Sorption and diffusion of 14C-atrazine through isolated plant cuticles

Chemosphere, Vol. 33, No. 6. pp. 995-1003,

Pergamon

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PII: SOO45-6535(96)00241-X

1996 Elsevier Britain.

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SORPTION AND DIFFUSION OF 14C-ATRAZINE THROUGH

ISOLATED

Saence

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PLANT

A. Charnel* and N. Vitton Laboratoire Transferts dans les Systemes VegCtaux DBMS, CEA/Grenoble. 17 rue des Martyrs 38054 Grenoble cedex 9. France m Germany

I8 April

1996: accepted

23 May

1996)

ABSTRACT

Sorption and penetration of atraxine through plant cuticles were investigated using enzymatically isolated tomato and pepper fruit cuticles. Sorption was rapid and equilibrium was reached within 4 - 6 hours for the two species. Mean values for cuticle/water partition coefftcients (K& were 140 and 131 for pepper and tomato cuticles, respectively. Sorption was not affected by pH solution between 3.5 and 8 but it was reduced at pH 1.7. The amount sorbed (mnol mg-‘) varied linearly with concentration from 1.3 tth4 to 0.1 mM. Atrazine was reversibly sorbed to cuticles and only 1 to 4% was still retained by cuticles after 48 hours washing. Diffusion of atrazine through cuticles increased linearly with time. Permeability coefficient values determined at 0.1 mM atrazine were 24.6 and 47.3 nm s“ for pepper and tomato fruit cuticles, respectively. Comparison with simazine showed that the cuticle/water partition coefficient was markedly lower for this herbicide. %, was 48.4 and 46.2 for pepper and tomato cuticles, respectively. Diffusion of simazine through cuticles was also lower particularly for pepper cuticles. Copyright 0 1996 Elsevier Science Ltd Key words:

isolated cuticles, cuticular sorption, cuticular permeability, atrazine, simazine

INTRODUCTION

Atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine) is one of the herbicides most widely used in the world (90000 tons/year). It is used as a pre- and post-emergency selective herbicide for weed control in maize crops, asparagus, pineapples, sorghum, sugarcane, forestry, grasslands, grass crops and as a non-selective herbicide for vegetation control in non-crop areas, In USA 40000 tons of the active ingredient are used every year (USEPA 1990); in France 5000 tons are spread annually mainly on maize cultivated on 5 million hectares. Atrazine is used in different european countries at concentrations varylng from 750 to 1500 g/ha. The uptake by the plants occurs primarily via the roots and partly through the leaves. Since the early 1980s an increasing number of countries have reported contamination of vulnerable 995

Ltd

reserved

CUTICLES

(Recened

1996

996

aquifers with triazines (i.e. atrazine and simazine). Most of these reports originate from the European Union. In the last decade much research has focused on the adsorption and degradation of atrazine and its metabolites in the soil but little information is available concerning its behaviour when reaching aerial parts of plants. The interface between the plant tissues and the environment is the cuticle, an extracellular lipoidal layer constituting the initial and main barrier to penetration of xenobiotics

deposited on plant

leaves. Moreover, it represents a compartment of the environment playing the role of reservoir for lipophilic xenobiotics. It is composed of the biopolymer cutin and embedded waxes with epicuticulat waxes on the outer surface. The transfer of water, nutrients

and pesticides through cuticles has already

been largely investigated using isolated cuticles of leaves and fruit. By using such model systems the behaviour of xenobiotics can be studied at cuticular barrier level without confounding effects due to underlying tissues. The purpose of the present study was to determine the behaviour of atrazine

in the

initial process of foliar penetration. Sorption and penetration were measured in vitro, using isolated cuticles, as previously described for other agrochemicals (Charnel 1986; Chaumat and Charnel 1990, 1991; Chaumat er al. 1992).

