Journal of Environmental Protection, 2010, 1, 410-419
doi:10.4236/jep.2010.14047 Published Online December 2010 (http://www.SciRP.org/journal/jep)
Kinetic of Transport of Technogenic Ecotoxicants
through Model Membranes
Elena A. Saratovskikh, Boris L. Psikha
Institute of Problems of Chemical Physics, Russian Academy of Sciences, Moscow, Russian.
Received January 19th, 2010; revised July 6th, 2010; accepted July 15th, 2010.
ABSTRACT
The kinetics of ecotoxicant transport through the membrane was studied. The ecotoxicants studied were pecticides
widely used in agriculture: lontrel (clopyralid), sencor (metribuzin), basagran (bentazon), roundup (glyphosate),
kusagard (alloxydim sodium), and sethoxydim, as well as lontrel complexes with cobalt and copper. All compounds
considered penetrate through the model phosphatidylcholine liposomal membranes. The transfer rate was monitored by
fluorescence quenching of
 -ATP inside the liposomes. The mathematical model for the process was proposed, and the
mass transfer rates were calculated. The octanol/water partition constants were determined. All the compounds con-
sidered were shown to accumulate in the fatty layer. The kinetics of their accumulation was studied, and the rates of
accumulation in the nonpolar phase were calculated, which correlate with the complexation constants of the same
compounds. Bioaccumulation of the toxicants is caused by the formation of complexes with the fatty phase (lipid part)
of the cellular membranes. The toxicants under study transferred into the nonpolar phase with a considerable rate dur-
ing the whole observation time, namely, 18 months.
Keywords: Ecotoxicant Pesticides, Liposomal Membranes, Mass Transfer Constants, Lipophilicity, Bioaccumulation,
Octanol/Water Partition (Koct/H2O)
1. Introduction
Technogenic ecotoxicant xenobiotics (foreign substances
for living cells), such as pesticides, are presently pre-
dominant contaminants of the environment [1,2]. They
also can accumulate in biological systems at almost all
trophic levels. In living organisms, the degree of bioac-
cumulation (ratio of the amount of the toxicant in the
tested subject to that in the reference sample) can be very
high. For instance, the accumulation of chloropyriphos in
the Pimephales promelas fish achieves 1.7·103 times and
that of dichlorodiphenyltrichloroethane is 1.3·105 times
[1]. Xenobiotics accumulate mainly in the lipid part of
cellular membranes by binding of the samples with
membrane proteins or lipids [3]. As a result, pesticides
influence on the structure and functions of the mem-
branes and can destruct the membrane systems [4,5].
Among various physicochemical properties of a pesticide
sample, lipophilicity exerts a determining effect on the
character of interaction of the sample with the lipid bi-
layer. Lipophilicity is usually characterized by the oc-
tanol/water partition coefficient (Koct/H2O).
To understand the mechanism of action of xenobiotic
pesticides, one should evaluate their ability to penetrate
inside the cell. Therefore, it is desirable to use for study-
ing pesticide transport through the cellular membrane
such systems and conditions under which the wholeness
of the membranes is retained. In most cases, model sys-
tems, viz., liposomes filled with various substances, are
widely used to investigate the transport of drugs, vita-
mins, hormones, viruses, and antibiotics through the
membranes [6]. In particular, permeability of the lipo-
somal membranes for pesticides and other toxicants of
technogenic origin were studied [7] to form concepts on
hazard of microcontaminants of water for human being.
A series of our studies [8-10] revealed the role of
complexation processes in the mechanism of action of
xenobiotics at the molecular level. It was shown, par-
ticularly, that mono- (in particular, ATP and its deriva-
tive ε-ATP), di-, and polynucleotides form complexes
with the pesticides, resulting in fluorescence energy loss
[8,9]. Therefore, in the present study we used liposomes
containing in the internal volume ε-ATP, whose fluores-

