- Email: [email protected]
Contribution to the Study of Nonextractable Pesticide Residues in Soils: Incorporation of Atrazine in Model Humic Acids Prepared from Catechol
CONTRIBUTION TO THE STUDY OF NONEXTRACTABLE PESTICIDE RESIDUES IN SOILS: INCORPORATION OF ATRAZINE IN MODEL HUMIC ACIDS PREPARED FROM CATECHOL G. BERTINI, M. SCHIAVONI*, F. ANDREUX2 and J.M. PORTAL2 lE.N.S.A.I.A., B.R. 172, 2Avenue de la Foret de Haye, 54505 Vandoeuvre les Nancy Cedex, France 2Centre de Pedologie Biologique, C.N.R.S., B.P. 5, 54501 Vandoeuvre les Nancy Cedex, France
ABSTRACT The fact that a portion of pesticide residues remains in soil after solvent extraction, particularly in organic soils, indicates that some of the pesticides may be incorporated into soil humus. Studies concerning the formation of these "bound" residues under field conditions are difficult, and one alternative is to use model humic acids for investigating pesticide behaviour in soils. A humic-like polymer was synthesized by oxidation of catechol in the presence of atrazine in a phosphate buffer (pH 6 or 7). The reaction mixture was then acidified to pH 1.5, precipitating "humic acids" while "fulvic acids" remained in solution. The different fractions were extracted with chloroform to remove extractable residues of atrazine, then submitted to carbon and nitrogen determination, spectroscopic analysis and chromatography. The results suggest that atrazine is partly adsorbed by the model humic acid, then extractable, and partly non-extractable with chloroform. The nature of the different binds or associations of atrazine with synthetic polymers are discussed. Keywords: bound residues, atrazine, catechol, synthetic polymer
INTRODUCTION
The synthetic organic products used for crop protection are easily bound to soil organic matter. A fraction of these pesticides is adsorbed and extractable using organic solvents, whereas certain residues remain unextractable. These latter are called "bound residues" (Schiavon et a1.,1977; Lichtenstein et a1.,1977; Khan, 1982; Capriel et a1.,1985). The problem of bound residues of xenobiotics has received increasing attention, but the mechanism of their binding to soil humus is still undefined. The use of model
* Corresponding author
humic acids might provide new information concerning the reactions which take place under natural conditions. This type of approach has already been envisaged by several authors using phenolic or quoidal monomers and xenobiotic molecules (Mathur and Morley, 1978; You et al., 1982; Saxena and Bartha, 1983) . In this paper, model compounds were obtained by chemical oxidation of catechol in the presence of atrazine. They were studied to determine the behaviour of atrazine in a mixture of polymeric compounds, simulating the fulvic and humic substances.
106
MATERIALS AND METHODS Preparation of the models The catechol (1,2-dihydroxybenzene) and atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) are introduced into 250 ml of phosphate buffer at the final concentrations of 0.06 M and 0.003 M respectively. The oxidation process is accelerated by an oxygen stream while the reaction mixture is shaken in the dark at 20° C. The pH of the phosphate buffer is adjusted at 6 or 7 so as to compare two polymerization conditions. Indeed, the rate of catechol oxidation is closely linked to the pH of the medium and tends to zero for pH values lower than 4 (Andreux et al., 1980). The reaction is much faster at neutral or slightly basic pH values and the state of equilibrium, characterized by the formation of a brown to black-coloured solution is reached in 7-8 days at pH 6 and in 4-5 days only at pH 7. Two controls were made. The synthetic polymer was prepared in the absence of atrazine, and atrazine was subjected to the same oxidation procedure in the absence of catechol. In the latter case, atrazine was totally recovered by a single extraction of the reaction medium with chloroform, indicating that atrazine was stable under the experimental conditions employed for polymerization of the catechol. Separation of the polymers The whole of each synthetic polymer obtained is separated following a "molecular weight" criterion. By acidifying the medium to pH 1.5 using concentrated HCl, the acidinsoluble fraction is precipitated. After centrifugation, the supernatant is removed and the precipitate is lyophilized.