MATERIAL AND METHODS

Cuticle isolation: Cuticle discs (diameter 1 cm) were isolated enzymatically as previously described (Charnel and Bougie 1977) from commercially supplied fruits of tomato (Lycopersicon esculenfum Mill.) and pepper (Capsicum annuum L.). Mature fruit was used for tomato; green pepper was at the stage ordinarily used for consumption.

After their isolation, cuticles were extensively washed in deionized

water, air-dried and stored for further use. The integrity of cuticles was checked using a light microscope before permeability studies. Cuticular waxes were removed by incubating cuticle discs in chloroform (5 mL per mg) in a water-bath shaker for 24 hours at 25 “C. Measurement of cuticular sorption: Cuticle discs (~(1. 10 mg dry weight) were immersed in glass vials in 3 mL of ?Xabelled

herbicidal solution. Vials were closed with screw caps and shaken horizontally in

a water-bath at 25 “C. Sorption was determined indirectly by the decrease in radioactivity of the solution, measured on aliquots of 100 FL, as a function of time. Lack of sorption on the vial walls was controlled with replicates containing the labelled solution without cuticle. All measurements were made with four or five replicates per treatment. Sorption is expressed by the ratio between concentration of the chemical in the cuticle (dpm or nmoles mg-‘) and concentration of the chemical in solution (dpm or nmoles mg-‘). At equilibrium, this ratio corresponds to the cuticle/water partition coefficient (Kcw). This coefficient reflects the solubility of the tested compound in the cuticle, thus illustrating its affinity for cuticular material. The reversibility of the phenomenon was measured at the end of the sorption, by immersing cuticle samples, under agitation, in 10 mL water with 2% ethanol renewed minutes, 3.6, 24 and 48 hours. Means are given with standard error.

after 5, 15, 25, 35, 45, 55

99-l Measurement

of cuticular

permeability:

Permeability was measured using a double water-

compartment system consisting of two glass cells fued on a central support made of austenitic steel (Charnel et al. 1992). A cuticle disc (diameter 1 cm) was inserted between the two identical compartments of the device with the external surface of the cuticle in contact with the donor solution. An equal volume of liquid (2.6 mL) was pipetted in each cell and stirred by a magnetic stirbar. At the beginning of each experiment the donor contained the buffered herbicide solution and the receiver contained only the buffer solution. An aliquot of 100 pL was withdrawn from the receiver solution at the start of the experiment and at various time intervals between 1 and 7 days and radioactivity measured. A volume of 100 pL of buffer was immediately added to the receiver after each withdrawal to maintain a constant volume. The measurements were taken with six or seven cuticles per treatment. Experiments were carried out in an air-conditioned room at 20 “C. The herbicide transfer across the cuticle was expressed by the amount diffused in relation to time and by the permeability coefficient (P) calculated by dividing the flux measured under steady-state conditions by the concentration difference across the cuticle according to: P= F/A (Cdm- C&) with P in nm i’, F is the flux in nmol se’, A is the cuticb area in contact with the solutions in nn?, Cdm and C, are the concentrations in donor and receiver solutions in mnol MI-‘. The P coefficient is a measure of the overall membrane permeability. Means are given with standard error. Herbicide characteristics:

Radioactive herbicides were atrazine-ring-UL-‘4C (sp. act. 288.6 MBq

mmol~‘, radiochemical purity > 98%, Sigma) and simazine-ring-UL-14C (sp. act. 429.2 MBq mmol~‘, radiochemical purety > 98%, Sigma). The purity of the herbicides used for the preparation of solutions was 99% (Chem Service). The standard herbicide solution consisted of 0.1 mM atrazine or 0.01 mM simazine in 2 mM MES buffer (2(Nmorpholino)ethane sulfonic acid) at pH 6.5 with 2’/- ethanol The solution radioactivity was 0.37 KBq ml-’ for sorption experiments and 3.7 KBq ml-’ for permeability experiments. Other buffers used were SADH, succinic acid-(2,2-dimethylhydrazide) and HEPES, N-(2hydroxyethyl)-piperazine-N-(2-ethanesulfonic acid). Radioactivity

measurements: The radioactivity measurements were carried out by liquid scintillation

spectrometry (Tricarb 4430, Packard) using Aquasafe 300 (Zmsser Analytic) either on 100 pL aliquots in sorption and permeability experiments or on 1 mL aliquots in reversibility experiments. Dpm values were obtained after correction for background and quenching.

