradecyl ether C14 E3 0.29
Pentaethylene glycol monotetradecyl ether C14 E5 0.44
Hexaethylene glycol monotetradecyl ether C14 E6 0.54
Octaethylene glycol monotetradecyl ether C14 E8 0.81
Triethylene glycol monohexadecyl ether C16 E3 0.024
Octaethylene glycol monohexadecyl ether C16 E8 0.068

Table 1. Common names, specific activities and molar volumes (MVs) of the 14 C-labelled compounds

Common Specific activity MVb

Compound namea (GBq mol−1 ) (cm3 mol−1 )

Benzoic acid Benzoic acid 740 93.2

(2,4-Dichlorophenoxy)acetic acid 2,4-D 581 137.6
4-Amino-6-tert-butyl-4,5-dihydro-3-(methylthio)-1,2,4-triazin-5-one Metribuzin 770 162.0
1-(4-Chlorophenoxy)-3,3-dimethyl-1-(1H-1,2,4-triazol-1-yl)butan-2-ol Triadimenol 584 218.8
1-(Biphenyl-4-yloxy)-3,3-dimethyl-1-(1H-1,2,4-triazol-1-yl)butan-2-ol Bitertanol 624 267.4
a Sources: benzoic acid, Du Pont de Nemours, Dreieich, Germany; 2,4-D, Sigma Chemie, Deisenhofen, Germany; metribuzin, triadimenol and
bitertanol, Bayer AG, Leverkusen, Germany.
b
Calculated according to Abraham and McGowan.26

138 Pest Manag Sci 62:137–147 (2006)


of the radiolabelled compound on a rotating bench
(24 h, 25 ◦ C). After equilibration, wax samples were
blotted on filter papers and dissolved in chloroform,
and the radioactivity in the wax samples and in the cor-
responding external donor solutions was determined
by liquid scintillation counting (Counter 1409, Wal-
lac, Turku, Finland) after adding 5 ml of scintillation
cocktail (Ultima Gold XR, Packard, Meriden, CT,
USA). Samples were counted for 20 min and neces-
sary quench corrections were carried out. Kww values
were calculated from
masssubstance /masswax
Kww = (1)
masssubstance /masswater
Polymer matrix membrane/water partition coeffi-
cients (Kmxw ) were determined in a comparable way
and calculated according to
masssubstance /massmx
Kmxw = (2)
masssubstance /masswater
Apparent partition coefficients (K pH ) measured with
the ionisable compounds 2,4-D (pKa = 2.85)28 and
benzoic acid (pKa = 4.19)29 at pH 3 were corrected
by the degree of ionisation according to
K = K pH (1 + 10pH−pKa ) (3)
Kww values of monodisperse AEs were determined
as described previously by Burghardt et al.22 using
concentrations ten times the CMCs. Since AEs were
not radiolabelled, amounts sorbed to the wax were
determined by gas chromatography. The external
concentration was given by the CMC, since only free
monomers can enter the cuticle.23 Above the CMC
the concentration in the wax remains constant, since
the free monomer concentration in the aqueous phase
stays constant. Thus Kww of monodisperse AEs is
given by
masssubstance /masswax
Kww = (4)
CMC
Kmxw values of AEs were calculated from a prediction
equation (Kmxw = −1.78 + 0.52C − 0.17E).23
2.6 Determination of diffusion coefficients
Diffusion coefficients (D) of pesticides in reconstituted
cuticular wax were determined according to a
method described previously.6 Reconstituted wax
samples loaded with the 14 C-labelled compounds