INCORPORATION OF ATRAZINE IN MODEL HUMIC ACIDS
The freeze-dried polymers and the aqueous solution of the low molecular weight compounds will hereafter be termed humic acids (HAs) and fulvic acids (FAs) respectively, by analogy with the terms used for soil organic matter. The index "s" indicates that we are dealing with synthetic polymers. Extraction of atrazine The "free" or weakly bound atrazine is extracted by shaking with chloroform for one hour at room temperature. The HAs fractions (250 to 400 mg according to the synthesis conditions) are extracted by 200 ml of chloroform, then filtered, dried and recovered. The chloroform extract is dried under vacuum then dissolved in 50 ml of methanol. The FAs fractions are concentrated under vacuum at 40° C then extracted with 200 ml of chloroform (1:1 v/v) . The organic phase is separated from the aqueous phase in a separatory funnel, evaporated to dryness under vacuum then dissolved in 50 ml of methanol. Each extraction is repeated until there remain no further "free" atrazine residues. Chemical analyses Elemental analysis (Carlo Erba 1104 autoanalyser): Determination were made of nitrogen and carbon contents of the HAs fractions chloroform extracted. The percentage ofnitrogen and the C/N ratios allow the calculation of the amount of atrazine incorporated in the polymers. Infra-red spectroscopy: A Beckman Model IR 10 spectrophotometer was used to obtain infrared spectra. The HAs samples were analyzed as KBr pellets using a 20:1 ratio of KBr to sample. Assigning of the ab-
INCORPORATION OF ATRAZINE IN MODEL HUMIC ACIDS
107
TABLE 1 Weight yield of the syntheses of the model humic acids (expressed as a percentage of the catechol introduced)
Catechol only Synthesis pH 6 Synthesis pH 7
11 24
Catechol + Atrazine 7 17
TABLE 2 Relative distribution of nonextractable residues of atrazine in the synthetic polymer fractions (percentage of the total atrazine introduced)
Synthetic polymer obtained at pH 6 Humic acids Fulvic acids Total
6 44 50
sorption bands was made according to Bellamy (1975). High-performance liquid chromatography: The chloroform extracts of the HAs and FAs fractions were analyzed by HPLC to determine the amount of atrazine extracted. HPLC was performed with a Spectra-Physics SP 8770 isocratic pump and a SpectraPhysics SP 8440 UI-Visible detector at 240 nm. The separation was achieved by a Spectra-Physics R-P 18 column. Samples were eluted with a water:acetonitrile mixture (6:4 v/v), at a flow rate of one ml miri1. RESULTS Yields of the polymerization The yield of the synthesis is defined as being the ratio between the weight of HAs fraction (minus the amount of atrazine incorporated) and the weight of catechol ini-
Synthetic polymer obtained at pH 7 11 48 59
tially present. The yields obtained at the two pH values were compared, both in the presence and absence of atrazine (Table 1) . The weight yield is about two times higher at pH 7 than at pH 6, in agreement with the difference of the oxidation rate of the catechol between these two pH values. Furthermore, the synthesis carried out in the presence of atrazine has a clearly lower yield which would tend to indicate that the pesticide disturbs the normal polymerization process. Overall analysis of atrazine incorporation Whatever the pH value at which the polymerization process takes place, the synthetic polymers contained 50 to 59% bound atrazine, expressed in percent of the total atrazine added. The distribution of atrazine among the HAs and the FAs fractions were respectively 12 and 88% at pH 6, and 19 and 81% at pH 7 (Table 2) .