RESULTS

Sorption measurements

Sorption of atrazine by tomato and pepper fruit cuticles was very rapid during the first hours (Fig. 1) and equilibrium was achieved within 4 hours for pepper and 6 hours for tomato cuticles. Cuticle/water partition coefficient (&J reached 138 for pepper and 124 for tomato cuticles at pH 6.5 and 25 “C. The

YYX

amount

of atrazine

sorbed by cuticles increased

1.3 PM to 0.1 mM (Fig. 2). Sorption it was signiticanlly atrazine

sorption.

cuticles,

respectively.

lower Kcw

were still retained independently

at pH

values

Atrazine by tomato

was not signiticantly

1.7 (Fig.

were

linear-ly with increasing

3). Dewaxing

Ih(J (se

was reversibly

affected

by pH solution

caused

3.X) and 144 (se:

between

4.7)

for pepper

in

dewaxed sorbed

after 48 hrs.

solution.

??

10

increase

of atrazine

but only 1.4 to 3.3%

from

3.5 and 8, hut

and tomato

sorbed to cuticles (Fig. 4). 66% and 74%

of the herbicide

alrazlne

-+-

pepper

---+-

torrlalo

--+-*

ww tomato

30

20 Time

concentration

a slight but not significant

and pepper cuticles after S min washing

of the initial concentration

initial atrazine

(h)

Figure 1. Time-course of sorption of [‘“Clatrazine and [“C]siumzine by tomato and pepper isolated cuticles. Assay conditions: error. Where

standard

0.01

mM

herbicide,

1% ethanol,

pH 6.5. 25 “C. Means

error values are not shown, they are smaller

Equlllbrlum

concentration

(nmol

with their standard

than the data symbol.

ml-l)

Figure 2. Sorption of [“Clatrazine by pepper and tomato fruit cuticles as a fktctiort of atrazirle concentration in the buffer solution. Assay conditions: replications.

2 “/- ethanol,

pH 6.5, 25 “C, 24 hrs. Means

of 5

999 200

F

25

E

3 =

6

Figure 3. Eflect ethanol,

buffers

25 “C. Means

100

of pH

(2mM

on

partihon coeflci~r~rsfor. omzirre.

SADH,

oC5 replications

pH 1,7 and 3.5; 2 mM MES,

0

offhe

durdorr

of the sorptiort

ofwashing.

24 hrs and for washing:

2 mM

0.

I mM atrazinc.

HEPES,

2000

of /“C]arrazine

Assay conditions

3000

(mln) by pepper

for sorption:

md

torrratofruit cuticles us a ficrtctim

0.01 IIIM atrazinc,

2’/- ethanol,

pH 6.5. 25 “C,

pure water with 2”/~~ethnnol.

Permeability measurements Diffusion rapidly

of atrazine so the hold-up

across cuticles

increased

time obtained

by extrapolaling

short and could not be accurately

2”/,

pH 8). 24 hrs,

Pepper Tomato

1000 Time

Figure 4. Reversibility

conditions:

and 6.5;

wilh suurdard error in parcn~hescs.

-0--Q-

0

Assay

pi-1 5

determined;

line‘arly with lime (Fig. 5); steady slate was reached very UIC steady swc

the linear regression

Permeability coefficients determined l’ortwo abazinc concenlralions

slope to lhe lime

axis was very

coeKicient was greater than 0.98. are reported

in Table

I.