Kinetic of Transport of Technogenic Ecotoxicants through Model Membranes 411
cence quenching reflects pesticide penetration through
the membrane.
The purpose of the present work is to detect and quan-
titatively characterize the transfer rate though the lipo-
somal membrane for a series of pesticides: lontrel, sencor,
basagran, roundup, and sethoxydim, as well as lontrel
complexes with cobalt and copper, and to determine the
partition constants in an octanol/water system and the
rates of transfer to the nonpolar phase and accumulation
in it.
2. Methodology
The names and chemical formulas of the used pesticides
and metal complexes are given in Table 1. The sub-
stances under study were isolated from commercial sam-
ples and purified according to an earlier developed
method [8]. The complexes of the herbicide lontrel with
different metals were synthesized according to a known
procedure [10-15]; the synthesis of -APT was carried
out by a previously proposed procedure [8]. The follow-
ing commercial preparations were used without addi-
tional purification: egg phosphatidylcholine, 10% solu-
tion in ethanol (Serva, Germany), purity at least 98.5%;
cholesterol (analytical purity grade, Reakhim, Russia).
To determine the octanol/water partition constant
(Koct/H2O), a solution of the pesticide under study (10 ml
in the initial concentration) was poured to octyl alcohol
(10 ml). The resulting mixture was shaken for 20 min
and left to settle. The concentrations of the studied pesti-
cide in water and octanol were determined on a Specord
UV-VIS spectrophotometer (Carl Zeiss Jena, Germany).
The initial concentrations, wavelength in the absorption
maximum, and molar absorption coefficients of the sub-
stances used are listed in Table 1. After the first meas-
urement, all mixtures were poured to specially con-
structed sealed vessels that made it possible to take sam-
ples from the upper fatty phase (with a pipette) and lower
aqueous phase (through a valve at the ground section in
the bottom of the vessel). To simulate the bioaccumula-
tion of the ecological toxicants in subjects of the envi-
ronment, the pesticide concentrations in water and oc-
tanol were repeatedly measured (followed by the calcula-
tion of Koct/H2O) in 15 days, 30 days, 3 months, 6, 12, and
18 months. The solutions were kept at room temperature
between measurements.
To prepare multulayer liposomes, a mixture of phos-
phatidylcholine (2 ml) and cholesterol (0.2 ml) (10% so-
lution in chloroform) was dried in an inert gas flow on a
rotary evaporator (Rotavapor EL 131 and Water Bath
Büchi 461, “Büchi” Switzerland). The resulting film was
poured with a buffer (2.5 ml of 0.02 M Tris-НСl buffer,
рН 7.2) and thoroughly stirred for lipid swelling. To ob-
tain monolayer liposomes, a suspension of multulayer
liposomes was ultrasonicated using a described proce-
dure [16] with a UZDN-1 ultrasonic low-frequency dis-
perser (USSR) at 22 kHz and 0.4 A for 20 min in the
presence of -ATP (in 0.02 mol·l-1 Tris-НСl buffer, рН
7.2) during 30 s followed by cooling for 60 s at 0-5℃ in
an argon atmosphere. The free label was separated from
the encapsulated label by gel filtration on columns filled
with the Sephadex G-50 sorbent (Pharmacia, Sweden)
controlling by a UNICOD Type4701A flow-type den-
simeter (LKB, Sweden). The size of the obtained lipo-
somes (60-100 nm) was determined with a JEM-100B
electron microscope (Japan) described earlier [16].
Etheno derivative of adenosinetriphosphoric acid with
the excitation spectrum at λ = 312 nm and fluorescence
maximum at λ = 420 nm was used as the fluorescent la-
bel [17]. The choice of ε-ATP as a label was caused by
the fact that its quantum yield of fluorescence is one of
the highest yields among other etheno derivatives of nu-
cleotides, being 0.59 in the 0.025 mol·l-1 phosphate
buffer [18] at pH 7.0. The concentration of ε-ATP inside
liposomes was determined on a Specord M-40 spectro-
photometer (Jena, Germany) by the band at λ = 275 nm
with the molar absorption coefficient [18] ε = 6·103. In
all experiments, the initial concentration of -ATP inside
the liposomes was 10-4 mol·l-1. After the experiment, a
solution of the liposomes was subjected to gel filtration
on a column packed with the Sephadex G-50 sorbent,
after which the concentration of free -ATP inside the
liposomes was determined again.
Fluorescence was measured on an Aminco-Bowman
spectrofluorimeter (USA) in a standard cell (1 × 1 cm) in
a liposome suspension (2.7 ml) in the 0.02 mol·l-1 Tris-
HCl buffer, pH 7.2. In all entries, the initial fluorescence
intensity I0 = 6.5 ± 0.5 was determined only by the con-
centration of -ATP and the volume of the liposome
suspension added to the working cell and remained ap-
proximately the same in all entries. Experiments were
carried out by adding the pesticides to the cell with the
liposome suspension to a volume of 3 ml with the final
pesticide concentrations 10-2, 10-3, and 10-4 mol·l-1. The
change in the fluorescence intensity was detected during
19-21 h. The fluorescence intensity changes weakly dur-
ing the first 5-10 min after the pesticide was added to the
cell containing a liposome solution. This fact indicates
that the wholeness of the liposomal membranes was not
perturbed and no ε-ATP molecules escaped from the li-
posomes to the external solution.
To be sure that the pesticides penetrate inside the li-
posomes and ε-ATP does not leak out, we carried out
control measurements with the copper and cobalt com-
plexes. In these experiments, 20-22 h after the beginning
of measurements, the suspension of the liposomes was