108
INCORPORATION OF ATRAZINE IN MODEL HUMIC ACIDS
TABLE 3 C and N determination of the synthetic polymers prepared from catechol and atrazine, in the humic acid fractions obtained after solvent extraction (% of dry weight)
Catechol + Atrazine (pH 6) Catechol + Atrazine (pH 7)
C
N
C/N
38.5 45.2
2.8 1.6
13.6 28.4
The great amount of atrazine found (with respect to the introduced quantities) in the polymers synthesized at pH 7 is related to the polymerization yield, which is higher at pH 7 than at pH 6. Indeed, if the C/N ratios are compared, an incorporation of atrazine higher at pH 6 (C/N = 13.6) than at pH 7 (C/N = 28.4) is noted. These values correspond with the incorporation of one atrazine molecule, respectively for 12 catechol molecules at Ph 6 and for 26 catechol molecules at Ph 7 (Table 3). Infra-red spectra The infra-red spectra of the polymers synthesized from catechol and atrazine were compared with those of the polymers synthesized only from catechol. The incorporation of atrazine causes several structural modifications of the HAs fractions, especially in the 3200-3600 cm-1 region (valence vibrations of the O-H groups) and in the band centred around 1620 cm1 (aromatic C = C bonds and quinonoid C = O bonds) which are more intense in the presence of atrazine. It is also noted that the band centered at 1720 cm 1 corresponding to the C = 0 bonds of the COOH groups is shifted to 1740 cm 1 in the spectra of the polymers synthesized from catechol and atrazine. These polymers show also bands which are specific of
atrazine and which are all attributable to vibrations of aliphatic groups, located at 2880, 1180 and 800 cm-1 (Figs. 1 and 2) . DISCUSSION AND CONCLUSIONS
To study the reactions of atrazine with soil organic matter, the utilization of a simple model seems promising. In fact, the results, even fragmentary, allow us to bring out some interesting points. A significant amount of atrazine is incorporated in the synthetic polymers and remains unextractable by an organic solvent such as chloroform, whatever the pH of the reaction mixture. On the other hand, the pH has an effect on the amount of herbicide incorporated, probably in relation to the rate of catechol oxidation, which is greater at pH 7 than at pH 6. Thus, the reaction of atrazine with phenolic or quinoidal monomers, for example, could be slower than the polymerization of catechol, which would explain the lower ratio catechol/ atrazine incorporated at pH 7 than at pH 6. The presence of atrazine in the system also disturbs the polycondensation process. This is indicated by the lower weight yield of the polymerization and by the structural modifications of the polymers obtained, as revealed by infra-red spectroscopy. The herbicide is thus "reactive" during the polymerization, which excludes a purely physi-
INCORPORATION OF ATRAZINE IN MODEL HUMIC ACIDS
109
100j c o .~~
E
U)
b
~
(4-
4C 50_ a, u ` a~~ a
iiii ii i i i 4000
I
iii
3000
I_1.1_i . I i i i i i 2000
I
i i ~~ i ~~ i i i i i 1000 (cm-
Fig. 1. Infra-red spectra of the humic acid fraction. 100 C o II)
In
E
ni c ~ (U
4-
4C
~~5 0-
(a~~ h..
II 4000
I ~~ I 3000
2000
Fig. 2. Infra-red spectra of atrazine.
cal incorporation. The infra-red spectra also seem to indicate certain modifications within functional groups of the synthetic polymers which might become engaged in bonds with atrazine. The exact nature of
these bonds remains unknown and more extended investigation is still necessary, especially concerning the first stages of the polycondensation process.
110
REFERENCES Andreux, F., D. Golebiowska and M. Metche, 1980. Polymerisation oxydative de O-diphenols en presence ou non d'amino-acides. Cas des systemes (catecholglycocolle) et (catechol-diglycylglycine). C.R. de 1'assemblee generate du groupe polyphenols, Logrono (Espagne). Bull. de Liaison, 9:178-188. Bellamy, L.J., 1975. The infra-red spectra of complex molecules, Chapman and Hall, London. Capriel, P., A. Haisch and S.U. Khan, 1985. Distribution and nature of bound (nonextractable) residues of atrazine in a mineral soil nine years after the herbicide application. J. Agric. Food Chem., 33:567-569. Kahn, S.U., 1982. Bound pesticide residues in soil and plants. Residue Rev., 84:1-25. Lichtenstein, E.P., J. Katan and B.N. Anderegg,
INCORPORATION OF ATRAZINE IN MODEL HUMIC ACIDS