IO00

Concentration

(rnM)

Pcrmeahilily

0. 1 0.0

I

ctclticienl

(nm s

’)

T0ma1o

Pepper

47.3 (2.7)

24.6 (1.X)

44.5 (3.7)

1x.t (1.3)

1

100 Time (h)

Figure 5. Permeability of iduterI ethanol,

pH 6.5,

21 “C. Means

shown, they arc smaller

cuticle.\ to [‘4C~c~t~uzir~c~. Assay conditions:

of 7 replicalcs

and their standard

error.

0.013

Where

mM

atrazinc,

standard

errors

similarities

with

2”/-

are not

than the data sy~nbol.

Comparison with sinlaziue Sorplion

of simazine

concerning coefficienl

by isolated

both lirnc-course values (K_J

were

tornnio

and pcppcr

and comparisorr signiticanlly

between

lower

II uit cuticles

the IWO plan1 species

for siniazine

45.2 (s.c.: 2.4) for pepper and lomalo culicles. respectively. amount

rc1aincd by cuticles

48 hrs washing DiKusion cocltkicnis

reached

of siniazine

was etiminnted

(Fig.

I). K,

Desorption

was atso bcller lhrough

lonialo

but cuticle/water

alrazinc partiliou

values were 4X.4 (s.c.: 1.X) and

was very rapid, about 45%

aticr 5 nun washing and rhe traction

a value as low as 0.3 and

were 38.9 (se.:

showed

remaining

of the

in cuticles after

I .9 o/nlor tomato and pepper cuticles,

respcctivcly.

as compared

Permeability

with pepper fruit cuticles.

5.3) and 2.X (se.: 0.4) nm s.’ lor 1omato and pepper cuticles,

rcspccrivcly.

1001

DISCUSSION

This investigation has confirmed that atrazine, like other lipophihc compounds, is rapidly sorbed by plant cuticles (Charnel 1986, Charnel et al. 1991, Chaumat et al. 1992). Mean values for the partition coefficient relating the equilibrium concentration of atrazine in the cuticle to that in the surrounding aqueous solution were 140 (log KW: 2.14) and 131 (log K,,.: 2.12) for pepper and tomato fruit cuticles, respectively. These values are within the range (2.12
I OO?

the corresponding

ratios calculated

for octanoVwater

partition

coefficients

(2.45-3.47).

Another

characteristic of atrazine sorption in cuticles is the reversibility of the process. Desorption is rapid during the first minutes then continues more slowly after this time. More than 50 percent of the total amount of atrazine sorbed in tomato or pepper cuticles could be desorbed within 30 minutes. In natural conditions atrazine might be temporarily accumulated in cuticles following

contamination of aerial parts of plants

with this herbicide, the fraction retained by cuticles and non absorbed in underlying tissues might return to the soil and aquifers under the effect of leaching caused by rain. The extraction of cuticular waxes did not significantly modify cuticle I water partition coefficients for atrazine, although several results from the literature (Charnel 1986) showed an increase in the value of this parameter for dewaxed cuticles. However, the results discussed here concerning tomato and pepper fruit cuticles are not surprising as waxes represented a very low percentage of the cuticle mass for these two species, 2.1% and 1.9% of the initial dry weight of tomato and pepper fruit cuticles, respectively. Sorption was linearly related to the external concentration

over a wide concentration

range. The cuticle/water

partition coefficient was

essentially constant within the studied range of concentrations ( 1.3 l.tM to 0.1 mM). Similar results were reported for other lipophilic herbicides like 2,4-D, 2,4-DB and diuron (Charnel et al. 1991; Chaumat and Chaumat

1990). Atrazine can diffuse through isolated cuticles like other herbicides (Charnel 1986,

