Comparative Diffusion of Atrazine inside Aqueous or Organic Matrices and inside Plant Seedlings

Pesticide Biochemistry and Physiology 65, 36–43 (1999) Article ID pest.1999.2422, available online at http://www.idealibrary.com on

Comparative Diffusion of Atrazine inside Aqueous or Organic Matrices and inside Plant Seedlings M. Raveton, A. Schneider,* C. Desprez-Durand,† P. Ravanel, and M. Tissut Laboratoire de Physiologie Cellulaire Ve´ge´tale, and †Laboratoire Mode´lisation et Calcul, Universite´ Joseph Fourier, BP 53X, 38041 Grenoble Cedex 09, France; and *Rhoˆne-Poulenc, Secteur Agro, BP 9163, F-69263 Lyon Cedex 09, France Received September 23, 1998; accepted May 20, 1999 Several different matrices (water, n-butanol, n-octanol, lecithin, waxes, and suber) were chosen to measure [14C]atrazine diffusion rate and evaluate the specific diffusion parameter of this molecule. A simple experimental device was conceived for this purpose and two methods of calculation, deduced from Fick’s law, were established and compared. The same device was used for diffusion measurements inside corn seedling fragments, either dead or alive. In inert matrices, the highest diffusion parameter found for atrazine was obtained for water (2.6 6 0.9) 10210 m2 s21. For more lipophilic matrices, the value of the specific parameter decreased markedly, reaching only (1.2 6 1) 10212 m2 s21 for glycerides and (2.5 6 2.4) 10213 m2 s21 for paraffin. For dead corn roots or coleoptile, the diffusion parameter was close to that in water: (3.7 6 2.1) 10210 m2 s21 and (9.6 6 4) 10210 m2 s21. In living material, the movement of 14C-labeled compounds was much lower: (7.4 6 4.6) 10211 m2 s21 and (1.6 6 1.5) 10211 m2 s21. This was explained by atrazine hydroxylation in the presence of benzoxazinones, leading to a derivative which was accumulated inside the vacuole. q1999 Academic Press

INTRODUCTION

distribution is at least partly controlled by an active process. For the other xenobiotic compounds used in agriculture, short-distance distribution appears to be passive and is therefore supposed to be subjected to the general laws of diffusion (Darcy’s or Fick’s laws). However, this passive distribution has to occur in a very complex structural network, composed of either symplasmic or apoplastic spaces and of several lipophilic or aqueous compartments. The purpose of this work was to find out more about passive atrazine transfer in plant material, either dead or alive, in comparison with its transfer inside pure chemical matrices. Originally, the question was: Is atrazine transfer occurring mostly inside plant lipids or inside plant water?

With the exception of surface fungicides or insecticides, xenobiotic compounds used in agriculture have to penetrate inside plants. As a consequence, only the biochemically active ingredients (a.i.) which are able to penetrate into plants are selected as effective products during the routine screening carried out for applied research. Penetration inside plants is a complex, little understood phenomenon (1, 2). First, it is a transfer from the plant surface (leaf or roots) to an apoplastic space, either lipophilic in the case of leaf cuticule (3) and root suberin or mostly hydrophilic in the case of unprotected cell walls (root hairs). Then, it is followed by a transfer into the inner symplasmic parts of the living cells in order to obtain a biological effect. A short-distance distribution occurs alone in several cases, such as distribution from cell to cell inside a leaf, inside stem parenchyma, inside the roots, from root hairs to Caspary cells, or inside the whole seedling. For some substances such as paraquat (4) or glyphosate (5, 6) the cell

MATERIALS AND METHODS

Experimental Device For such experiments, a simple and efficient experimental device was necessary and a calculation method had to be chosen. The experimental device which was conceived here (Fig. 1A) 36

0048-3575/99 $30.00 Copyright q 1999 by Academic Press All rights of reproduction in any form reserved.