1977. Binding of "persistent" and "nonpersistent" C labelled insecticides in an agricultural soil. J. Agric. Food Chem., 25:43-47. Mathur, S.P. and H.V. Morley, 1978. Incorporation of methoxychlor-14C in model humic acids prepared from hydroquinone. Bull. Environ. Contam. Toxicol., 20:268-274. Saxena, A. and R. Bartha, 1983. Modeling of the covalent attachment of chloroaniline residues to quinoidal sites of soil humus. Bull. Environ. Contam. Toxicol., 30:485-491. Schiavon, M., F. Jacquin and C. Goussault, 1977. Blocage de molecules s-triaziniques par la matire organique. In: Soil Organic Matter Studies, I.A.E.A., Vienna, pp. 327-332. You, 1.-S., R.A. Jones and R. Bartha, 1982. Evaluation of a chemically defined model for the attachment of 3,4-dichloroaniline to humus. Bull. Environ. Contam. Toxicol., 29:476-482. 14
ABSTRACT The fact that a portion of pesticide residues remains in soil after solvent extraction, particularly in organic soils, indicates that some of the pesticides may be incorporated into soil humus. Studies concerning the formation of these "bound" residues under field conditions are difficult, and one alternative is to use model humic acids for investigating pesticide behaviour in soils. A humic-like polymer was synthesized by oxidation of catechol in the presence of atrazine in a phosphate buffer (pH 6 or 7). The reaction mixture was then acidified to pH 1.5, precipitating "humic acids" while "fulvic acids" remained in solution. The different fractions were extracted with chloroform to remove extractable residues of atrazine, then submitted to carbon and nitrogen determination, spectroscopic analysis and chromatography. The results suggest that atrazine is partly adsorbed by the model humic acid, then extractable, and partly non-extractable with chloroform. The nature of the different binds or associations of atrazine with synthetic polymers are discussed. Keywords: bound residues, atrazine, catechol, synthetic polymer
INTRODUCTION
The synthetic organic products used for crop protection are easily bound to soil organic matter. A fraction of these pesticides is adsorbed and extractable using organic solvents, whereas certain residues remain unextractable. These latter are called "bound residues" (Schiavon et a1.,1977; Lichtenstein et a1.,1977; Khan, 1982; Capriel et a1.,1985). The problem of bound residues of xenobiotics has received increasing attention, but the mechanism of their binding to soil humus is still undefined. The use of model
* Corresponding author
humic acids might provide new information concerning the reactions which take place under natural conditions. This type of approach has already been envisaged by several authors using phenolic or quoidal monomers and xenobiotic molecules (Mathur and Morley, 1978; You et al., 1982; Saxena and Bartha, 1983) . In this paper, model compounds were obtained by chemical oxidation of catechol in the presence of atrazine. They were studied to determine the behaviour of atrazine in a mixture of polymeric compounds, simulating the fulvic and humic substances.
106
MATERIALS AND METHODS Preparation of the models The catechol (1,2-dihydroxybenzene) and atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) are introduced into 250 ml of phosphate buffer at the final concentrations of 0.06 M and 0.003 M respectively. The oxidation process is accelerated by an oxygen stream while the reaction mixture is shaken in the dark at 20° C. The pH of the phosphate buffer is adjusted at 6 or 7 so as to compare two polymerization conditions. Indeed, the rate of catechol oxidation is closely linked to the pH of the medium and tends to zero for pH values lower than 4 (Andreux et al., 1980). The reaction is much faster at neutral or slightly basic pH values and the state of equilibrium, characterized by the formation of a brown to black-coloured solution is reached in 7-8 days at pH 6 and in 4-5 days only at pH 7. Two controls were made. The synthetic polymer was prepared in the absence of atrazine, and atrazine was subjected to the same oxidation procedure in the absence of catechol. In the latter case, atrazine was totally recovered by a single extraction of the reaction medium with chloroform, indicating that atrazine was stable under the experimental conditions employed for polymerization of the catechol. Separation of the polymers The whole of each synthetic polymer obtained is separated following a "molecular weight" criterion. By acidifying the medium to pH 1.5 using concentrated HCl, the acidinsoluble fraction is precipitated. After centrifugation, the supernatant is removed and the precipitate is lyophilized.