Chaumat et al. 1992). Values of permeability coefficients were not very different from those previously reported for diuron (40.1 and 14.3 nm s-’ for tomato and pepper fruit cuticles, respectively). In the same study a greater permeability was also observed for tomato cuticles (Chaumat et al. 1992). The cuticular permeability of these two species to atrazine differed by a factor equal to 2 and 2.5 for the two concentrations considered (Table 1) in spite of relatively similar cuticle/water partition coefftcients (Figs 1 and 2). This variation is attributed to a lower mobility (or diffusion) of atrazine in pepper fruit cuticles which might be related to the fme microstructure comparison

between

and chemical composition

atrazine and simazine concerning

cuticular permeability

of these cuticles. The showed

a significant

difference between the two herbicides only with pepper cuticles which suggests that the structural characteristics

of the cuticles are of major importance and must be given particular attention when

attempting to establish models to predict the transfer of xenobiotics through plant cuticles (Gouret et al. 1993, Viougeas et al. 1995). Data reported on retention and transfer of atrazine through cuticles should be useful for validating models which will have to be developed to estimate the behavior of environmental chemicals in atmospheric environment/ plant systems (Trapp and MC Farlane, 1995).

REFERENCES

Charnel A. (1986) Foliar absorption of herbicides: study of the cuticular penetration cuticles. Physiol. V&J., 24.491-508.

using isolated

1003

Chamel A. (1988) Behaviour of chemicals applied to leaves studied with entire plants and isolated cuticles. In Plant growth and leaf-applied chemicals, P.M. Neumann, ed., CRC, Boca Raton, 2750. Chamel A. and Bougie B. (1977) Absorption foliaire du cuivre: etude de la fixation et de la p&r&ration cuticulaims. Physiol. Kg., 15.679-693. Charnel A., Gambonnet B. and Charnel A. (1992) Effects of two ethoxylated nonylphenols on sorption and penetration of [‘4C]isoproturon through isolated plant cuticles. Plant Physiol. Biochem., 30, 713-721. Charnel A., Gambonnet B., Arnaud L. and Charnel A. (1991) Foliar absorption of [‘4C]paclobutrazol: Study of cuticular sorption and penetration using isolated cuticles. Plant Physiol. Biochem.. 29, 395-401.

Chaumat E. and Charnel A. (1990) Study of the cuticular retention and permeation of diuron using isolated cuticles of plants. Plant Physiol. Biochem., 28,7 19-726. Chaumat E. and Charnel A. (1991) Sorption and permeation to phenylurea herbicides of isolated cuticles of fruit and leaves. Effects of cuticular characteristics and climatic parameters. Chemosphere, 22, 85-97.

Chaumat E., Charnel A., Taillandier G. and Tiisut M. (1992) Quantitative relationships between structure and penetration of phenylurea herbicides through isolated plant cuticles. Chemosphere, 24, 189200.

Gouret E ., Rohr R. and Charnel A. (1993) Ultrastructure and chemical composition of some isolated plant cuticles in relation to their permeability to the herbicide, diuron. New Phytologist, 124, 423431.

Kerler F. and SchUnherr J. (1988) Accumulation of lipophilic chemicals in plant cuticles: Prediction from OctanoYwaterpartition coefficients. Arch. Environ. Contam Toxicol., 17, l-6. Montgomery J.H. (1993) Agrochemicals desk reference, Environmental data, Lewis publishers, Boca Raton. Riederer M. and Schonherr J. (1984) Accumulation and transport of (2,4-dichlorophenoxy) acetic acid in plant cuticles. I. Sorption in the cuticular membrane and its components. Ecotoxicol. Environ. Safety, 8, 236-247.

Trapp S. and MC Farlane J.C. (1995) Plant contamination.Modeling and simulation of organic chemical processes. Lewis Publishers, Boca Raton.

USEPA (1990) Environmental fact sheet: atrazine label amendments. USEPA Oftice

of Pesticide

Programs, Washington D.C. Viougeas M.A., Rohr R. and Charnel A. (1995) Structural changes and permeability of ivy (Hedera helix L.) leaf cuticles in relation to leaf development and after selective chemical treatments. New Phytologist, 130, 337-348.