ATRAZINE BIOAVAILABILITY IN CORN

37

FIG. 1. Experimental device allowing the measurement of the diffusion transfer rate, in the case of a homogeneous matrix or in the case of plant fragments (A). (B) Structure of the studied column.

allowed measurement of the amount of the studied product moving through a 5-mm-long zone B in the studied matrix (water, n-octanol, waxes, plant material), from a donor compartment A in which the product was present at a C0 concentration, to a receiver compartment C in which the product was absent at the beginning of the experiment (T0) and in which the concentration C1 of the studied product increased with time. In the case of glycerides, suberin, and liquid organic solvents, a stable concentration in the donor compartment (A) of the column was obtained, through a transfer of atrazine from a water solution for at least 6 h. With glycerids and suberin (solid column), the water solution was stirred. In the case of liquid phases (n-butanol or n-octanol), the equilibrium was obtained

through diffusion from water to the organic solvent, without stirring. In the case of paraffin, the exchanges from water to the column were very slow and another technique was used in which atrazine was directly dissolved in melted paraffin and immediately added to the bottom of the solid column. Under these conditions, the diffusion processes observed in the different types of columns showed no important convection movement inside each liquid or solid phase. A similar experimental device was used in experiments with corn material, the column system being replaced by a 2-cm-long excised fragment of coleoptiles or roots. At the time chosen for measurement, the polypropylene tube containing the solid matrices or the plant material was carefully cut in order to

38

RAVETON ET AL.

isolate the compartments A, B, and C. In the case of the liquid columns, the various solvents were collected with a syringe. Calculation Principle The calculation principle chosen here was deduced from the simplifed Fick’s law. (C 2 C1) dq 5D 0 S dt hb D measurements were carried out when C0 was stable after equilibration with the aqueous compartment. C1 was measured in the 5-mm-long receiver compartment situated just above compartment B. In contrast with C0, C1 changed with time. Two ways of calculation were considered for estimating the diffusion parameter value (D). A rough estimation of D value was obtained, when C1 changed from time 1 to time 2, using the average value of C1 corresponding to [C1(t1) 1 C1(t2)] 4 2. Under such conditions, the value of D was obtained through Eq. [1]: D5

F

dq hb

G

1 C1(t2)) (C S dt C0 2 1(t1) 2

[1]

A more accurate estimation was obtained when considering the continuous change of C1 from time 1 to time 2 (Eq. [2]). qz 5

dq S dt

D dC1 52 dt C0 2 C1 hchb

1

Determination of the Radioactivity Content in Columns and Plant Material Root and coleoptile samples were weighed separately, disrupted with a mortar, and dispersed in an ethanol/water solution (1/1, v/v). The quantities of radioactivity present in column slices (5 mm thickness) and crude extracts were measured by liquid scintillation procedure (Intertechnique Scintillatior Model SL 4000, Ready Safe liquid scintillation from Beckman) and expressed as “atrazine equivalent” amounts. Analysis of Corn Benzoxazinones and [14C]Atrazine Metabolites

dC1 qz S D(C0 2 C1) 5 5 dt V hc hb

2

C 2 C1(t2) hchb , Ln 0 dt C0 2 C1(t1)

D52

is the interval of time (s) between time 2 to time 1; S is the section area (m2); hb is the length of compartment B (m); hc is the length of compartment C (m); V is the volume of compartment C (m3); C0 is the concentration of product in the compartment A (mol m23); C1 is the concentration of product in the compartment C (mol m23); and D is the diffusion coefficient. In order to evaluate the two formulas shown under Materials and Methods, the values obtained when using each of them were compared. This comparison was carried out using the appropriate Student’s test for paired replicates (7). When considering a 5% risk threshold, the value of t for this test was 1.70 and the corresponding value of Student’s table was 2.16. This demonstrated that, with such a 5% risk, the results of the two formulas did not differ significantly.