were either desorbed with phospholipid suspension
(PLS, 10 g kg−1 , soybean lecithin; Roth, Karlsruhe,
Germany) serving as a standard17 or with the different
AE solutions. The amounts of radioactivity desorbed
at each time period and the amounts remaining in
the wax sample at the end of the experiment were
determined by liquid scintillation counting. Diffusion
coefficients were calculated by plotting the relative
amount desorbed (Mt /M0 ) versus the square root of
Pest Manag Sci 62:137–147 (2006)
Diffusion of pesticides in cuticular wax
time (t), applying the adequate solution of the second
Fickian law:30
Mt /M0 = (4/l)(Dt/π )1/2 (5)
The thickness of the wax layer (l) was calculated from
the amounts of wax reconstituted on the aluminium
discs, assuming a wax density of 0.9 g cm−3 .31
2.7 Gas chromatography
The amounts of monodisperse AEs sorbed in the
cuticular wax were measured by gas chromatography
after dissolution of the wax samples in chloroform. For
derivatisation the samples were treated with N,N-bis-
trimethylsilyltrifluoroacetamide (BSTFA, 100 µl mg−1
wax or Genapol C-050; Machery-Nagel, Düren, Ger-
many) in dry pyridine (1:1 v/v; Merck, Darmstadt,
Germany) for 30 min at 70 ◦ C. A gas chromatograph
equipped with a flame ionisation detector and an
on-column injector (HP 5890 II, Hewlett Packard,
Avondale, PA, USA) was used. The compounds were
separated on a fused silica column (DB-1, J&W Sci-
entific, Folsom, CA, USA) using the chromatographic
conditions described previously.22 The peak areas
were integrated electronically and quantification was
by internal standard (n-dotriacontane, purity 99%;
Fluka). Correction coefficients of the AEs were deter-
mined experimentally by analysing known amounts
together with the internal standard.
2.8 Statistics
Diffusion coefficients and partition coefficients are
given as means of ten replicates with 95% confidence
intervals. Regression analysis was carried out using
SigmaPlot 7.0 (Systat Software, Point Richmond, CA,
USA).
3 RESULTS
3.1 Partition coefficients
Wax/water partition coefficients of the pesticides
for cuticular wax of C. album ranged from 3.7
for metribuzin to 2090 for bitertanol (Table 3).
The corresponding polymer matrix membrane/water
partition coefficients were up to one order of
magnitude higher and ranged from 49 for metribuzin
to 10 100 for bitertanol (Table 3). Wax/water partition
coefficients of monodisperse AEs ranged from 109 for
C12 E8 to 231 000 for C16 E3 (Table 4). In homologous
series the wax/water partition coefficient increased
with increasing alkyl chain length and decreased with
increasing ethylene oxide chain length. Polymer matrix
membrane/water partition coefficients were about one
order of magnitude higher (Table 4).
3.2 Diffusion coefficients
Values of D ranged from 1.1 × 10−18 to 2.7 ×
10−17 m2 s−1 , decreasing with increasing size of the
compound (Table 5). The monodisperse AE C12 E3
increased D by factors between 9.6 and 174.
139
M Burghardt et al.

