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Journal of Environmental Quality 30:520-525 (2001)
© 2001 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America

TECHNICAL REPORT
ORGANIC COMPOUNDS IN THE ENVIRONMENT

On the Interaction Mechanisms of Atrazine and Hydroxyatrazine with Humic Substances

Ladislau Martin-Netoa, Dinis Gomes Traghettab, Carlos M.P. Vaza, Silvio Crestanaa and Garrison Spositoc

a Embrapa Instrumentação Agropecuária, C.P. 741, 13560-970 São Carlos (SP), Brazil
b Centro Universitário Positivo, Av. Nossa Senhora Aparecida, 174, 80440-000 Curitiba (PR), Brazil
c Ecosystem Sciences, Hilgard Hall #3110, Univ. of California, Berkeley, CA 94720-3110

Corresponding author (martin{at}cnpdia.embrapa.br)

Received for publication March 20, 2000.

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Atrazine (6-chloro-N2-ethyl-N4-isopropyl-1,3,5-triazine-2,4-diamine) is retained against leaching losses in soils principally by sorption to organic matter, but the mechanism of sorption has been a matter of controversy. Conflicting evidence exists for proton transfer, electron transfer, and hydrophobic interactions between atrazine and soil humus, but no data are conclusive. In this paper we add to the database by investigating the role of (i) hydroxyatrazine (6-hydroxy-N2-ethyl-N4-isopropyl-1,3,5-triazine-2,4-diamine) and (ii) hydrophobicity in the sorption of atrazine by Brazilian soil humic substances. We demonstrate, apparently for the first time, that hydroxyatrazine readily forms electron-transfer complexes with humic substances. These complexes probably are the cause of the well-known strong adsorption by humic acids and they may be the undetected cause of apparent electron-transfer complexes between soil organic matter and atrazine, whose transformation to the hydroxy form is facile. We also present evidence that supports the important contribution of hydrophobic interactions to the pH-dependent sorption of atrazine by humic substances.

Abbreviations: AT, atrazine • ESR, electron spin resonance • FA, fulvic acid • FTIR, Fourier-transform infrared • HA, humic acid • HYAT, hydroxyatrazine


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ATRAZINE is used widely for the control of broadleaf and grassy weeds on both agricultural and nonagricultural land. Unfortunately, it is also very widely detected in water supplies (Thurman et al., 1991, 1992; Ritter et al., 1994; Tindall and Vencill, 1995; Goolsby et al., 1997; Kolpin et al., 1998; Clark et al., 1999). For this reason, atrazine is considered to be an important environmental contaminant, with potential carcinogenic effects of s-triazines being of growing concern in water quality management (Birardar and Rayburn, 1995). Off-site movement of atrazine can be prevented, however, through an understanding of its fate in the soil environment (Racke et al., 1997), which includes both degradation (20–150 d half-life) and retention by soil humus (Koskinen and Clay, 1997).

The mechanism of atrazine sorption by soil organic matter has been a topic of considerable controversy. Early work (Weber et al., 1969; Hayes, 1970) showed that sorption was inhibited by the low pKa (1.68) of the herbicide molecule, with proton transfer between it and carboxyl groups at low pH proposed as the probable mechanism of retention by organic colloids. Wang et al. (1990)( 1991) presented adsorption envelopes for atrazine on humic substances that support this mechanism, in that they exhibited sharp peaks at pH <= 3, depended strongly on the extent of carboxyl protonation, and were diminished in the presence of metal cations (see also Gamble et al., 1994). Martin-Neto et al. (1994) and Sposito et al. (1996) provided additional experimental support through infrared spectra demonstrating an increased content of carboxylate species in atrazine–humic acid adducts. Moreover, they also concluded, in agreement with theoretical studies by Welhouse and Bleam (1993a)(b), that the electron-donating capability of atrazine usually was not sufficient to engage an electron-transfer complexation mechanism with humic acids.