Kinetic of Transport of Technogenic Ecotoxicants through Model Membranes 412
Table 1. Names, initial concentrations, absorption wavelength maxima, and chemical formulas for the compounds used.
№п/п
Pesticide name:
1. industrial;
2. nomenclature of acting substance
Сinit, M λ, nm CHEMICAL FORMULA
1 Lontrel (Clopyralid) 3,6-dichloropicolinic acid 1·10-2 283
N
Cl
Cl COOH
2. Sencor (Metribuzin)
4-amino-6-tert-butyl-3-methylthio-1,2,4-triazinone-5 1·10-2 292
NN
NNH2
O
SCH 3
(CH 3)3C
3. Basagran (Bentazon)
3-isopropylbenzo-2,1,3-thiadiazinone-4-dioxide-2,2 1·10-3 332 NCH(CH3)2
SO2
N
H
O
4. Roundup (Glyphosate)
N-phosphonomethylglycine 1·10-2 212 (OH)2POCH2NHCH2COOH
5. Kusagard (Sodium salt alloxydime) 2.4·10-2 289
_
O
C
H
3
CH
3
H
3
C
C
O
O
O
C
CH
2
CH
2
CH
3
NO
H
CH
2
CH CH
2
Na
+
6.
Sethoxydim
2-1-(ethoxyimino)butyl-5-2-(ethylthio)propyl-3-h ydro xy-
2-cyclohexen-1-odine
1·10-3 286 C
OH
H7
3
C
NOC2H5
O
2
HCHC
3
HC
S
5
H
2
C
7. Lontrel metal complexes: Cu(lontrel)2; Cо(lontrel)2 1·10-2 281 N
C
O
Me t
O
O
N
C
O
C
L
CL
CL
CL
repeatedly passed through the separation column (Sepha-
dex G-50) and elemental analysis of the separated lipo-
some suspension was carried out to determine the content
of metals (cobalt and copper).
In each series of experiments, three pesticide concen-
trations were used, and reproducibility of data in each
entry was checked for three independently prepared
samples of the liposomes.
3. Results and Discussion
3.1. Determination of Wholeness of the
Liposomes
The results obtained in preliminary experiments showed
that the cobalt content inside the liposomes corresponds
to ~5·10-5 mol·l-1 Co(lontrel)2-ATP and the copper con-
tent corresponds to ~6.2·10-5 mol·l-1 Cu(lontrel)2-ATP,
which agrees well with the experimental data and predic-
tions of the model on the content of the reaction products.
Thus, it was experimentally shown for these two cases
that quenching occurs directly inside the liposomes. An
additional confirmation that the wholeness of the lipo-
somes was retained was obtained in a series of experi-
ments, when 20 h after the beginning of measurement of
the quenching kinetics, the pesiticide was additionally
added in a high concentration (their concentration in the
suspension increased by 10 times). In these entries, no
noticeable changes in the fluorescence intensity and its
quenching rate occurred.
3.2. Mass Transfer Rate
The studied process consists of two stages: 1) penetration
of pesticide through the liposome membrane; 2) interac-
tion of pesticide with -ATP. Earlier it was shown by us
[8] that the interaction of pesticides with -ATP occurs
instantaneously and is characterized by a complexation
constant (Кc/form). Therefore the transport of pesticide
through a membrane is the limiting stage and can be

Kinetic of Transport of Technogenic Ecotoxicants through Model Membranes 413
evaluated from the -ATP fluorescence quenching rate
inside liposomes. The -ATP concentration inside lipo-
somes is not stationary during experiment, but decreases
in time due to complexation of -ATP with pesticide.
The results of measurements of the relative fluores-
cence intensity (I(t)/I(0)) in time for all the substances
studied at three initial pesticide concentrations are pre-
sented in Figures 1 and 2. In all entries, the -ATP fluo-
rescence was quenched within a rather prolonged time
(20 h) during which the fluorescence intensity decreased
to 70-60% of the initial value.
We considered the following model of the studied
process for the quantitative analysis of the experimental
results. The pesticide molecules (P) from the cell with
the volume V1 penetrate in the internal volume of the
liposomes (V2) with the rate j(t) and enter into the equi-
librium reaction with the -ATP molecules (E) to form
product П, which is incapable of fluorescing
K
PE П
where K is the equilibrium constant of the formation of
product П.
The system of kinetic equations describing the change
in the reactant concentrations inside the liposomes takes
the form






dP Пjt
dt
 (1)






KP EП  (2)




0
ПEE (3)


  
112 00
PVP ПVPV   (4)
Here E0 is the initial concentration of
 -ATP inside the
liposomes, [P1] is the pesticide concentration in the cell
outside the liposomes, and P0 and V0 are the concentra-
tion and volume of the pesticide solution introduced into
the cell before the beginning of experiment, respectively.
Equation (1) with allowance for Equations (2) and (3)
can be rewritten with respect to the concentration [E] in
the form




2
0
2
KE EdEjt
dt
KE


 (5)
Since the  -ATP concentration inside the liposomes [E]
is related to the experimentally measured fluorescence
intensity I(t) through the simple relation




00EEItI , (6)
Equation (5) can be used for the calculation of the
transfer rate of the pesticide through the lipid membrane
j(t). To calculate the transport rate of the pesticide inside
the liposomes, we used the earlier obtained by us [8]
02004006008001000 120014001600
0.0
0.2
0.4
0.6
0.8
1.0
5
2
- 6
- 5
- 4
- 3
- 2
- 1
I(t)/I(0)
Time, min
Figure 1. Change in the fluorescence intensity of ε-ATP
encapsulated into the liposomes after the addition of lontrel
(1-3) and Co(lontrel)2 complex (4-6). Concentration of
ε-ATP is 1·10-4 mol/l. Concentration of the pesticides in the
cell: 1·10-4 (1, 4); 1·10-3 (2, 5); 1·10-2 (3, 6) mol/l. Here and in
Figure 2 the initial fluorescence intensity remained appro-
ximately the same and equal to I0 = 6.5 ± 0.5. Solid lines (2,
5) are theoretical curves plotted by Equation (5) with the
parameters given in Table 2.
0200 400 600 800100012001400
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1
2
3
4
5
6
I(t)/I(o)
Time, min
Figure 2. The same as in Figure 1, for the pesticide concen-
tration 1·10-3 mol/l. Points indicate the experimental data
with various pesticides: 1, basagran; 2, sencor; 3, kusagard;
4, roundup; 5, sethoxydim; 6, copper complex of lontrel
herbicide (Cu(L)2). Solid lines are theoretical curves plotted
by Equation (5) with the parameters given in Table 2.
formation constants of the  -ATP–pesticide complexes
presented in Table 2.
The expression for the rate of pesticide penetration
from the external volume inside the liposomes j(t) de-
pends on the mechanism of pesticide transfer through the