Table 3. Wax/water partition coefficients (Kww ) for cuticular wax of Table 6. Diffusion coefficients (D) of benzoic acid and bitertanol in
Chenopodium album and polymer matrix membrane/water partition reconstituted cuticular wax of Chenopodium album leaves and effects
coefficients (Kmxw ) of the pesticides and benzoic acid of the monodisperse alcohol ethoxylates (AEs) on D

Compound Kww Kmxw Benzoic acid Bitertanol

D × 10−16 D × 10−16
Benzoic acid 8.7 (±0.9) 58 (±8) AE (m2 s−1 ) Effect (m2 s−1 ) Effect
2,4-D 80 (±13) 494 (±67)
Metribuzin 3.7 (±0.6) 49 (±12) Control 0.27 (±0.03) 1 0.011 (±0.004) 1
Triadimenol 248 (±45) 1 330 (±190) C12 E3 2.59 (±0.45) 9.6 1.91 (±0.22) 174
Bitertanol 2090 (±350) 10 100 (±2200) C12 E5 — — 0.96 (±0.29) 87
C12 E6 1.29 (±0.36) 4.8 0.89 (±0.17) 81
C12 E7 1.00 (±0.20) 3.7 0.68 (±0.26) 62
Table 4. Maximum concentrations in the wax (cmax wax ) and wax/water
C12 E8 0.97 (±0.29) 3.6 0.39 (±0.14) 35
partition coefficients (Kww ) for cuticular wax of Chenopodium album
C14 E3 — — 1.95 (±0.29) 177
and polymer matrix membrane/water partition coefficients (Kmxw ) of
C14 E5 — — 1.26 (±0.43) 118
the monodisperse alcohol ethoxylates (AEs)
C14 E6 — — 0.72 (±0.15) 65
−1 C14 E8 — — 0.35 (±0.10) 32
AE cmax
wax (mmol kg ) Kww Kmxw
C16 E8 0.82 (±0.23) 3.0 0.38 (±0.11) 35
C12 E3 56 (±7) 1 610 (±190) 8 910
C12 E5 36 (±10) 687 (±180) 4 070
C12 E6 17 (±4) 268 (±67) 2 750
and the number of ethylene oxide units (E) of the
C12 E7 16 (±7) 203 (±90) 1 860
C12 E8 11 (±2) 109 (±20) 1 260 monodisperse AEs:
C14 E3 57 (±8) 19 700 (±2900) 97 700
C14 E5 35 (±12) 8 040 (±2210) 44 700 log Kww = −2.43(±0.38) + 0.53(±0.03)C
C14 E6 20 (±2) 3 700 (±360) 30 200
C14 E8 12 (±2) 1 410 (±250) 13 800 − 0.24(±0.02)E (6)
C16 E3 55 (±15) 231 000 (±62 000) 1 070 000
C16 E8 9.3 (±2.7) 13 700 (±4000) 151 000
It is noteworthy that the contribution of the alkyl
chain length in Eqn (6) to Kww and to the CMC
Desorption in the presence of the polydisperse AE (see Table 2) has the same value with opposite signs.
Genapol C-050 enhanced D by factors between 6.2 The reason for this phenomenon is plausible from
and 68 (Table 5). The highest effects were observed a thermodynamic point of view, as the low water
with the largest compound (bitertanol) and the lowest solubility and the resulting tendency of an alkyl chain
with the smallest compound (benzoic acid). The to escape the aqueous environment are considered
effects of a wider range of monodisperse AEs were as the main driving forces for both sorption in a
measured with benzoic acid and bitertanol. D of lipophilic phase32 and micelle formation.33 Therefore
benzoic acid was enhanced by factors between 3.0 the influence of the alkyl chain length on the maximum
−1
(C16 E8 ) and 9.6 (C12 E3 ) and the effects on D of concentration in the wax (cmax wax , mmol kg ), which
bitertanol were in the range between 32 (C14 E8 ) is given by the product of Kww and the CMC, is
and 177 (C14 E3 ). Effects decreased with increasing cancelled, leading to Eqn (7) describing sorption in
number of ethylene oxide groups (Table 6). the wax solely as a function of the number of ethylene
oxide units (E) (Fig. 1B; r 2 = 0.97):

4 DISCUSSION
4.1 Sorption of monodisperse alcohol
ethoxylates to cuticular wax
Equation (6), obtained from a multiple regression
analysis, describes a quantitative structure–property
relationship (Fig. 1A; r 2 = 0.997) characterising the
dependence of Kww on the alkyl chain length (C)
wax = 2.21(±0.12) − 0.15(±0.02)E
log cmax (7)
Similar quantitative structure–property relation-
ships have been described for the sorption of AEs
to cuticular wax of Hordeum vulgare L.22 and to cutic-
ular polymer matrix membranes of Citrus aurantium

Table 5. Diffusion coefficients (D) of the pesticides and benzoic acid in reconstituted cuticular wax of Chenopodium album leaves and effects of the
alcohol ethoxylates C12 E3 and Genapol C-050

Control C12 E3 Genapol C-050

Compound D × 10−16 (m2 s−1 ) D × 10−16 (m2 s−1 ) Effect D × 10−16 (m2 s−1 ) Effect

Benzoic acid 0.27 (±0.03) 2.59 (±0.45) 9.6 1.67 (±0.49) 6.2
2,4-D 0.16 (±0.04) 2.30 (±0.47) 14 1.04 (±0.24) 6.5
Metribuzin 0.084 (±0.015) 2.53 (±0.63) 30 0.88 (±0.19) 10
Triadimenol 0.020 (±0.006) 1.86 (±0.57) 93 0.79 (±0.20) 40
Bitertanol 0.011 (±0.004) 1.91 (±0.21) 174 0.75 (±0.22) 68