Senesi (1992), on the other hand, had proposed electron transfer between the triazine ring (or the amino groups) in atrazine and quinone-like moieties in humic acid as a principal mechanism of sorption, reasoning that such complexes would be stabilized as semiquinone species by the complex molecular structure of humic acid. Piccolo et al. (1992), Senesi et al. (1995), and Sposito et al. (1996) indeed have found this mechanism (as evidenced usually by an increase of semiquinone free radical content in atrazine–humic acid adducts) to operate in some instances. Sposito et al. (1996) have hypothesized that, for humic acids low in carboxyl groups but high in semiquinone free-radical species (indicative of advanced humification), there is a possibility of atrazine engaging in electron-transfer reactions.

In this paper, we extend the experimental database concerning the mechanisms through which atrazine is sorbed by humic acids by investigating two hypotheses: (i) that previous observations of electron-transfer reactions between atrazine and organic matter may actually have involved hydroxyatrazine instead and (i) that hydrophobic interactions also play an important role in the sorption of atrazine by organic matter. In respect to (i), the proton-promoted hydrolysis of atrazine sorbed by organic matter is facile (Li and Felbeck, 1972; Wolfe et al., 1990; Martin-Neto et al., 1994) and hydroxyatrazine is very strongly complexed by humic substances (Clay and Koskinen, 1990; Moreau and Mouvet, 1997). Its high basicity should facilitate electron-transfer reactions with organic matter (Sposito et al., 1996), although this mechanism has not been studied spectroscopically. In respect to (ii), the importance of hydrophobic interactions in the sorption of atrazine by humic substances is often conjectured (Gamble et al., 1994; Bottero et al., 1994; Lerch et al., 1997; Celis et al., 1997; Piccolo et al., 1998). Of particular relevance are recent studies by Graber and Borisover (1998), who found that atrazine was effectively blocked by water molecules from adsorbing onto the external sites of a peat sample at pH > 6, and by Chien et al. (1997), who showed that atrazine is solubilized by humic acid through partitioning into its hydrophobic domains. Chien and Bleam (1997) showed further that atrazine was stabilized in these domains by hydrogen bonding. Hydrophobic domains in humic acid are revealed clearly in recent 13C NMR data (Hu et al., 2000) and in comprehensive molecular models (Schulten and Schnitzer, 1997).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Humic Substance Samples
The humic acid (HA) and fulvic acid (FA) samples used in this investigation were extracted from a Brazilian Oxisol and from a peat site in the central region of São Paulo State. Conventional extraction and purification methods (Schnitzer, 1982) were employed to obtain humic substances of low ash content (21–23 g kg-1). Table 1 lists the elemental and functional group analysis of the humic substances (Vieira, 1996). The data differ from data typical of temperate-zone soil humic substances, but nonetheless fall into the range of observed composition for these materials worldwide (Rice and MacCarthy, 1991).


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  		Table 1. Chemical composition of the humic substance samples.{dagger}

 
Atrazine and Hydroxyatrazine Complexes
Reagent-grade atrazine and hydroxyatrazine (98% pure; Ciba-Geigy, Greensboro, NC) were dissolved in Milli-Q water (Millipore, Bedford, MA) to prepare 30 mg L-1 atrazine and 108 mg L-1 water–ethanol (50:50 v/v) hydroxyatrazine stock solutions. Exactly 15 mg of purified HA and FA were reacted with 25 mL of atrazine (AT) or hydroxyatrazine (HYAT) stock solution at fixed pH values in the range 2 to 8. The sample solutions (denoted HAAT, FAAT, HAHYAT, or FAHYAT) were mixed gently on a shaker at room temperature for 4 d in the absence of light to avoid photoreactions (Pelizzeti et al., 1990). Solid-phase products for use in spectroscopic experiments were obtained by freeze-drying the solutions. Reference HA and FA samples without AT or HYAT were prepared using the same procedures.

