The binding of atrazine and its dealkylated derivatives to humic-like polymers derived from catechol

The Science of the Total Environment, 117/118 (1992) 207-217 Elsevier Science Publishers B.V., Amsterdam

207

The binding of atrazine and its dealkylated derivatives to humic-like polymers derived from catechol Francis G. Andreux a, J.M. Portal a, Michel Schiavon b and Gilles Bertin b aCentre de P~dologie Biologique, CN.R.S., B.P. 5, 54501 Vandoeuvre-lks-Nancy Ckdex. France bService de Protection des Cultures, E.N.S.A.LA., B.P. 172, 54505 Vandoeuvre-lbs-Nancy C~dex, France

ABSTRACT Polymeric humic-like substances were prepared by oxidizing catechol under abiotic conditions at pH 6-8 in the presence of the following compounds: glycine, diglycine, triglycine, glucosamine, atrazine, de-ethylatrazine, de-isopropylatrazine and diaminoatrazine. A transient red quinoid pigment first appeared, then a brown color developed. After 5 days the dark reaction solutions were separated into fulvic acid and humic acid-type polymers. The weight yields of polymers ranged from 5 to 45°/% and the C/N ratios ranged from about 18 (glucosamine) to 4 (triglycine). Atrazine and its mono-dealkylated metabolites produced a decrease in polymer yields and were predominantly bound to the fulvic acid-type fractions. Large amounts bound to the humic acid-type fractions were observed only in the case of diaminoatrazine. Key words." polymeric humic-like substances; atrazine; dealkylated derivatives; catechol;

polymer yields

INTRODUCTION The most popular theories on the formation of humic substances are based on the oxidative polymerization of the numerous phenolic molecules released by plant residues and soil microorganisms [1]. Due to the chemical complexity of such molecules, several attempts were made in the past to synthesize 'model' humic polymers from representative phenolic precursors by chemical or enzymic oxidation and in the presence o f a nitrogen source, generally ammonia, amino acids, or proteins [2-4]. In the same period, similar reactions involving pesticides and their derivatives were carried out with the aim of studying their stabilization in soils, especially in the case of chlorophenols [5], methoxychlor [6] and chloroanilines [7-9].

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F.G. ANDREUX ET AL.

Aromatic amines are among the most reactive molecules and can interact with soil humus through either reversible adsorption reactions or irreversible covalent bonds [10]. The chemical structure of addition products between phenolics and NH2-bearing pollutants such as 4-chloroaniline has been established [11-13] and their incorporation into humic- like polymers demonstrated [14]. Such compounds can be considered as structural models of so-called 'pesticide-bound residues'. In the case of atrazine, incorporation in humic-like polymers was found to take place during the oxidation of catechol, but little is known about the mechanisms involved in this reaction [15,16]. The study reported here focuses on the incorporation of atrazine and its dealkylated derivatives in humic-like polymers prepared from catechol, and on the influence of the experimental conditions, especially the presence of amino acids and peptides, on the yield and composition of the polymeric materials obtained. MATERIAL AND METHODS

Preparation of polymers The general procedure described by Andreux et al. [17,18] and Adrian et al. [19] was applied in the case of the polymer prepared from catechol alone, which was used as a reference. Specific changes were incorporated according to the nature of the nitrogen source added.

From catechol and biomolecular compounds Catechol was recrystallized in methanol and dissolved in sodium phosphate buffer (pH 6-8) to obtain a 0.06 M concentration. Each solution was then mixed with an equal volume of buffer solution containing one of the following compounds at 0.06 M concentration: glycine, diglycine, triglycine and glucosamine. The mixture was then allowed to react in the dark at 25°C and under a constant flux of oxygen for 4 - 5 days. In the assays with catechol alone, the medium took on a greyish-green colour within less than 24 h due to the formation of semi-quinone, then a dark brown colour appeared, which attained a maximum after about 100 h at pH 8. In the presence of an NH2-bearing molecule, an intense red colour due to the formation of N-substituted quinones developed after a few minutes and persisted for several hours, until the brown colour developed. Kinetic studies of the absorption spectra in UV-visible light showed a progressive decrease of the absorption band of catechol at 280 nm, and a tendency to a monotone spectrum, as in humic substances [4,17,18]. At the end of the reaction, the solutions were transferred to dialysis bags of 1000 or 4000 Da cut-off, to remove the sodium phosphate, together with oligomeric com-