INCORPORATION OF ATRAZINE IN MODEL HUMIC ACIDS
The freeze-dried polymers and the aqueous solution of the low molecular weight compounds will hereafter be termed humic acids (HAs) and fulvic acids (FAs) respectively, by analogy with the terms used for soil organic matter. The index "s" indicates that we are dealing with synthetic polymers. Extraction of atrazine The "free" or weakly bound atrazine is extracted by shaking with chloroform for one hour at room temperature. The HAs fractions (250 to 400 mg according to the synthesis conditions) are extracted by 200 ml of chloroform, then filtered, dried and recovered. The chloroform extract is dried under vacuum then dissolved in 50 ml of methanol. The FAs fractions are concentrated under vacuum at 40° C then extracted with 200 ml of chloroform (1:1 v/v) . The organic phase is separated from the aqueous phase in a separatory funnel, evaporated to dryness under vacuum then dissolved in 50 ml of methanol. Each extraction is repeated until there remain no further "free" atrazine residues. Chemical analyses Elemental analysis (Carlo Erba 1104 autoanalyser): Determination were made of nitrogen and carbon contents of the HAs fractions chloroform extracted. The percentage ofnitrogen and the C/N ratios allow the calculation of the amount of atrazine incorporated in the polymers. Infra-red spectroscopy: A Beckman Model IR 10 spectrophotometer was used to obtain infrared spectra. The HAs samples were analyzed as KBr pellets using a 20:1 ratio of KBr to sample. Assigning of the ab-
INCORPORATION OF ATRAZINE IN MODEL HUMIC ACIDS
107
TABLE 1 Weight yield of the syntheses of the model humic acids (expressed as a percentage of the catechol introduced)
Catechol only Synthesis pH 6 Synthesis pH 7
11 24
Catechol + Atrazine 7 17
TABLE 2 Relative distribution of nonextractable residues of atrazine in the synthetic polymer fractions (percentage of the total atrazine introduced)
Synthetic polymer obtained at pH 6 Humic acids Fulvic acids Total
6 44 50
sorption bands was made according to Bellamy (1975). High-performance liquid chromatography: The chloroform extracts of the HAs and FAs fractions were analyzed by HPLC to determine the amount of atrazine extracted. HPLC was performed with a Spectra-Physics SP 8770 isocratic pump and a SpectraPhysics SP 8440 UI-Visible detector at 240 nm. The separation was achieved by a Spectra-Physics R-P 18 column. Samples were eluted with a water:acetonitrile mixture (6:4 v/v), at a flow rate of one ml miri1. RESULTS Yields of the polymerization The yield of the synthesis is defined as being the ratio between the weight of HAs fraction (minus the amount of atrazine incorporated) and the weight of catechol ini-
Synthetic polymer obtained at pH 7 11 48 59
tially present. The yields obtained at the two pH values were compared, both in the presence and absence of atrazine (Table 1) . The weight yield is about two times higher at pH 7 than at pH 6, in agreement with the difference of the oxidation rate of the catechol between these two pH values. Furthermore, the synthesis carried out in the presence of atrazine has a clearly lower yield which would tend to indicate that the pesticide disturbs the normal polymerization process. Overall analysis of atrazine incorporation Whatever the pH value at which the polymerization process takes place, the synthetic polymers contained 50 to 59% bound atrazine, expressed in percent of the total atrazine added. The distribution of atrazine among the HAs and the FAs fractions were respectively 12 and 88% at pH 6, and 19 and 81% at pH 7 (Table 2) .
108
INCORPORATION OF ATRAZINE IN MODEL HUMIC ACIDS
TABLE 3 C and N determination of the synthetic polymers prepared from catechol and atrazine, in the humic acid fractions obtained after solvent extraction (% of dry weight)
Catechol + Atrazine (pH 6) Catechol + Atrazine (pH 7)
C
N
C/N
38.5 45.2
2.8 1.6
13.6 28.4