Electron Spin Resonance Spectra
Electron spin resonance (ESR) spectra of the freeze-dried HAAT, FAAT, HAHYAT, FAHYAT, HA, and FA samples were obtained with a Varian (Palo Alto, CA) ESR spectrometer operating at X-band frequency (9 GHz) with the sample at room temperature. Semiquinone free radicals were quantified using the approximation: intensity x line width2 (I x {Delta}H2) (Poole and Farach, 1972) based on a strong pitch standard combined with a ruby secondary standard containing Cr(III) (Martin-Neto et al., 1991). The secondary standard produces more accurate spin quantitation than the conventional calibration with the strong pitch reference because it can detect very small variations of the Q factor in the microwave cavity during an experiment. Variations of the Q factor affect the intensity of the ESR signal, causing errors in spin quantitation. Experimental conditions were carefully checked using low microwave power (ca. 1 mW), thus avoiding saturation of the semiquinone signal, providing adequate modulation amplitude (normally 1 G [10-4 T] peak-to-peak) and avoiding distortion of the signal by increasing signal linewidth (Poole and Farach, 1972).

Ultraviolet-Visible Spectra
Ultraviolet (UV)-visible spectra were obtained scanning the wavelengths between 200 and 400 nm with a Shimadzu (Kyoto, Japan) UV-visible spectrophotometer. Measurements were performed with the samples just after preparation and after shaking them for 4 d. Atrazine has a principal absorption maximum at 223 nm, and that for hydroxyatrazine is at 240 nm (Martin-Neto et al., 1994). Samples were prepared for analysis by dissolving 200 µL atrazine– or hydroxyatrazine–humic substance adducts in 3.5 mL water. To detect changes in the atrazine and hydroxyatrazine bands, all measurements were performed with HA or FA samples as references.

Fourier-Transform Infrared Spectra
Experiments were performed in transmission mode with a Bomem (Quebec, Canada) Fourier-Transform Infrared (FTIR) spectrometer using 30 mg KBr pellets. Two milligrams of HA, FA, HAAT, or FAAT were mixed with 100 mg KBr (Martin-Neto et al., 1994). The bands for carboxyl and carboxylate groups (1720 and 1610 cm-1, respectively) were carefully monitored.

Polarographic Analysis
Electrochemical analysis of AT was carried out using a 348B EG&G PARC Polarographic Analyzer (Princeton Applied Research, Princeton, NJ) connected to a 303A Hg electrode. A saturated Ag/AgCl electrode was used as a reference electrode, while a Pt wire served as sensing electrode. The sample solution of AT was placed in a 15-mL polarographic cell and analyzed by differential pulse polarography (DPP).

Samples for analysis were prepared by diluting 300 µL of the atrazine– and hydroxyatrazine–humic substance adduct solutions 20 times in Milli-Q water to obtain 6-mL samples. The pH value was adjusted to 2.3 just before analysis, as suggested by Vaz et al. (1996), so as to obtain a maximum DPP peak current. Hydroxyatrazine is not electroactive, but under our experimental conditions, the peak obtained can be attributed to the reduction of protonated atrazine (Vaz et al., 1996). This method is suitable only for detecting free electroactive AT molecules in solution (Bourque et al., 1989). Parameters for the experiment were: pulse intensity, 50 mV; scan velocity, 2 mV s-1; nitrogen gas purging, 15 min; and medium-sized Hg drops.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Table 2 presents ESR data for HA or FA and their atrazine complexes at various pH values. There was no change in the content of semiquinone free radicals after HA or FA reaction with AT. These data confirm our previous results, indicating that AT is reluctant to undergo electron-transfer reactions with humic substances. It is noteworthy that the peat HA has both low carboxyl and high free radical content, but still did not show an increase of semiquinone free radicals in its adduct with atrazine. This observation differs from our previous results with an Entisol HA sample from California that had a composition similar to the peat sample (Sposito et al., 1996).