Kinetic of Transport of Technogenic Ecotoxicants through Model Membranes 414
Table 2. Octanol/water partition constants, mass transfer rate constants, and complexation constants of the pesticides and
metal complexes.
Pesticide concentration
10-2 M 10-3 M 10-4 M Substance
J·108, M·s-1
Average value J·108, М·s-1 Кc/form·10-3, with -ATP according to [7]
1. sencor 2.1 7.6 2.8 4.2  1.7 26.5  3.3
2. lontrel 4.1 5.2 2.8 4.1  0.7 15.9  2.0
3. kusagard 14.7 14.7 14.5 14.6  0.1 9.7  0.5
4. roundup 10.9 13.8 9.7 11.5  1.2 8.2  1.2
5. sethoxydim 40.5 41.2 39.6 40.4  0.5 5.0  0.3
6. basagran 5.9 6.1 5.1 5.7  0.3 4.7  0.4
7. Cu(lontrel)2 18.0 15.6 21.2 18.3  1.6 851  82
8. Co(lontrel)2 7.0 6.2 8.0 7.1  0.5 600  200
lipid membrane. At first we considered the most probable
mechanism of simple diffusion for which the rate of pes-
ticide transfer is proportional to the difference of its con-
centrations outside [P1] and inside [P] the liposomes:
j(t) =  ([P1] – [P])
where β is the mass transfer coefficient or membrane
permeability [11]. The  parameter quantitatively char-
acterizes the substance transfer through the membrane,
and the task was to determine the  value from the ex-
perimentally measured dependence of the ε-ATP con-
centration [E]exp on time t. The problem was solved as
follows. In each entry, the  value that minimizes the
functional






2
exp
1
n
calc
ii
i
ФEtE t 



where n is the number of points in the entry and [E]calc(ti)
is the ε-ATP concentration inside the liposomes at the
time moment ti calculated from the system of Equations
(1)-(4) at the given  value, was determined from the
experimental kinetic curve [E]exp(t) using a computer. It
turned out, however, that the  mass transfer coefficient
value depends strongly on the initial pesticide concentra-
tion P0: for each pesticide in three entries at the initial
pesticide concentrations 10-4, 10-3, and 10-2 mol we ob-
tained three  values, which differed from each other by
~10 and ~100 times (inversely proportional to the initial
concentrations).
This unexpected result is understandable from the
formal point of view. As can be seen from the data
shown in Figure 1, the experimentally observed quench-
ing rate (and hence, the rate of pesticide penetration in-
side the liposomes) is virtually independent of the initial
pesticide concentration. Taking into account the experi-
mental error, the relative fluorescence is independent of
the initial quencher concentration in entries with both
lontrel (group of experimental points 1-3) and its com-
plex with cobalt Co(lontrel)2 (group of experimental
points 4-6), as well as for all other toxicants and metal
complexes studied in this work. Thus, the assumption
that the pesticide is transferred through the liposomal
membrane via the mechanism j(t) =  ([P1] – [P]) contra-
dicts the experimental data.
Based on the observed specific feature of the experi-
ment and taking into account the results of calculations,
we repeated experimental data processing assuming that
the transfer rate of the pesticide inside the liposomes is
independent of the gradient of its concentrations, accept-
ing formally that j(t) =  , i.e., j = const. The results of
calculation are presented in Table 2: the j values ob-
tained at different initial concentrations of each pesticide
are given in columns 4-6, and their arithmetical mean is
given in column 7. It can be seen from the data in Table
2 that the j values calculated at different concentrations
of each pesticide do not already depend explicitly on
these concentrations. This makes it possible to average
the obtained j values and characterize each pesticide by
the average value of the transfer rate through the lipid
membrane.
The theoretical quenching curves were calculated by
the determined j values and compared with the experi-
mental ones. As follows from the data in Figures 1-2, the
calculation performed using Formula (5) describes satis-
factorily the available experimental data.
Let us analyze the data presented in Table 2. Seth-
oxydim and kusagard exhibited the highest rate. Both
these compounds have complex structure and bear large
organic fatty fragments. Roundup follows kusagard, and