140 Pest Manag Sci 62:137–147 (2006)

 Diffusion of pesticides in cuticular wax

6 2.4
C16 A Bitertanol
2.2
5
C14 2.0

log(effect)
log K ww

4
1.8
C12
3
1.6

2 1.4 A

1.0 Benzoic acid

1.8 B
(mmol kg−1)

1.5

log(effect)
0.8

1.2
max
log c wax

0.6
0.9
B
0.6 0.4
2 4 6 8 10
1 2 3 4 5 6 7 8 9
E
E
ž
Figure 2. Correlation of the effects of ( ) monodisperse alcohol

Figure 1. Correlation of (A) wax/water partition coefficients (Kww )
and (B) maximum concentrations of monodisperse alcohol
ethoxylates in Chenopodium album wax (cmax wax ) with the number of
ethylene oxide units (E).
L.23 Kww and Kmxw are related by a linear free energy
relationship:
log Kww = −1.04(±0.27) + 1.05(±0.07) log Kmxw
(8)
This principal relationship was described in the past
for pesticides7 and monodisperse AEs.22,24 However,
the findings presented here demonstrate that both
classes of chemically very different compounds fall
on one and the same regression line. As indicated by
the negative value of the intercept of the regression
equation, the sorption capacity of the wax is distinctly
lower than that of the amorphous cutin polymer, which
can be attributed to the semicrystalline structure of the
wax.34 The congruent behaviour of pesticides and AEs
indicates that, in spite of the pronounced effect of the
AEs on D in the cuticular wax, no additional sorption
sites are formed and the sorption capacity of the wax
remains unchanged.
4.2 Enhancement of diffusion coefficients of
pesticides by alcohol ethoxylates
Plotting the logarithm of the effects of AEs on D
of benzoic acid and bitertanol versus the degree of
°
ethoxylates and ( ) the polydisperse alcohol ethoxylate Genapol
C-050 on diffusion coefficients of (A) bitertanol and (B) benzoic acid in
cuticular wax of Chenopodium album with the number of ethylene
oxide units (E). Regression equations: bitertanol,
log(effect) = −0.14E + 2.7 (r 2 = 0.96); benzoic acid,
log(effect) = −0.094E + 1.3 (r 2 = 0.97).
ethoxylation reveals a linear dependence (Fig. 2).
Since maximum concentrations in the wax are directly
related to the logarithm of the ethoxylation (Eqn (7)),
a linear dependence of the effects on the maximum
concentrations exists (Fig. 3). This result strongly
supports the conclusion that AEs exhibit a non-
specific plasticising effect on cuticular waxes, which
is independent of their chemical structure (alkyl chain
length or number of ethylene oxide groups) and
depends only on the amounts sorbed to the wax.22,23
Even the effect of the polydisperse AE Genapol C-050
fitted this trend of the monodisperse AEs when the
mean alkyl chain length (Cmean = 13.5) and the mean
degree of ethoxylation (Emean = 4.6) were used (Figs 2
and 3).
4.3 Size dependence of diffusion coefficients
The values of D for pesticides in reconstituted cutic-
ular wax of C. album were size-dependent (Figs 4
and 5). AEs enhanced the mobility of the compounds
by up to two orders of magnitude, and size selectivity
was largely reduced. Size dependence is a charac-
teristic feature of diffusion across solid phase barri-
ers, as has been reported for reconstituted cuticular

Pest Manag Sci 62:137–147 (2006) 141

M Burghardt et al.

200 -15
C12E3
Bitertanol
160
-16

log D (m2 s-1)

120 Genapol C-050
effect

-17 1
80
2
3
40 -18 control
4
A 5
0 A
-19
12 1.9 2.0 2.1 2.2 2.3 2.4 2.5
Benzoic acid
log MV (cm 3
mol−1)

9 Figure 4. Correlation of the logarithm of diffusion coefficients (D) of

the pesticides with the logarithm of molar volumes (MVs) according to
effect

the Stokes–Einstein law (Eqn (11)). Values of D were determined using

an inert control desorption medium (PLS) and two active desorption
6 media (micellar solutions of Genapol C-050 and C12 E3 ). Regression
equations: control, log D = −3.2 log MV − 10.1 (r 2 = 0.95); Genapol
C-050, log D = −0.75 log MV − 14.4 (r 2 = 0.90); C12 E3 ,
3 log D = −0.33 log MV − 14.9 (r 2 = 0.75). Compounds: 1 = benzoic
acid; 2 = 2, 4-D; 3 = metribuzin; 4 = triadimenol; 5 = bitertanol.
B
0
0 10 20 30 40 50 60 -15
max
cwax (mmol kg-1) C12E3