[2]

where qz is the diffusion rate of the molecule (mol m22 s21); dq is the amount of compound transfered (moles) through B compartment; dt

Before and after diffusion experiments, seedling fragments were extracted by acetone/water (4/1, v/v). After partition with petrol ether, the hydroacetonic solution was concentrated in vacuo and submitted to TLC on silica gel plates (Merck 60F 254) with ethyl acetate/acetic acid/ formic acid/water (40/2/2/4, v/v/v/v) as a solvent. Under such conditions, Rf of the studied compounds were as follows: atrazine (0.80), OH-atrazine (0.40), glutathion conjugate (0.05), DIMBOA (0.88), glucosyl-2-DIMBOA (0.17), and heat transformed inactive benzoxazinone (0.96).

39

ATRAZINE BIOAVAILABILITY IN CORN

Benzoxazinones were detected under UV light (254 and 366 nm). [14C]Atrazine and its derivatives were located and their amounts were measured with the use of a Berthold 14C analyzer. Chemicals Atrazine, hydroxyatrazine (OH-Atr), deethylatrazine (DEA), deisopropylatrazine (DIA), and didealkylatrazine DiDA) (purity grades $99%) were purchased from Cluzeau (Interchim, Ste Foy la Grande, France). Paraffin and glyceride mixture were purchased from Aldrich and 14Cring UL atrazine (radiochemical purity $98%, sp act 0.92 GBq mmol21) was purchased from Sigma. Atrazine, DEA, DIA, and DiDA stock solutions were prepared with ethanol as solvent whereas OH-atrazine was dissolved in water/ acetic acid (19/1, v/v). RESULTS

Diffusion Parameter Values (D) in the Case of Atrazine Diffusing either in Water or in n-Butanol In a first attempt, it was necessary to check if the D value remained constant for different values of C0 or when C1 changed. In the case of the water–gelose columns, C0 remained almost constant (close to 4 mM) during the experiment and C1 varied from 0 to 2.57 mM. Eleven measurements of D in this case during three separate experiments gave an average value D 5 (2.6 6 0.9) 10210 m2 s21 (value calculated with Eq. [2]). Under comparable conditions, with the nbutanol column, the C0 value was 13.7 6 0.8 mM and C1 varied from 0 to 5.6 mM. Table 1 shows the reproductibility of results of six

measures obtained in two sets of experiments with n-butanol. Taking into account 17 measures obtained in four sets of experiments, the average value for D 6 ts8/=n was (3.8 6 1.7) 10211 m2 s21 (calculation with Eq. [2]). This value was clearly lower than the D value measured with water 1 gelose columns. Furthermore, it hardly changed when the concentration C0 was increased from 4 mM to six times more, showing that Fick’s law remained valid in a large range of concentrations, below the solubility limit. Determination of D for Different Pure Matrices Table 2 shows the values of D obtained with the same experimental device for several matrices of increasing lipophilicity. First, n-octanol was studied and then, a solid mixture of glycerides with dominant C12 saturated fatty acids. Paraffin waxes were studied afterward and finally suberin (cork). The matrix was in a solid state for the last three components. The accuracy of the D determination in each case was sufficient to show that the D value decreased markedly when the lipophilicity of the matrix increased. The lowest values of D were obtained for saturated glycerides and for paraffin waxes, for which the average value of D was, respectively, 220 and 1040 times lower than that for water– gelose (calculation with Eq. [2]). The decrease of D values for atrazine which seemed to be related to the increase in lipophilicity of the matrix was not submitted to a drastic change when this matrix, which was first liquid with n-octanol, became solid with saturated

TABLE 1 D Coefficient Values of Atrazine Diffusion in a N-Butanol Column when Changing C0 and C1 Concentrations Time (h)

C0 (mM) C1 (mM) D (m2 s21)

22.5

23

42

46

48

70

12.9 0.8 2.1 10211

13.6 1.7 5.6 10211

13.5 3.5 6.1 10211

13.9 3.6 6.7 10211

13.3 3.8 3.4 10211

15.1 5.6 5.6 10211

40

RAVETON ET AL. TABLE 2 D Coefficient Values of Atrazine Diffusion inside Different Matrices

Component Water–gelose n-Butanol n-Octanol Glycerides Paraffin Corka Corn roots Corn coleoptiles Dead roots Dead coleoptiles a b