BINDING OF ATRAZINE AND DEALKYLATED DERIVATIVES TO HUMIC-LIKE POLYMERS

CL

209

CL

NH

I

N

C.2H5

I iC3H 7

I

N

H

I

iC3H 7

(a)

(b)

eL

eL

NH

N C2H 5

I

I

H

H

(c)

N

I H

(d)

Fig. 1. Chemical structures of atrazine (a) and its dealkylated derivatives de-ethylatrazine (b), de-isopropylatrazine (c), and diaminoatrazine (d).

pounds. The remaining polymer solution was then decationized on a Dowex 50 (H +) resin bed, dialysed again, freeze-dried, and carefully weighed.

From pesticides and pesticide derivatives The chemical structure of atrazine and its dealkylated derivatives is given in Fig. 1. Atrazine (2-chloro-4-ethylamino-6-isopropylamino-l,3,5-triazine) was added to the buffer solution at four concentrations to obtain atrazine/ catechol molar ratios of 1:160, 1:80, 1:40 and 1:20. The reaction was generally run at pH 7. Atrazine-containing polymers were also prepared from mixtures of catechol + glycine, diglycine, or triglycine at equimolar 0.03 M concentration. ~4C ring-labeled atrazine was added so that the molar ratio was 1:20 and the specific radioactivity was 70 MBq per mole of atrazine. In this case, the reaction was run at pH 7 only, for 4 days. Mono-dealkylated atrazine (de-ethyl- and de-isopropylatrazine) and diaminoatrazine (de-alkyl-, de-isopropylatrazine) were previously dissolved in a few drops of methanol and added to a 0.06 M solution of catechol in phosphate buffer at pH 6 at a molar ratio of 1:20. A precipitate was formed at the m o m e n t o f addition, but vanished before the end of the reaction.

210

F,G. ANDREUX ET AL.

The dark solutions obtained in the presence o f pesticide and the reference solutions without pesticide were acidified to pH 1.5 with a few drops of 6.0 N HC1 and allowed to stand overnight at 4°C. The acid-insoluble and acidsoluble fractions were separated by centrifugation and termed 'humic acids' (HA) and 'fulvic acids' (FA), respectively. The unreacted or weakly bound atrazine and metabolites were repeatedly extracted with chloroform from the HA precipitate (250 ml, 400 mg) and from the F A solution (1:1 v/v). The chloroform extracts were redissolved in methanol and the chloroform-free HA and F A residues were freeze-dried.

Analytical procedures The elemental composition of the polymer powders and of the corresponding H A and F A fractions was determined by dry combustion in Carlo Erba 1104 and 1106 analyzers. The nitrogen content o f the ash-free polymers was used to estimate the molar ratios between N-containing compounds and catechol. Organic carbon in water solutions o f polymers and FA was determined using a Carlo Erba T C M 400 analyzer. The radioactivity in the materials prepared from ~4C-labeled molecules was measured in a Packard liquid scintillation spectrometer, and in the presence o f 'lumagel'. The amounts of labeled atrazine incorporated in the polymers were calculated on the basis of their specific radioactivity. The atrazine residues present in the chloroform extracts of HA and F A were determined by high performance liquid chromatography, with SpectraPhysics equipment, including an isocratic pump, a UV-visible detector, a SP 100 RP 18 column, and a mobile phase o f water-acetonitrile (60:40 v/v, at 1 ml min -~ flow rate). TABLE 1 Elemental analysis and molar composition of model humic polymers prepared from catechol and N-containing compounds C (%) N (%) H (°/'o) O (°/o) C/N a H/C b O/C b Mole per mole of catechol Catechol Cat/glycine Cat/diglycine Cat/triglycine Cat/glucosamine

52.80 0.00 2 . 9 0 49.50 3.10 2 . 9 0 45.77 8.21 3.20 46.70 11.10 3 . 7 0 48.16 2.66 3 . 9 2

44.30 0.0 0.66 0.63 44.50 16.0 0.70 0.66 42.80 5.6 0.84 0.70 38.50 4.2 0.95 0 . 6 2 45.26 18.1 0.98 0.70

aC/N ratios on percent weight basis. bH/C and O/C ratios on atomic number basis.