ž
Figure 3. Correlation of the effects of ( ) monodisperse alcohol -16
°
log D (m 2 s−1)

ethoxylates and ( ) the polydisperse alcohol ethoxylate Genapol 1

C-050 on diffusion coefficients of (A) bitertanol and (B) benzoic acid in 2 Genapol C-050
cuticular wax of Chenopodium album with the maximum 3
concentration of the alcohol ethoxylates in the wax (cmax
wax ). The
-17
maximum concentration of Genapol C-050 was calculated according control 4
wax +
to Eqn (7). Regression equations: bitertanol, effect = 2.9cmax 5
8.7 (r 2 = 0.94); benzoic acid, effect = 0.13cmax
wax + 1.9 (r = 0.98).
2
-18

waxes,7,35 isolated cuticular membranes,9,19,20 biologi- B

cal membranes,36 the stratum corneum of mammalian
skin37,38 and synthetic polymers.39 – 42 The size depen-
dence of D in reconstituted cuticular wax of C. album
will be discussed on the basis of three different models.
The Stokes–Einstein relationship and the free volume
theory will be used to highlight mechanistic aspects.
A quantitative analysis of the interaction between
pesticides, accelerators and cuticular waxes will be
conducted using an extended free volume theory.
4.4 Stokes–Einstein law
The classical approach correlating D with the size of
the diffusing compounds is the Stokes–Einstein law,
which states that the diffusion coefficient D is inversely
proportional to the friction force (f ):
D = kT /f
where T is the absolute temperature and k is the
Boltzmann constant. According to the law of Stokes,
the friction force depends on the viscosity of the
-19
100 150 200 250
3 −1
MV (cm mol )
Figure 5. Correlation of the logarithm of diffusion coefficients (D) of
the pesticides in cuticular wax of Chenopodium album with molar
volumes (MVs) according to the free volume theory (Eqn (12)). Values
of D were determined using an inert control desorption medium (PLS)
and two active desorption media (micellar solutions of Genapol C-050
and C12 E3 ). Regression equations: control, ln D = −0.012MV
− 36.2 (r 2 = 0.98); Genapol C-050, ln D = −0.0042MV
− 36.1 (r 2 = 0.78); C12 E3 , ln D = −0.0019MV − 35.7 (r 2 = 0.77).
Compounds: 1 = benzoic acid; 2 = 2, 4-D; 3 = metribuzin;
4 = triadimenol; 5 = bitertanol.
medium (η) and the radius of the diffusing molecule
(r), assuming a spherical shape:
(9)
f = 6π ηr (10)
Combining Eqns (9) and (10) and taking the volume
of the diffusing molecule (molar volume MV =