Kinetic of Transport of Technogenic Ecotoxicants through Model Membranes 415
the transfer rates of basagran and lontrel are close. The
series considered is closed by sencor, which is the most
reactive complexing agent according to [8]. Among the
considered compounds, the maximum penetrating ability
was manifested by ketones, cyclohexanone derivatives,
and then derivatives of arylphosphoric acid, pyridine, and
triazine. The pesticides can be arranged in the following
order by the mass transfer rate: sencor  lontrel  basa-
gran  roundup  kusagard  sethoxydim. The mass tran-
sfer rate of the Cu(lontrel)2 complex is higher than that of
Co(lontrel)2.
Since the result obtained (transfer rate of the pesticides
through the lipid membrane is independent of their con-
centration) differs from the commonly accepted concepts
on the diffusion mechanism of the process, we attempted
to reveal reasons for the observed phenomenon. First, we
checked whether the phenomenon is related to the solu-
bility of the studied pesticides in the fatty phase or not.
For this purpose, we measured the octanol/water partition
coefficients Koct/H2O (Table 3, column 3). Sencor and
basagran manifest the highest ability to be dissolved in
an organic solvent. This ability indirectly reflects lipo-
philicity of chemical compounds. According to the lipo-
philicity values, the pesticides studied can be arranged in
the following order: sencor > basagran > sethoxydim >
lontrel > roundup > kusagard. As can be seen, this se-
quence differs from the above series for the mass transfer
rate.
3.3. Schemes of Interact with Phospholipid
Molecules
At the same time, we have shown previously that the
pesticides considered can react with organic compounds
to form complexes characterized by high stability con-
stants [8,9]. Some relation between the transfer rates J
and complexation rate constants (Кc/form) of the pesticides
with functional structures of the living cell, viz., ATP,
NADH, DNA, and RNA, is observed. This is exempli-
fied in Table 2 by the Кc/form constants of the pesticides
with -ATP. It can be seen that the compounds (except
for basagran that possesses intrinsic fluorescence and the
lontrel metal complexes that have their own sequence of
biological activity) with high Кc/form values are character-
ized by the low penetration rate through the cell mem-
branes, i.e., the mass transfer rate constants have an in-
verse relation to the complexation constants with -ATP:
their ability to form complexes [8,9]. The presence of
such a relation allows us to assume that the transfer of
the pesticides through the lipid membrane is not a purely
diffusional process but is limited by the chemical interac-
tion with some elements of the lipid membrane analo-
gously to the phenomenon of negative active diffusion
[12].
It was shown by NMR, 1H NMR, and introduction of
fluorescent labels that the pesticides are entrapped into
the hydrocarbon part of the lipid bilayer. It was also as-
sumed in several studies that the pesticide is arranged
parallel to the hydrocarbon chains and thus can form a 1:
1 complex with phosphatidylcholine [3,13,14].
In the case of lontrel (3,6-DCPA), whose acidity is
caused by the chlorine atoms, the formation of a complex
with lipids in the model liposomal or cellular membrane
can occur with covalent bonding upon the elimination of
water and HCl, as shown in Scheme 1(b).
The orientation of the 3,6-DCPA molecule parallel to
the phosphatidylcholine molecule is possible due to the
electronegative oxygen atom of the phosphate group of
lecithin and the N atom of the pyridine ring bearing a
partial positive charge (δ+), as well as due to the electro-
static interaction of the chlorine atom of 3,6-DCPA and
positively charged N atom of phosphatidylcholine.
The chemical structure of a phosphatidylcholine mole-
cule is shown in Scheme 1(a). The structure of a possible
3,6-DCPA complex with two phosphatidylcholine mole-
cules is presented in Scheme 1(c).
A sencor molecule can also interact with one or two
phospholipid molecules. As shown in Scheme 2(a), co-
valent bonds can be formed due to both the highly reac-
tive sulfur atom and lone electron pairs of the nitrogen-
atom of the heterocycle. In this case, a stable five-mem-
Table 3. Octanol/water partition constants, bioaccumulation coefficients, and accumulation rates of the pesticides in octanol.
Names
of substances
Koct/H2O
20 min
Koct/H2O
18 months BC V1·10-3, M·h-1 20 min
V2·10-8, M·h-1
6 months
V3·10-8, M·h-1
18 months Кc/form·10-3, M-1
sencor 66.57 ± 2.0 100.4 ± 3.0 0.99 ± 0.01 32.84 0.37 0.37 26.5 ± 3.3
lontrel 2.56 ± 0.1 3.38 ± 0.1 0.77 ± 0.03 23.97 4.06 4.06 15.9 ± 2.0
kusagard 0.30 ± 0.01 0.77 ± 0.03 0.44 ± 0.01 18.47 46.10 32.60 9.7 ± 0.5
roundup 0.46 ± 0.014 2.27 ± 0.07 0.69 ± 0.02 10.50 42.67 18.90 8.2 ± 1.2
sethoxydim 7.83 ± 0.23 25.00 ± 0.8 0.96 ± 0.03 2.96 0.58 0.58 5.0 ± 0.3
basagran 52.19 ± 1.8 70.4 ± 2.12 0.99 ± 0.01 3.27 0.04 0.04 4.7 ± 0.4

Kinetic of Transport of Technogenic Ecotoxicants through Model Membranes 416
2 OCH
CH 2
CH 2 OR
3
POCH 2 CH 2 N(CH 3 ) 3
-
O+
OR
1
R
O
a
(a)
b
N
CN CH 3
O
O-
PO C
H
H
H
H
CH
3
CH 3
+
Cl C
HO
O
Cl
+
-
-
-
HCl
H
2
O
-
P OCCNCH
Cl
N
C
O
3
CH
3
CH
3
H
H
O
O
-
(b)
P O
O
O
CCNCH 2
CH NCCOP
N
Cl C
Cl
O
3
H
H
H
H
CH
3
CH
3
CH
3
H
H
H
H
CH
3
O
O
(c)
Scheme 1. Structure of a phosphatidylcholine molecule (a)
and possible lontrel complexes with one (b) and two (c)
phospholipid molecules.
bered ring can be formed between sencor and phosphol-
ipid.
The oxygen of the free keto group releases its lone
electron pair to form the ether bond with the second
phospholipid molecule, as if hanging by the ends of the
fatty acids (see Scheme 2(b)). Evidently, the reactivity
possibilities of the sencor molecule are not exhausted by
the both types of interactions. The enhanced electron
density on the nitrogen atoms of the sencor heterocycle
can allow sencor to react with one more (third) or even
two (third and fourth) phosphatidylcholine molecules.
The roundup molecule has three hypothetical addition
sites: the terminal hydroxy groups and carboxy group, as
well as the nitrogen atom localized in the middle of its
linear structure. Naturally, this molecule can readily re-
alize its possibilities reacting with one or two phosphate-
dylcholine molecules (Scheme 3).
The analysis of the pesticides used in the work from
the viewpoint of reactive chemical substituents in their
structure prone to covalent bonding with phosphatidyl-
choline suggests that this affinity decreases in the series:
lontrel > sencor > lontrel metal complexes > roundup >
kusagard > sethoxydim > tachigaren (hymexazol) >
basagran. This order agrees with the experimental data
on the octanol/water partition constants and, naturally, is
inversed to the order of mass transfer rates through the
POC CNCH
3
O
O
HH
HH
CH
3
CH
3
C
O
NH
H
SCH
3
N
N
N
CH
3
H
3
C
H
3
C
NH
3 -
-
CH
4
PO
O
O
CCN(CH
3
)
3
HH
S
(CH
3
)
3
CNN
N
O
a
(a)
POC CN
O
O
HH(CH
3
)
3
N
CH
3
S
C(CH
3
)
3
N
N
N
OC
CH
3
H
CCO P
O
O
H
2
H
2
b
(b)
Scheme 2. Scheme of possible complex formation of sencor
with one (a) and two (b) phospholipid molecules.
(CH3)3
H2
POC CN
O
O
HH
POCNC C
N CC O P
O
H2
C
H2
(CH3)2
H2H2
O
O
Scheme 3. Scheme of possible complex formation of round-
up with two phosphatidylcholine molecules.
liposomal membranes.
3.4. Lipophilicity
More detailed elucidation of the mechanism of interac-
tion between the studied substances and the lipid bilayer
requires additional investigation. However, it is evident
that the formation of complexes of the toxicants with the
nonpolar phase of cellular membranes in vivo results in
their accumulation inside the membrane. To determine
the rate of this process, we determined the concentrations
of the substances in octanol and water during a very pro-
longed time, namely, 18 months.
The octanol/water partition constants (Koct/H2O), de-
termined 20 min after mixing of an aqueous solution of
the pesticide with octyl alcohol and in 18 months at the
end of observation, are presented in Table 3. Sencor and
basagran manifest the highest lipophilicity (ability to be
dissolved, to a great extent, in the fatty phase octanol).
The process considered in the present work, namely,
transfer of the pesticides from water to octanol as the
nonpolar phase, was characterized in time by several
steps with different transfer rates. The kinetic data of
pesticide accumulation in n-octanol are shown in Figure
3. All the compounds considered exhibit pronounced two
(and three for some compounds) regions with different
rates of the process. During the first 20 min the relative
rate of the concentration change for all compounds is