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  		Table 2. Semiquinone content for humic acid (HA) or fulvic acid (FA) and their adducts with atrazine (HAAT or FAAT)

 
Table 3 presents ESR parameters for the HA and FA samples and their hydroxyatrazine complexes. In the hydroxyatrazine–HA adducts, there was an observable increase of stable free radical content. For samples with high spin content, such as the peat HA, the percentage increase was quite remarkable, with a maximum of 37% at pH 4.5. These results indicate that hydroxyatrazine indeed reacts through an electron-transfer mechanism with HA and FA from the Oxisol, but especially with HA from the peat, our most humified sample. The behavior of hydroxyatrazine is similar to other s-triazine herbicides, such as prometone [6-methoxy-N,N'-bis(1-methylethyl)-1,3,5-triazine-2,4-diamine], which has a significant basicity that renders it highly effective in engaging electron-transfer mechanisms to complex HA (Senesi et al., 1987).


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  		Table 3. Semiquinone content in fulvic acid (FA) or humic acid (HA) and their adducts with hydroxyatrazine (HYAT)

 
Figure 1 shows the pH dependence of AT sorption to the Oxisol HA and FA, as obtained from a combination of UV-visible spectrophotometry and polarographic methods. The polarograms showed a principal reduction peak from the presence of atrazine (peak potential = -0.9 V), but no peak from hydroxyatrazine (Vaz et al., 1996). The UV-visible spectra showed formation of hydroxyatrazine, mainly at low pH (Martin-Neto et al., 1994). (Sorption was determined by subtracting the amount of hydroxyatrazine [as determined by the UV-visible method] from that of the atrazine lost from solution as determined by polarography [i.e., sorbed amount is amount lost less that degraded to hydroxyatrazine].) Figure 1 indicates that the AT sorption capacity for HA and FA is very low for an initial AT concentration of 0.1 mol m-3. The HA showed higher sorption capacity than the FA, in agreement with the results obtained by Wang et al. (1990)(1991). The maximum sorption of AT was at pH {approx} 3 for both HA and FA, and markedly decreased at pH values other than 3. At the pH values commonly observed in soil solutions, sorption of AT by the humic substances was very low, in accordance with the data obtained by Wang et al. (1990)( 1991).



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  		Fig. 1. Graph showing adsorption envelopes for atrazine (AT; 0.14 mmol L-1) on Oxisol humic acid (HA) and fulvic acid (FA), both at a concentration of 600 mg L-1

 
The atrazine aqueous solubility dependence with pH is shown in Fig. 2 . In this experiment, starting with a saturated aqueous atrazine, the UV-visible absorption spectrum at 223 nm was monitored (Martin-Neto et al., 1994) and an increase of herbicide solubility was detected for pHs below 3.



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  		Fig. 2. The pH dependence of atrazine solubility in water as detected by UV-visible spectroscopy, monitoring the absorption band at 223 nm. The initial concentration of the saturated aqueous solution of atrazine was 0.28 mmol L-1. Bars indicate standard deviation (SD)

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Given the hypothesis that high basicity of s-triazines promotes electron-transfer reactions with humic substances (Sposito et al., 1996), an enhanced semiquinone content of HYAT–humic substance adducts relative to AT–humic substance adducts (Tables 2 and 3) is expected. This reaction mechanism can explain the much stronger adsorption of HYAT by soils (Clay and Koskinen, 1990; Celis et al., 1997, 1998; Moreau and Mouvet, 1997), and may be related to the decreasing extractability of AT from field soils in which its conversion to the hydroxy form is possible, both abiotically and microbially (Pignatello and Huang, 1991; Kruger et al., 1997). Moreover, inadvertent conversion of atrazine to hydroxyatrazine may explain some of the electron-transfer products detected in studies of AT–HA interactions, since AT conversion to HYAT is facile at low water contents, even in laboratory samples (Evangelou and Wang, 1993; Gamble et al., 1994; Celis et al., 1997, 1998).