142 Pest Manag Sci 62:137–147 (2006)


4π r 3 /3) into account, the following relationship is
obtained:
D × MV1/3 = constant (11)
According to Eqn (11), a plot of the logarithm
of D versus the logarithm of molar volumes should
result in a regression line with a slope of −0.33.
The Stokes–Einstein relationship describes diffusion
in liquids, and slopes normally vary between −0.5
and −0.33.36 In cuticular wax, which is solid and
partially crystalline at room temperature, double-
logarithmic plots were characterised by negative slopes
varying between −2.9 and −15.7.6 However, in the
presence of Genapol C-050 and C12 E3 the slopes
were very close to −0.33 (Fig. 4). This indicates that,
in the presence of effective accelerators, diffusion in
cuticular waxes is comparable to that in fluid media.43
Therefore the activity of accelerators is described by
a plasticising action increasing the fluidity of cuticular
waxes. Experimental evidence for this mechanism
has been obtained by electron spin resonance (ESR)
spectroscopy,24 nuclear magnetic resonance (NMR)
spectroscopy5 and calorimetric measurements.5,44
4.5 Free volume theory
An alternative approach to the Stokes–Einstein rela-
tionship was developed taking into account the much
steeper size dependence of non-Stokesian diffusion
in solid media.36 The free volume theory describes
an exponential decrease in the diffusion coefficient D
with increasing molar volume (MV):9,38,45
D = D0 e−β×MV
where D0 is the diffusion coefficient of a hypothetical
compound having a molar volume of zero and β is
the size selectivity. The reciprocal value of the size
selectivity has the meaning of the mean free volume
available for diffusion (Vf ):
Vf = 1/β
Plotting the logarithm of D versus the molar volumes
of the pesticides shows that the free volume theory
is applicable for the description of diffusion in
reconstituted cuticular wax of C. album (Fig. 5).
The advantage of this approach is related to the
fact that the parameters D0 and β of Eqn (12)
have a direct physical meaning, which can be
related to the reconstituted wax in which diffusion
Table 7. Size selectivities (β), mean free volumes available for diffusion (Vf ), diffusion coefficients of a hypothetical compound having a molar
volume of zero (D0 ) and correlation coefficients (r 2 ) obtained by plotting the logarithm of diffusion coefficients (D) of pesticides versus the molar
volumes for cuticular wax of Chenopodium album
Desorption
medium β (mol cm−3 )
Control 0.020 (±0.005)
Genapol C-050 0.0042 (±0.0040)
C12 E3 0.0020 (±0.0019)
Pest Manag Sci 62:137–147 (2006)
Diffusion of pesticides in cuticular wax
is measured. With the Stokes–Einstein approach,
which is phenomenological, this is not possible. The
significance of the free volume parameters (β, Vf , D0 )
can be illustrated by a simple model of the molecular
structure of cuticular waxes, dividing the wax into
a crystalline and an amorphous fraction.34 The size
selectivity characterises the transport properties of
the amorphous wax fraction where diffusion takes
place. According to the free volume theory, diffusion
can only occur within voids of the same size as
or larger than the molar volume of the diffusing
molecule. Molecules are considered to be large if
the quotient Vf /MV < 1, and they are considered
small if Vf /MV > 1.46 Free volume arises from
the segmental motion4 and diffusion of the wax
molecules.18 Size selectivity of the wax of C. album was
0.020 mol cm−3 and this fits size selectivities reported
for reconstituted cuticular waxes,7,18,22 ranging from
0.015 to 0.036 mol cm−3 , and for isolated cuticular
membranes of six different plant species,20 with an
average of 0.022 mol cm−3 . Crystalline areas form
an excluded volume for diffusion, since the chain
motions of the wax molecules are much more limited
owing to the dense packing of the molecules.4 D0
forms a measure for the tortuous pathway around
the inaccessible crystalline areas and represents a
rough measure for crystallinity of the cuticular wax.
Comparison of cuticular membranes and polymer
matrix membranes revealed tortuosity factors between
200 and 2000,9,14,19 and tortuosity was considered as
the main reason for differences in solute mobilities
between different plant species.20
(12) Since the values of D0 for cuticular wax of C.
album are not significantly different in the absence
and presence of the AEs C12 E3 and Genapol C-
050 (Table 7), the tortuous pathway is not shortened
by the action of AEs, and wax crystallinity remains
unchanged. However, size selectivity of the amorphous
wax fraction decreased from 0.020 mol cm−3 in the
absence of any accelerator to 0.0020 mol cm−3 in the
(13) presence of C12 E3 (Table 7). This corresponds to
an increase in the mean free volume available for
diffusion from 50 up to 500 cm3 mol−1 (Table 7).
Similar effects of AEs were found for reconstituted wax
of H. vulgare22 and for isolated cuticular membranes of
C. aurantium and Pyrus communis L.16 This indicates
that the accelerators exhibited the same mode of action
despite different wax compositions.
The free volume theory expressed by Eqns (12)
and (13) is based on a simple probability
Vf ln D0 D0 × 10−16
(cm3 mol−1 ) (m2 s−1 ) (m2 s−1 ) r2
50.0 −15.72 (±0.41) 1.90 0.98
238 −15.69 (±0.33) 2.04 0.76
500 −15.50 (±0.16) 3.16 0.75
143
M Burghardt et al.