Kinetic of Transport of Technogenic Ecotoxicants through Model Membranes 417
Figure 3. Kinetic curves of pesticide accumulation in oc-
tanol. 1 (○) – lontrel; 2 (□ ) – sencor; 3 (▼) – basagran; 4 (X)
– roundup; 5 (∆) – kusagard; 6 (■) – sethoxydim. a – from 0
to 20 min; b – from 20 min to 18 months.
maximum: about 98% sencor and basagran and 90%
sethoxydim transfer from the aqueous phase to n-octanol.
In the next time region from 20 min to 6 months, the rate
of pesticide transfer from water to the nonpolar phase
decreases substantially and the process approaches to
saturation. The kinetic curves of sethoxydim and round-
up have the third break after 6 months when the diffusion
rate decreases once more (more than twofold for round-
up).
During one year of measurements, 5% lontrel are line-
arly transformed into n-octanol. Up to 25% kusagard and
30% roundup are accumulated in the nonpolar phase
during 18 months. However, for all the considered com-
pounds, their accumulation in the nonpolar phase does
not reach the equilibrium stationary state even after 18
months, and the transfer of the substances from an aque-
ous solution to octanol evidently continues further.
The transfer rates of the substances to the nonpolar
phase in all the three regions are collected in Table 3.
According to the accumulation rates of the pesticides in
octanol (V, mol/h) in the first region (V1), all the com-
pounds considered can be arranged in the following order:
sencor  lontrel  kusagard  roundup  basargan 
sethoxydim.
3.5. Bioaccumulation
To model the bioaccumulation process, the measure-
ments of Koct/H2O were repeated in 15 and 30 days, 3, 6,
12, and 18 months. The ratio of the maximum pesticide
concentration in octanol to the initial concentration in an
aqueous solution was designed as the bioaccumulation
coefficient (BC):




18oct init
BC CC.
The calculated BC values are given in Table 3. The
minimum BC value equal to 0.44 was obtained for kusa-
gard, and the maximum BC value (0.99) was obtained for
sencor and basagran. The sequence of the substances by
decreasing BC is as follows: sencor = basagran  seth-
oxydim  lontrel  roundup  kusagard. This sequence
corresponds to the order of these substances by their
lipophilicity values (or octanol/water partition constants
Koct/H2O) and remains unchanged for the all compounds
considered during the whole observation period.
The process of toxicant transfer from water to the fatty
phase has no upper concentration limit, i.e., does not
reach the equilibrium state of the toxicant concentrations
in the polar and nonpolar phases but continues to transfer
to the nonpolar phase continuously during a very pro-
longed time (in our specific case, during the whole ob-
servation time of 18 months).
Published data are available only for three of the
compounds studied by us, namely, basagran, sencor, and
roundup. The toxicity values of these substances with
respect to various types of multicellular organisms ob-
tained for the “accumulation” parameter are listed in Ta-
ble 4. First, it is noteworthy that the same toxic dose of
basagran is accumulated in organisms of different types:
in Crustacea, water insects, animal plankton, or algae.
The toxic effect occurs when this compound is accumu-
lated in an amount of 86·10-6 g/l (0.358·10-6 mol/l).
Not only one basagran was checked for fishes and al-
gae. As can be seen from the data in Table 4, with re-
spect to algae sencor accumulates the concentration toxic
for Chlorella fusca (51·10-6 g/l, 0.238·10-6 mol/l) for 24 h,
whereas basagran accumulates its (somewhat higher)
toxicity dose for only 30 days. Sencor and roundup were
studied for different fish species. Sencor was studied for
Leuciscus idus melanotus, and roundup was examined
for Cyprinus carpio. Nevertheless, when comparing for
one period of action (3 days), the accumulated toxic dose
of roundup is by 6 (in g/ml) or 10 (in mol/l) times higher
than that for sencor. Thus, the toxicity of these three
compounds by the “accumulation” parameter generally
decreases in the sequence: sencor > basagran > roundup.
The comparison of this series with the data in Table 3
shows that the Koct/H2O values decrease in the same order.
Therefore, the above series can hypothetically be ex-
trapolated if assuming that the accumulation value of the
studied compounds decreases in the series of decreasing
their Koct/H2O: sencor > basagran > sethoxydim > lontrel >
roundup > kusagard.
4. Conclusions
In our opinion, it is important that the rate of penetration
of the metal complexes through the biological membrane
is higher than that for the initial pesticide (for instance,