The great amount of atrazine found (with respect to the introduced quantities) in the polymers synthesized at pH 7 is related to the polymerization yield, which is higher at pH 7 than at pH 6. Indeed, if the C/N ratios are compared, an incorporation of atrazine higher at pH 6 (C/N = 13.6) than at pH 7 (C/N = 28.4) is noted. These values correspond with the incorporation of one atrazine molecule, respectively for 12 catechol molecules at Ph 6 and for 26 catechol molecules at Ph 7 (Table 3). Infra-red spectra The infra-red spectra of the polymers synthesized from catechol and atrazine were compared with those of the polymers synthesized only from catechol. The incorporation of atrazine causes several structural modifications of the HAs fractions, especially in the 3200-3600 cm-1 region (valence vibrations of the O-H groups) and in the band centred around 1620 cm1 (aromatic C = C bonds and quinonoid C = O bonds) which are more intense in the presence of atrazine. It is also noted that the band centered at 1720 cm 1 corresponding to the C = 0 bonds of the COOH groups is shifted to 1740 cm 1 in the spectra of the polymers synthesized from catechol and atrazine. These polymers show also bands which are specific of
atrazine and which are all attributable to vibrations of aliphatic groups, located at 2880, 1180 and 800 cm-1 (Figs. 1 and 2) . DISCUSSION AND CONCLUSIONS
To study the reactions of atrazine with soil organic matter, the utilization of a simple model seems promising. In fact, the results, even fragmentary, allow us to bring out some interesting points. A significant amount of atrazine is incorporated in the synthetic polymers and remains unextractable by an organic solvent such as chloroform, whatever the pH of the reaction mixture. On the other hand, the pH has an effect on the amount of herbicide incorporated, probably in relation to the rate of catechol oxidation, which is greater at pH 7 than at pH 6. Thus, the reaction of atrazine with phenolic or quinoidal monomers, for example, could be slower than the polymerization of catechol, which would explain the lower ratio catechol/ atrazine incorporated at pH 7 than at pH 6. The presence of atrazine in the system also disturbs the polycondensation process. This is indicated by the lower weight yield of the polymerization and by the structural modifications of the polymers obtained, as revealed by infra-red spectroscopy. The herbicide is thus "reactive" during the polymerization, which excludes a purely physi-
INCORPORATION OF ATRAZINE IN MODEL HUMIC ACIDS
109
100j c o .~~
E
U)
b
~
(4-
4C 50_ a, u ` a~~ a
iiii ii i i i 4000
I
iii
3000
I_1.1_i . I i i i i i 2000
I
i i ~~ i ~~ i i i i i 1000 (cm-
Fig. 1. Infra-red spectra of the humic acid fraction. 100 C o II)
In
E
ni c ~ (U
4-
4C
~~5 0-
(a~~ h..
II 4000
I ~~ I 3000
2000
Fig. 2. Infra-red spectra of atrazine.
cal incorporation. The infra-red spectra also seem to indicate certain modifications within functional groups of the synthetic polymers which might become engaged in bonds with atrazine. The exact nature of
these bonds remains unknown and more extended investigation is still necessary, especially concerning the first stages of the polycondensation process.
110
REFERENCES Andreux, F., D. Golebiowska and M. Metche, 1980. Polymerisation oxydative de O-diphenols en presence ou non d'amino-acides. Cas des systemes (catecholglycocolle) et (catechol-diglycylglycine). C.R. de 1'assemblee generate du groupe polyphenols, Logrono (Espagne). Bull. de Liaison, 9:178-188. Bellamy, L.J., 1975. The infra-red spectra of complex molecules, Chapman and Hall, London. Capriel, P., A. Haisch and S.U. Khan, 1985. Distribution and nature of bound (nonextractable) residues of atrazine in a mineral soil nine years after the herbicide application. J. Agric. Food Chem., 33:567-569. Kahn, S.U., 1982. Bound pesticide residues in soil and plants. Residue Rev., 84:1-25. Lichtenstein, E.P., J. Katan and B.N. Anderegg,
INCORPORATION OF ATRAZINE IN MODEL HUMIC ACIDS
1977. Binding of "persistent" and "nonpersistent" C labelled insecticides in an agricultural soil. J. Agric. Food Chem., 25:43-47. Mathur, S.P. and H.V. Morley, 1978. Incorporation of methoxychlor-14C in model humic acids prepared from hydroquinone. Bull. Environ. Contam. Toxicol., 20:268-274. Saxena, A. and R. Bartha, 1983. Modeling of the covalent attachment of chloroaniline residues to quinoidal sites of soil humus. Bull. Environ. Contam. Toxicol., 30:485-491. Schiavon, M., F. Jacquin and C. Goussault, 1977. Blocage de molecules s-triaziniques par la matire organique. In: Soil Organic Matter Studies, I.A.E.A., Vienna, pp. 327-332. You, 1.-S., R.A. Jones and R. Bartha, 1982. Evaluation of a chemically defined model for the attachment of 3,4-dichloroaniline to humus. Bull. Environ. Contam. Toxicol., 29:476-482. 14