100 180
control C12E3
Genapol C-050 150 5
80
C12E3
120
60
p (%)

effect
90
4
40
60
3
20 30 2
1
Genapol C-050
0 0
0 300 600 900 1200 1500 0 2 4 6 8 10
−1 1/Dcontrol × 1016 (s m−2)
MV (cm3 mol )

Figure 6. The probability (p) calculated according to Eqn (14) that,
adjacent to a diffusing molecule with a certain molar volume (MV), a
hole of proper size allows diffusional movement in the cuticular wax of
Chenopodium album.
calculation.36,45 The size selectivity results from the
exponential distribution of the hole size in the
amorphous wax fraction. The probability (p) that a
compound of a certain molar volume (MV) finds a
free volume (Vf ) in its adjacent environment which is
large enough for a diffusional jump is given by
p = e−MV/Vf (14)
For molecules with large molar volumes the number
of holes of sufficient size is much lower than for
smaller molecules (Fig. 6). An increase in the mean
free volume by the plasticising action of accelerators
raises the probability for a diffusing compound
to find a fitting void. Therefore effects on D of
large compounds are distinctly higher than in the
case of smaller compounds (Tables 5 and 6). With
effective accelerators, size selectivity becomes much
less pronounced (Figs 4 and 5). Therefore good
correlations were observed when plotting the effects
of C12 E3 and Genapol C-050 on diffusion versus
the reciprocal of D measured in the absence of any
accelerator (Fig. 7). Similar observations have been
reported for solute mobilities and effects of C8 E4
in the transport-limiting skin of isolated cuticular
membranes of C. aurantium,21 illustrating again that
similar results are obtained using the model systems
of reconstituted cuticular wax and isolated cuticular
membrane.
4.6 Extended free volume theory
In order to develop a more general model for the
prediction of D of pesticides in cuticular waxes in the
absence and presence of AEs, the free volume theory
has to be extended. Corresponding to the action of
plasticisers in technical polymers,40,42,47 the effect of
AEs on the free volume of cuticular waxes can be
described by
Vf = Vf0 + αcmax (15)
wax
Figure 7. Correlation of the effects of alcohol ethoxylates Genapol
C-50 and C12 E3 on diffusion coefficients (D) of pesticides in cuticular
wax of Chenopodium album with the reciprocal of D measured in the
absence of the accelerators (Dcontrol ). Regression equations: Genapol
C-050, effect = 7.3 × 10−16 × 1/Dcontrol + 2.5 (r 2 = 0.999); C12 E3 ,
effect = 18.5 × 10−16 × 1/Dcontrol + 3.9 (r 2 = 0.998). Compounds:
1 = benzoic acid; 2 = 2, 4-D; 3 = metribuzin; 4 = triadimenol;
5 = bitertanol.
where Vf0 is the mean free volume of the pure wax,
α is a coefficient describing the efficiency of an
AE for increasing the free volume, and cmax
wax is the
maximum concentration of the AE in the wax. Since
sorption of the AEs occurred from micellar solutions,
maximum concentrations in the wax (cmax wax ) can be
used. From Eqns (12) and (13) and the definition of
the accelerator effect, i.e.
effect = Daccelerator /Dcontrol (16)
Eqn (17) is obtained:
MV V0 1
= f max + Vf0 (17)
ln(effect) α cwax
Plotting MV/ln(effect) versus 1/cmax
wax gives a straight
line (Fig. 8). From the slope and the intercept of
the regression equation the values of Vf0 and α can
be calculated (Table 8). The good correlation and
the mean free volume of the pure wax obtained by
this approach, which is not significantly different
from the value obtained by the simple free volume
model (Table 7), are a good indication for the
validity of Eqn (17). Baur et al.15 used an equation
of similar form to describe the self-accelerating effect
of chlorfenvinphos on its own mobility within the
transport-limiting wax barrier of isolated cuticular
membranes of P. communis:
1 B 1 1
= + (18)
ln(effect) AX A

144 Pest Manag Sci 62:137–147 (2006)

 Diffusion of pesticides in cuticular wax

100 -15

90
MV/ln(effect) (cm3 mol−1)

80 -16

log D (m2 s−1)

70
-17
60

50
-18
40

30 -19
0 20 40 60 80 100 120 0 1 2 3 4 5 6 7
−1 MV/(V 0f + ac wax
max
1/c max
wax (kg mol ) )