D values 6 ts’/=n (m2 s21) Eq [1] (1.7 (1.1 (3.4 (6.9 (3.6 (1.8 (6.4 b (8 (1.9 b (1.8 (9 b (9 (4.4 b (10.8

6 6 6 6 6 6 6 6 6 6 6 6 6 6

1.3) 1029 0.4) 10210 2.5) 10211 5.6) 10213 3) 10213 1.3) 10212 3) 10211 4.5) 10211 1.1) 10211 0.7) 10211 6) 10210 7.8) 10210 2.3) 10210 2.5) 10210

D values 6 ts’/=n (m2 s21) Eq [2] (2.6 (3.8 (1.3 (1.2 (2.5 (7.8 (5.4 b (7.4 (2.3 b (1.6 (3.7 b (3.4 (9.6 b (4.3

6 6 6 6 6 6 6 6 6 6 6 6 6 6

0.9) 10210 1.7) 10211 0.7) 10211 1.0) 10212 2.4) 10213 6.3) 10212 3.2) 10211 4.6) 10211 2.1) 10211 1.5) 10211 2.1) 10210 2.2) 10210 4) 10210 2.5) 10210

Presence in the suberin matrix of air bubbles which can disturb the atrazine diffusion process. Concentration of treatment 5 25 mM.

glycerides. Moreover, in the suberin matrix which represents a relatively hydrophilic solid polymer with a nonnegligible number of hydroxyles, the D value was found to be higher than for paraffin waxes but lower than for saturated glycerides. Estimation of the Diffusion Rate Expressed by D in Plant Material Our experimental device was chosen because it had approximately the same shape and length as 2-cm-long corn coleoptile segments or first root segments. When maintaining a saturating moisture in the experimental system, a true diffusion process could be observed inside this plant material. Such a plant material is a complex mixture of lipophilic and hydrophilic, either apoplastic or symplasmic, spaces. Moreover, one of these spaces is liquid (water), another one is known to be semi-fluid (the biological membranes), and some others are solid (cell wall, cuticle, starch). However, the quantitatively preeminent space is water. In a first attempt, we tried to use dead plant material in which we were sure that biological activities could not hinder the diffusion process. After several inconclusive assays, using dead material

after chemical treatments (uncouplers) or frozen material, we chose a 5-min immersion of plant fragments in boiling water in order to kill them. After this treatment, wall polymers remained mostly unchanged and biological membranes were submitted to a severe chaotropic effect but the whole lipid content of the fragments was shown to remain the same; most of the proteins were coagulated through heating and the hydrophilic components were diluted with water (loss of salts, soluble carbohydrates and organic acids, most of the benzoxazinones, especially the 2glucosides. . .). Especially in corn tissues, the heat treatment destroyed a specific secondary metabolite (DIMBOA) which detoxifies atrazine through hydroxylation (8, 9). This aglycone was transformed into MBOA after heating (Formula I); the latter was inactive on atrazine hydroxylation (10–12). In such a dead material, be it with roots or with coleoptiles, the values of D for atrazine were comparable to the water–gelose column values (Table 2). This uniform and high D value was also the same at 4 mM and at 25 mM atrazine for C0, suggesting that it was a pure diffusion phenomenon. When repeating the experiment with living material, as shown in Table 2, the

41

ATRAZINE BIOAVAILABILITY IN CORN

FORMULA I. Structure of DIMBOA and MBOA.

values of D decreased by 7 times for roots and 40 times for coleoptiles in comparison with boiled material. This difference remained the same with 4 and 25 mM atrazine. Therefore, it could be concluded that a major change in xenobiotic transfer occurred from dead to living corn material. Such a discrepancy might originate either from the presence of the cytostructure existing in the living cell only or from their thermolabile biochemical activities. The TLC and chemical analysis of the labeled components originating from [14C]atrazine and present in the living material at the end of the experiment (Fig. 2) showed that this second hypothesis was true and that OH-atrazine accumulated inside the living material. As detailed elsewhere, 2-OH-atrazine was the main metabolite of atrazine in young corn seedlings and its formation resulted from a pure, although thermolabile, chemical process (9). The experiment was repeated with oat seedlings living fragments and in this case the D value (D 5 (6 6 3.2) 10210 m2 s21) atrazine was close to that measured in water–gelose columns.