0 0.29 0.53 0.58 0.32

BINDING OF ATRAZINE AND DEALKYLATED DERIVATIVES TO HUMIC-LIKE POLYMERS

21 l

RESULTS A N D DISCUSSION

Incorporation of biomolecules in catechol polymers The ash-free reaction products contained from 45 to 53% C and from 2.9 to 3.9% H (Table 1). The polymer prepared from catechol alone had the lowest H/C ratio, corresponding to a highly aromatic material. This value was only slightly modified with glycine, and was much higher with triglycine and glucosamine. The oxygen contents and the O/C ratios showed only slight differences from one polymer to another. Only in the polymer prepared from triglycine was the oxygen content clearly lower than in the other cases, probably because of the high nitrogen content of this polymer. The C/N ratios showed high variations, from 4.2 in the latter case to 18.1 for the polymer prepared in the presence of glucosamine. The biomolecule/catechol molar ratios indicated that about one mole of N-containing molecule was combined with two moles of catechol in the case of di- and triglycine, and to three moles of catechol in the case of glycine and glucosamine.

Incorporation of atrazine in catechol polymers Influence of the relative concentration of atrazine Changes in the atrazine/catechol molar ratio in the reaction medium were TABLE 2 Influence of atrazine/catechol molar ratio on the incorporation of atrazine into the catechol polymer prepared at pH 7. The initial concentration of catechol was 0.06 M Atrazine/catechol molar ratio

Atrazine in polymer (mg g-1 of atrazine added to the medium) Atrazine in the humic acid fraction (mg g-l of humic acids) Fulvic acid/humic acid (ratio of atrazine contents) Fulvic acid/humic acid (ratio of total carbon contents)

1:160

1:80

1:40

1:20

320

350

300

550

18

33

37

49

0.25

0.54

1.5

4.0

6.1

9.0

9.0

6.7

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F.G. ANDREUX ET AL.

expected to affect the incorporation of atrazine into the polymers and the polymerization reaction itself. The data of Table 2 show that nearly 320 mg g-1 of reacted atrazine were incorporated into the final polymer when the molar ratio varied from 1:160 to 1:40. When it was only 1:20, the resulting incorporation of atrazine was nearly two times higher. This indicates that the concentrations of atrazine used in this experiment did not limit its reaction with catechol derivatives. Table 2 also shows that the incorporation of atrazine in the HA-type fraction increased moderately with increasing molar ratio, whereas the ratio between atrazine in FA and atrazine in HA increased 16 times. The FA/HA ratios, based on total carbon contents, did not vary in the same proportions, although a clear tendency toward an increasing proportion of FA with increasing molar ratio was noticed. This suggests that the accumulation of atrazine in the FA-type fractions probably hindered the polymerization processes.

TABLE 3 Influence of the presence of biomolecules on the incorporation of atrazine into the catechol polymer prepared at pH 7. The initial concencentrations of catechol and of biomolecule were 0.03 M. The initial atrazine/catechol molar ratio was 1:20 Biomolecule added to catechol

Atrazine in polymer (mg g-1 of atrazine added to medium) Atrazine in the humic acid fraction (mg g-i of humic acid) Fulvic acid/humic acid (ratio of atrazine contents) Weight yield of humic acid (mg g-1 of initial reactants) Atrazine/catechol in humic acid (molar ratio) aNot determined.

None

Glycine

Diglycine

Triglycine

268

263

251

245

58.8

27.8

34.5

17.7

2.8

7.3

2.7

4.9

105

78

71

73

0.031

0.017

ND a

0.017

213

BINDING OF ATRAZINE AND DEALKYLATED DERIVATIVES TO HUMIC-LIKE POLYMERS

Influence of biomolecular compounds In the absence of the biomolecular compound, and at atrazine/catechol molar ratios of 1:20, comparison of Tables 2 and 3 indicates that the incorporation of atrazine was two times lower when the concentration of catechol in the reaction medium decreased from 0.06 to 0.03 M. The presence of biomolecular compounds (Table 3) had no effect on the proportion of atrazine incorporated into the polymers, but reduced significantly its proportion in the HA-type fraction in contrast to that in the FA-type fraction. This effect was stronger with triglycine than with glycine, and was curiously weaker with diglycine. These results suggest that biomolecules competed favorably with atrazine for incorporation into the polymers, resulting in a lower amount of atrazine in the HA-type and in the accumulation of atrazine-FA products.