Figure 8. Plot according to the free volume theory (Eqn (17)) relating
the effects of monodisperse alcohol ethoxylates on the diffusion
coefficients of pesticides to the molar volume of the diffusing
compounds (MV) and the maximum concentration of the alcohol
ethoxylates sorbed in the wax (cmaxwax ) of Chenopodium album. The
intercept of the regression line is equal to the mean free volume of the
wax (Vf0 ). From the slope of the regression line the constant α
describing the contribution of alcohol ethoxylates to the increase in
the mean free volume of the wax can be calculated (r 2 = 0.89).
Table 8. Free volume parameters calculated according to Eqns (17)
and (19) for cuticular waxes of Chenopodium album and Hordeum
vulgare. Vf0 is the mean free volume of the pure wax in the absence of
alcohol ethoxylates (AEs), D0 is the diffusion coefficient of a
hypothetical molecule having a molar volume of zero, and the
constant α describes the concentration-dependent effect of the AEs
on the mean free volume of the wax
Vf0
D0
Wax (cm mol−1 )
3 (m2 s−1 )
Figure 9. Plot according to the free volume theory (Eqn (19)) relating
the diffusion coefficients (D) of the pesticides in Chenopodium album
wax to the molar volume of the diffusing compounds (MV) divided by
Vf0 + αcmax
wax . The intercept of the regression line represents D of a
hypothetical compound having zero molar volume (D0 ). The slope of
the regression line is not significantly different from −1 (r 2 = 0.96).
This is in agreement with the ‘rule of mole fractions’
in the plasticisation of synthetic polymers, which states
that the plasticising effect is independent of size,
chemical nature and structure of the plasticisers and
depends only on the mole fractions in the polymer.48
Data from a previous study using barley wax22 were
also successfully modelled using Eqn (19) (Table 8).
Using the general form of the free volume model
successfully enables the prediction of D of pesticides
α
in the absence and presence of AEs in reconstituted
(cm3 kg−1 mol−2 )

C. album 44.3 3.10 × 10−16 5810 30

H. vulgare 47.0 3.82 × 10−16 5460
Dmeasurement × 10−17 (m2 s −1)

25

This equation is equivalent to Eqn (17) if the constant 20

A is replaced by MV/Vf0 and the constant B is replaced
by MV/α. The simple free volume model (Eqn (12)) 15
can be written in a general form combining Eqns (12)
and (15):
10
D = D0 e−MV/(Vf +αcwax )
0 max
(19)
5
According to Eqn (19), a plot of the logarithm of D
versus MV/(Vf0 + αcmax
wax ) should give a straight line with
0
a slope of −1 and an intercept representing log D0 . For
the diffusion of pesticides in reconstituted cuticular 0 5 10 15 20 25 30
wax of C. album, such a linearity is indeed obtained −17 −1
Dprediction × 10 2
(m s )
(Fig. 9) and the value of D0 obtained by this procedure
(Table 8) is not different from the value obtained by Figure 10. Correlation of experimentally determined diffusion
the simple free volume model (Table 7). coefficients (Dmeasurement ) of pesticides in cuticular wax of
Taking all parameters together, D of pesticides in Chenopodium album versus predicted diffusion coefficients
the absence and presence of AEs can be successfully (Dprediction ) according to the free volume theory described by
Eqn (19). The free volume parameters listed in Table 8 and the
predicted (Fig. 10) using the general form of the
maximum concentrations of alcohol ethoxylates sorbed in the wax
free volume model (Eqn (19)). Modelling of D calculated by Eqn (7) were used. The slope of the regression line is
in reconstituted wax was only possible when the not significantly different from unity and the intercept of the
accelerator concentration was given in molar units. regression line is not significantly different from zero (r 2 = 0.88).

Pest Manag Sci 62:137–147 (2006) 145

M Burghardt et al.
cuticular wax. However, D values of AEs themselves
are only rarely available. So far, only D for C12 E8
has been reported,19,49 and there is good evidence
that both accelerators and pesticides have to penetrate
at the same rate in order to enhance foliar uptake.9
Therefore modelling D of AEs in cuticular waxes
according to the free volume theory will be necessary
in future. This should allow prediction of the best
combinations of accelerators and pesticides in the
formulation of plant protection agents.
ACKNOWLEDGEMENTS
This work was supported by Novartis Crop Protection
AG (Basel) and by the DFG.
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