FIG. 2. 14C scanning of extracts of dead corn seedling fragments treated with atrazine (A) and of extracts of the same, but live fragments (B).

DISCUSSION

Atrazine is a herbicide acting on photosynthesis through its binding to D1 protein (13, 14). It has no target in the seedlings. This compound is characterized by a relatively high affinity for lipids, compared to water, which is measured by the conventional parameter log P (P being the partition parameter between n-octanol and water). The value of log P for atrazine is close to 2.5 (15). We verified this point and obtained a P value of 287 6 10, leading to a log P value of 2.45 under our conditions. Such a value suggests that atrazine is able to concentrate inside lipophilic matrices such as n-butanol, n-octanol, glycerides, or waxes, as observed during our experiments. This concentration process also occurred in the case of isolated organelles such as potato mitochondria obtained as described in (16). Lipids, mostly polar lipids constituting the biological membranes of these organelles, amount to 1.42% of their fresh weight. With atrazine at 1 mM, the concentration factor inside these lipids compared to the concentration in the matrix water was 155, giving a log P lipid/water of 2.19, not far from log P octanol/water (2.5). In addition to this concentration process, this study demonstrates that diffusion occurred in each of the studied matrices but that the rate of diffusion was significantly higher inside water than inside more lipophilic matrices. The D value in water spaces was close to the value found in killed corn material and very close to the value found for living material in the case of oat seedlings. In the case of living corn seedlings, the rate of the metabolization process was so high that it fully changed the rate

42

RAVETON ET AL.

of 14C distribution inside this material, because atrazine was readily transformed into 2-OH-atrazine in the presence of DIMBOA and this atrazine metabolite was segregated inside the vacuole (8, 9). Our results show that when no metabolization occurs, a compound with a moderate lipophilicity such as atrazine is transfered from cell to cell through a diffusion process first concerning the hydrophilic spaces and mostly water itself. Figure 3 is a scheme attempting to illustrate this point. In the hypothesis of atrazine remaining as a true aqueous solution in the living material, the

high rate of its diffusion in nonmetabolizing species was most likely based on two facts: (1) the high value of D in water and (2) the high content of water in the plant (95%). However, in living symplasmic material, the structure of which is based on a succession of water spaces separated by biological membranes, one cannot postulate that atrazine transfer was exclusively based on diffusion inside water or hydrophilic spaces. The most likely hypothesis is that the transfer through a biological membrane is not a ratelimiting step. Two ways of explaining this point may be considered. First, atrazine and similar lipophilic compounds dissolved inside water

FIG. 3. Hypothetical scheme illustrating the atrazine transfer rate from cell to cell either in metabolizing or non metabolizing plants. The diffusion flux occurs mostly through hydrophilic spaces. In corn, the intense transformation of atrazine into OH-2-atrazine, which is segregated inside the vacuole, greatly decreases the diffusion rate of atrazine. Atrazine accumulation occurs in the lipophilic spaces through partitioning but the diffusion rate is low inside these spaces.