To explain the adsorption envelope for atrazine reacted with humic substances, Wang et al. (1990)(1991) proposed that conformational changes of humic molecules determine this curve. Martin-Neto et al. (1994) and Sposito et al. (1996) observed changes in the ESR signal for semiquinone free radicals in humic substances at different pH values that could be attributed to conformational changes. Both the Canadian Spodosol HA and tropical HA investigated by Sposito et al. (1996) and Martin-Neto et al. (1994) gave strong evidence of highly inhomogeneous spin-relaxation processes for pH < 3. Fundamentally, this means that a broad distribution of local effective magnetic fields exists for the free-radical species in HA, presumably because of different local molecular environments (Czoch and Francik, 1989). In our experiments (Table 3), the linewidth of the semiquinone free radical signal decreased with decreasing pH for both the Oxisol and peat humic substances, providing evidence that the relaxation time of the excited-state semiquinone signal of these humic substances is longer at low pH. This result, in turn, means that, at low pH, there are molecular environments, including protected sites of significant hydrophobicity, which disappear at high pH because of conformational changes (induced by acidic functional group deprotonation). In these protected sites, the interaction with the external environment is less effective, resulting in an increase in the relaxation time of the semiquinone free radical. In agreement with this perspective, the peat HA (i.e., the most humified sample in our study, with high semiquinone content) had the smallest linewidth, whereas the Oxisol FA, which is the least humified sample, had the largest linewidth. Evidently, in peat HA, the sites where semiquinone radicals exist are better protected against interactions than in FA or HA of lower humification.

Senesi et al. (1996) using scanning electron microscopy proposed a globular structure for HA at pH < 4, whereas at pH > 6, an elongated structure was proposed. Globular clusters of humic substances at pH ~ 5 were observed by Ikai and Österberg (1996) and by Namjesnik-Dejanovic and Maurice (1997) using atomic force microscopy. Conformational changes in HA driven by pH also are suggested by the 2D NMR data of Chien and Bleam (1999) and by the size-exclusion chromatrography data of Piccolo et al. (1996), Engebretson and Wandruszka (1997), and Conte and Piccolo (1999), who view HA as an assembly of small molecules held together mainly by hydrophobic interactions. These results are associated with the existence of conformational changes in humic substances dependent on parameters such as pH, ionic strength, and hydrophobic interactions. These conformational changes influence mechanisms of pesticides sorption.

However, changing conformation alone does not explain decreasing atrazine sorption at pH < 3 (Fig. 1; see also Wang et al., 1991). Starting with a saturated aqueous solution of AT, we determined the water solubility of AT at different pH values by monitoring its UV-visible absorption spectrum at 223 nm. As can be seen in Fig. 2 for pH < 3, there is a noticeable increase in the intensity of the band at 223 nm, indicating an increase in solubility. As pH is decreased, AT becomes more protonated , and this mitigates the hydrophobic interaction of AT with HA and increases competition from H+ for carboxyl sites. Our simple result provides a partial explanation for the low-pH branch of the adsorption envelope of atrazine, and gives indirect support for hydrophobic mechanisms in AT complexation reactions with humic substances. Our FTIR spectra (data not shown) displayed an increase in the carboxylate band (1610 cm-1) at pH < 4, indicating that proton transfer was operating between both HA and FA and AT, similar to the results obtained by Senesi et al. (1987), Martin-Neto et al. (1994), and Sposito et al. (1996). However, the total adsorption envelope for AT (Fig. 1) cannot result solely from this mechanism, because there is a decrease in sorption for pH < 3 despite an increase in the carboxylate band as detected by FTIR.


   ACKNOWLEDGMENTS
 
The research reported in this paper was supported in part by USDA Project W-82, "Pesticides and Other Toxic Organics in Soil and Their Potential for Ground and Surface Water Contamination" and in part by PADCT/CNPq (Brazilian Agency) Project "Application of Spectroscopic Methods and Development of Equipment to Agriculture" (No. 620324/98-8). Thanks to Prof. Otaciro R. Nascimento, USP-IFSC, São Carlos (SP), Brazil, for permitting use of the Varian ESR spectrometer, and to Prof. Luiz A. Avaca, USP-IQSC, São Carlos (SP), Brazil, for permitting use of the EG&G PARC Polarographic Analyzer. Gratitude is expressed to three anonymous referees for very helpful reviews, and to Angela Zabel for excellent preparation of the typescript.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 




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