Incorporation of dealkylated derivatives in catechol polymers The incorporation of atrazine into the polymers was compared with that of its three dealkylated derivatives (Table 4). In the case of atrazine, it is interesting to compare the results obtained at pH 6 with those obtained at pH 7 (Table 2). The proportion of atrazine incorporated in the polymer was

TABLE 4 Incorporation of atrazine (A) and its de-alkylated metabolites into the catechol polymer prepared at pH 6. The initial concentration of catechol was 0.06 M. The initial pesticide/ catechol molar ratio was 1:20 Pesticide or metabolite added

Pesticide in polymer (rag g-i of pesticide added to medium) Pesticide in the humic acid fraction (rag g-1 of humic acid) Fulvic acid/humic acid (ratio of pesticide contents) Pesticide/catechol in humic acid (molar ratio)

Atrazine

De-ethyl-A

De-isopropyl-A

Diamino-A

750

790

860

995

74

65

72

813

36.5

35.5

42

0.53

0.041

0.044

0.045

3.27

214

F.G. A N D R E U X ET AL,

about 1.5-times higher, but the incorporation in FA, relative to HA, was 9 times higher at pH 6 than at pH 7. The atrazine/catechol molar ratio in the HA-type fraction was similar to that in the reaction medium (0.050). The reaction of de-ethyl and de-isopropylatrazine followed a similar pattern as atrazine, although the amounts incorporated and their molar ratios in the HA fraction were higher than those of atrazine. In contrast, diaminoatrazine was almost entirely incorporated into the HA fraction, and its molar ratio was > 3, which suggested the existence of a very active copolymerization reaction with catechol. Free NH~- groups in the dealkylated metabolites should normally give rise to the formation of C - N covalent bonds with catechol-derived quinones, as shown in the case of aromatic amines [7-14]. Examples of the structures

CL

HoON¢ OH

OH

OH

(a)

CL

CL

N'J'N

NJ'N

~

NH iC3H7

HN C2H5

N~

HO

OH

OH

OH (b)

(c)

Fig. 2. Proposed structures for the reaction products of catechol with dealkylated derivatives of atrazine, diamino atrazine (a), de-ethylatrazine (b), and de-isopropylatrazine (c).

BINDING OF ATRAZ1NE AND DEALKYLATED DERIVATIVES TO HUMIC-LIKE POLYMERS

2[5

proposed for such addition products are given in Fig. 2. The presence of unreacted material was investigated in the chloroform extracts, but no free metabolite was detected. In the case of atrazine, a small proportion of this molecule was found, together with an unidentified compound which could be an oligomeric addition product of atrazine and catechol [15]. The solubility of the molecule and the reaction pH were decisive factors: monodealkylated metabolites were more soluble in water than atrazine, but still reacted slowly compared with di-dealkylated atrazine. It is likely that these reactions would have proceeded faster at higher pH. CONCLUSION

The need for improved knowledge on natural humic substances and the mechanisms of their interactions with non-humic compounds has stimulated increasing interest in the synthesis of model humic polymers. Many of these non-humic compounds contain reactive NH2- and N H - groups, especially biomolecules and numerous pesticides, that can compete and occupy free sites in the polymers. This study confirms that atrazine binds readily to humic-like polymers by reacting during the oxidative polymerization of phenols, in spite of its relatively low water solubility. Only a limited proportion was incorporated into humic acid-type polymers, and the main proportion was bound to fulvic acid-type polymers, or to chloroform-soluble catechol derivatives. This was more marked when the atrazine/catechol molar ratio increased, and in the presence of NHz-bearing biomolecules. It is likely that these fulvic acid-type and oligomeric forms favoured the migration of atrazine under field conditions. The chemical nature of the bonds between atrazine and the polymeric material was not established, but non-covalent processes such as ionexchange and charge-transfer complexation probably prevailed, as already shown by other authors [20-22]. In contrast, de-alkylated metabolites of atrazine, with their free NH2groups and in spite of their higher water solubility, probably promoted the formation of covalent bonds with oxidized polyphenols, leading to their incorporation into humic acid-type polymers. It appears, therefore, that active dealkylation of atrazine represents a key step in the formation of high molecular weight and non-extractable 'bound' atrazine residues. ACKNOWLEDGEMENTS

The authors are indebted to Mrs Bernadette Gerard and Mres Th6r&e Orel for technical assistance with the organic elemental analysis of humic compounds.

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F.G. ANDREUX ET AL.

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BINDING OF ATRAZINE AND DEALKYLATED DERIVATIVES TO HUMIC-LIKE POLYMERS

19

20 21

22

2 ]7

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