ATRAZINE BIOAVAILABILITY IN CORN

could follow the rapid water transfer through the membrane, possibly with the use of aquapores where they exist (17). Second and more probably, the partition process between water and membrane may be characterized by a rate of exchange between water and lipids, which we estimate as very high since the membrane is very thin. In accordance with this hypothesis, it is only in compact thick lipophilic spaces that the diffusion rate inside such a matrix could greatly lower the global transfer of a lipophilic product inside plants. We have now very good examples of such a scheme with seeds and thick cuticles. In the case of compounds which are much more lipophilic than atrazine, such as substituted anilides (18) for instance or pentachlorophenol (19), the rate of the comparative transfer through water or through the lipophilic spaces remains to be studied. REFERENCES 1. M. G. T. Shone, B. O. Barlett, and A. V. Wood, A comparison of the uptake and translocation of some organic herbicides and a systemic fungicide by barley. 2-Relationship between uptake by roots and translocation to shoots. J. Exp. Bot. 25, 401 (1974). 2. M. Tissut and F. Se´verin, “Plantes, Herbicides et De´sherbage, Bases Scientifiques et Techniques,” ACTA, 1984. 3. A. Chamel, Foliar adsorption of herbicides: Study of the cuticular penetration using isolated cuticles Physiol. Ve´g. 24, 491 (1986). 4. J. J. Hart, J. M. Di Tomaso, and L. Y. Kochian, Characterization of paraquat in transport in protoplasts from maize (Zea Mays L.) suspension cells. Plant Physiol. 103, 963 (1993). 5. M. H. Denis and S. Delrot, Carrier-mediated uptake of glyphosate in broad bean (Vicia faba) via a phosphate transporter. Physiol. Plant. 87, 569 (1993). 6. F. Morin, V. Vera, F. Nurit, M. Tissut, and G. Marigo, Glyphosate uptake in Catharanthus roseus cells: Role of a phosphate transporter. Pestic. Biochem. Physiol. 58, 13 (1997).

43

7. B. Rosner, “Fundamentals of Biostatistics,” 4th ed., 1994. 8. M. Raveton, P. Ravanel, A. M. Serre, F. Nurit, and M. Tissut, Kinetics of uptake and metabolism of atrazine in model plant systems. Pestic. Sci. 49, 157 (1997). 9. M. Raveton, P. Ravanel, M. Kaouadji, J. Bastide, and M. Tissut, The chemical transformation of atrazine in corn seedlings. Pestic. Biochem. Physiol. 58, 199 (1997). 10. J. B. S. Brendenberg, E. Honkanen, and A. I. Virtanen, The kinetics and mechanism of decomposition of 2,4dihydroxy-1,4-benzoxazin-3-one. Acta Chem. Scand. 16, 135 (1962). 11. M. D. Woodward, L. S. Corcuera, J. P. Helgeson, and C. D. Upper, Decomposition of 2,4-dihydroxy-7 methoxy-2H-1,4-benzoxazin-2(4H)-one in aqueous solutions. Plant Physiol. 61, 796 (1978). 12. P. Kuwar, D. E. Moreland, and W. S. Chilton, 2H-1,4benzoxazin-3(4H)-one, an intermediate in the biosynthesis of cyclic hydroxyamic acids in maize. Phytochemistry 36, 893 (1994). 13. W. Oettmeier, K. Masson, and U. Johanningmeier, Evidence for two different herbicide-binding proteins at the reducing side of photosystem II. Biochim. Biophys. Acta 679, 376 (1982). 14. A. Trebst, and W. Draber, Inhibitors of photosystem II and the topology of the herbicide and QB binding polypeptide in the thylakoid membrane. Photosynth. Res. 10, 381 (1986). 15. A. Finizio, A. Di Guardo, A. Bassoli, and M. Vighi, Erbicidi triazini: Confronto tra metodi di stima del coefficiente di ripartizione n-ottanolo/acqua (KoW). in “I Diserbanti” (G. Biaggini-Lucca, Ed). 88 Simposio chimica degli antiparassitari (Piacenza), 1991. 16. C. Balland, R. Naigre, P. Kalck, M. Tissut, and P. Ravanel, Methyl-limonate induces a restricted chaos in plant mitochondrial membranes. Phytochemistry 46, 65 (1977). 17. C. Maurel, Aquaporine and water permeability of plant membranes. Annu. Rev. Plant Physiol. 48, 399 (1997). 18. F. Nurit, E. Gomez de Melo, P. Ravanel, and M. Tissut, The use of cultured Acer cells as a screening tool for mitotic inhibitors. I - References herbicides. Plant Growth Regul. 9, 47 (1990). 19. A. Albertin, S. Chiron, F. Nurit, P. Ravanel, and M. Tissut, Penetration, distribution and effects of [14C] pentachlorophenol inside cultured Acer cells. Phytochemistry 30, 3553 (1991).