Kinetic of Transport of Technogenic Ecotoxicants through Model Membranes 418
Table 4. Toxicity doses for basagran, sencor, and roundup accumulated in various organisms.
Toxic dose, 10-6
Substance Common name Scientific name Living stageTime
g/l mol/l
Reference
Crustacea
basagran Fiddler crab Uca minax not indic. 11 days 86 0.358 [19]
Water insects
basagran Southern house mosquito Culex quinquefasciatus not indic. 30 days 86 0.358 [19]
Animal plankton
basagran Water flea Daphnia magna 30 days 86 0.358 [19]
Algae
sencor Green algae Chlorella fusca 24 h 51 0.238 [20]
Chlorella fusca vacuolata 24 h 50 0.233 [21]
basagran Algae, algal mat Algae 30 days 86 0.358 [19]
Fishes
sencor Carp Leuciscus idus melanotus5-6 cm, 1.5 g3 days 54 0.225 [21]
Ide, silver or golden orfe Leuciscus idus 2-5 g 3 days 54 0.225 [20]
roundup Cyprinus carpio Cyprinus carpio 3.5-4.0 cm 0.5 day 531 3.14 [22]
Cyprinus carpio 3.5-4.0 cm 1 day 508 3.004 -“”-
Cyprinus carpio 3.5-4.0 cm 2 days 518 3.063 -“”-
Cyprinus carpio 3.5-4.0 cm 3 days 344 2.034 -“”-
Cyprinus carpio 3.5-4.0 cm 5 days 264 1.561 -“”-
Cyprinus carpio 3.5-4.0 cm 7 days 110 0.651 -“”-
Cyprinus carpio 3.5-4.0 cm 14 days 151 0.893 -“”-
lontrel) (Table 2). We have earlier [10,15] found a pos-
sibility of easy formation of the lontrel complexes with
metals in environmental subjects and their high stability
in the non-dissociated form in aqueous solutions. It is
also shown that thus formed complexes of various metals
are highly reactive as well, and their reactivity is compa-
rable with that of the initial pesticide or even higher [8,9].
The data obtained in the present study on the mass trans-
fer rates of the copper and cobalt complexes of the lon-
trel herbicide revealed that the transfer rate of the com
plexes is much higher than that for the initial pesticide:
for Co(lontrel)2 by three times, and for Cu(lontrel)2 by
almost 10 times. It can be assumed that ecological danger
from the toxic action of the studied environmental con-
taminants increases when they are entrapped into cells in
the metal-bound state.
When studying the step of pesticide penetration inside
the liposomes, one can assume that fluorescence is
quenched due to the escape of -ATP from the liposomes.
However, the performed measurements of the concentra-
tions of the copper and cobalt complexes and the pres-
ence of residual amounts of -ATP inside the liposomes
after 20-24 h of measurements prove that all the com
pounds considered penetrate through the model lipo-
somal membranes without their complete destruction.
The flow of a xenobiotic through the membrane is inde
pendent of the initial toxicant concentration and is de-
termined, to a great extent, by its chemical structure. The
mass transfer constants of the compounds vary in a wide
range from 2·10-8 to 40·10-8 mol·l-1 s
-1. The toxicants
studied by us can conventionally be classified as 1)
highly reactive but with a low membrane permeability
(sencor and lontrel), 2) poorly reactive but with a high
permeability of the membranes (basagran, sethoxydim),
and 3) “resonance,” i.e., drastically enhancing their toxi-
cological properties due to the chemical interaction with
other environmental contaminants, in particular, with the
formation of metal complexes.

Kinetic of Transport of Technogenic Ecotoxicants through Model Membranes 419
REFERENCES
[1] A. V. Yablokov, “Poisonous Dressing,” Mysl., Moscow,
1990, p. 27. (in Russian)
[2] E. A. Saratovskikh, N. B. Kozlova, V. G. Papin and E. V.
Shtamm, “Decomposition of Herbicide Lontrel by Bio-
logical and Photochemical Methods,” Applied Biochem-
istry and Microbiology, Vol. 42, No. 1, 2006, pp. 38-44.
(in Russian)
[3] V. N. Golubev, “Mechanisms of Interaction of Pesticides
with the Lipid Bilayer of Cellular Membranes,” Russian
Chemical Reviews, Vol. 62, No. 7, 1993, pp. 726-734. (in
Russian)
[4] B. T. Bowman and W. W. Sans, “Determination of Oc-
tanol-Water Partitioning Coefficient (Kow) of 61 Organo-
phosphorus and Carbamate Insecticides and Their Rela-
tionship to Respective Water Solubility (S) Values,”
Journal of Environmental Science and Health, Vol. B18,
No. 6, 1983, pp. 667-683.
[5] H. Miyoshi, T. Nishioka and T. Fujita, “Quantitative
Analysis of Effect of Substituted Phenols on Membrane
Characteristics of Lecithin Liposomes,” Bulletin of the
Chemical Society of Japan, Vol. 5, 1986, pp. 1099-1107.
[6] G. Gregoriadis and A. M. Allison (Eds.), “Liposomes in
Biological Systems,” Meditsina, Moscow, 1983. (in Rus-
sian)
[7] Y. Utsuvi, K. Kiyoshige, S. Shimbara and A. Hamada,
“Comparative Studies on Cytotoxicity of Micropollutants
in Water – Principle of Cytotoxicity Matrix,” Journal
Seek Entry for Environmental Toxicology and Water
Quality, Vol. 9, No. 4, 1994, pp. 333-339.
[8] E. A. Saratovskikh, T. A. Kondrat’eva, B. L. Psikha, R. I.
Gvozdev and V. G. Kartsev, “Complex-Formation of
Some Pesticides with Adenosine Triphosphoric Acid,”
The Bulletin of the Russian Academy of the Sciences. Di-
vision of Chemical Science, Vol. 37, 1988, pp. 2252-
2258.
[9] E. A. Saratovskikh, M. V. Lichina, B. L. Psikha and R. I.
Gvozdev, “Character of the Reaction of Dinucleotides
and Polynucleotides with Some Pesticides,” The Bulletin
of the Russian Academy of the Sciences, Division of
Chemical Science, Vol. 38, 1989, pp. 1822-1827.
[10] Z. G. Aliev, L. O. Atovmyan, E. A. Saratovskikh, V. I.
Krinichnyi and V. G. Kartsev, “Synthesis, Structure, and
Spectral Characteristics of Copper-Complexes with Pi-
Colinic-Acid Derivatives,” The Bulletin of the Russian
Academy of the Sciences. Division of Chemical Science,
Vol. 37, 1988, pp. 2246-2252.
[11] Kotyk and K. Yanachek, “Membrane Transport,” Mir.
Moscow, 1980. (in Russian)
[12] D. A. Frank-Kamenetskii, “Diffusion and Heat Transfer
in Chemical Kinetics,” Nauka, Moscow, 1987, pp. 103-
104. (in Russian)
[13] A. Leo, “Partitioning in Pesticide Mode of Action and
Environmental Problems,” In: P. S. Magee, G. K. Kohn
and J. J. Menn, Eds., Pesticide Synthesis through Rational
Approaches, American Chemical Society, Washington,
1984, pp. 213-218.
[14] W. M. Arold, U. Zimmerman, W. Pauli, M. Benzing, C.
Niehns and J. Ahlers, “The Comparative Influence of
Substituted Phenols (Especially Chlorophenols) on Yeast
Cells Assayed by Electro-Rotation and Other Methods,”
Journal of Biochemica et Biophysica Acta, Vol. 942, No.
1, 1988, pp. 83-95.
[15] E. A. Saratovskikh, “Synthesis of Bidentate Complexes
of 3,6-dichloropicolinic Acid,” The Bulletin of the Rus-
sian Academy of the Sciences. Division of Chemical Sci-
ence, Vol. 38, 1989, pp. 2140-2141.
[16] M. V. Genkin, B. P. Ulanov, O. E. Dotsenko and R. M.
Davydov, “Specific Features of Spectra of Liposome
Turbidity,” Russian Journal of Physics Chemistry, Vol.
61, No. 1, 1987, pp. 220-224. (in Russian)
[17] T. A. Kondrat’eva, R. I. Gvozdev and L. V. Tat’yanenko,
“Spectral Properties of 8-Bromoetheno Derivatives of
Adenine Nucleotides,” Chemistry of heterocyclic Com-
pounds, No. 7, 1983, pp. 987-992. (in Russian)
[18] J. R. Barrio, J. A. Secrist, N. J. Leonard and G. Weber,
“Fluorescent Modification of Adenosine-Containing Co-
enzymes. Biological Activities and Spectroscopic Proper-
ties,” Biochemistry, Vol. 19, No. 19, 1972, pp. 3499-
3506.
[19] G. M. Booth, C. C. Yu and D. J. Hansen, “Fate, Metabo-
lism, and Toxicity of 3-Isopro-Pyl-1H-2,1,3-Benzo-
Thiadiazin-4(3H)-1-2,2-Dioxide in a Model Ecosystem,”
Journal of Environmental Quality, Vol. 2, No. 3, 1973, pp.
408-411.
[20] F. Korte, D. Freitag, H. Geyer, W. Klein, A. G. Kraus and
E. A. Lahaniatis, “A Concept for Establishing Ecotoxi-
cologic Priority Lists for Chemicals,” Chemosphere, Vol.
7, No. 1, 1978, pp. 79-102.
[21] D. Freitag, H. Geyer, A. Kraus, R. Viswanathan, D.
Kotzias, A. Attar, W. Klein and F. Korte, “Ecotoxi-Co-
logical Profile Analysis VII. Screening Chemicals for
Their Environmental Behavior by Comparative Evalua-
tion,” Ecotoxicology and Environmental Safety, Vol. 6,
1982, pp. 60-81.
[22] Y. S. Wang, C. G. Jaw and Y. L. Chen, “Accumulation of
2,4-D and Glyphosate in Fish and Water Hyacinth,” Wa-
ter, Air and Soil Pollution, Vol. 74, No. 3-4, 1994, pp.
397-403.