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Published online 7 November 2005
Published in J Environ Qual 34:2187-2196 (2005)
DOI: 10.2134/jeq2004.0409
© 2005 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America
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Rhizosphere Conference

Effect of Corn Root Exudates on the Degradation of Atrazine and Its Chlorinated Metabolites in Soils

K. Wengera,e,*, L. Biglerb, M. J.-F. Suterc, R. Schönenbergerc, S. K. Guptad and R. Schuline

a Department of Microbiology, Cornell University, Wing Hall, Ithaca, NY 14853
b University of Zurich, Institute of Organic Chemistry, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland
c Swiss Federal Institute for Environmental Science and Technology, Ueberlandstrasse 133, CH-8600 Dubendorf, Switzerland
d FAL) Reckenholz, Swiss Federal Research Station for Agroecology and Agriculture, Reckenholzstrasse 191, CH-8046 Zurich, Switzerland
e Institute of Terrestrial Ecology (ITO), ETH Zurich, Grabenstrasse 3, CH-8952 Schlieren, Switzerland

* Corresponding author (kmw44{at}cornell.edu)

Received for publication November 4, 2004.

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION AND CONCLUSIONS
 REFERENCES
 
DIMBOA (3,4-dihydro-2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3-one), a major benzoxazinone of Poaceae plants, was isolated and purified from corn seedlings. The effect of isolated and purified DIMBOA on the degradation of atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine], and its toxic breakdown products, desethylatrazine [2-chloro-4-amino-6-(isopropylamino)-s-triazine; DEA] and desisopropylatrazine [2-chloro-4-(ethylamino)-6-amino-s-triazine; DIA], was studied in the absence of plants using batch experiments, while the effect of corn root exudates on these compounds was determined in hydroponic experiments. Degradation experiments were performed in the presence and absence of 50 µM, 1 mM, or 5 mM DIMBOA resulting in ratios of DIMBOA to pesticide of 1:1, 20:1, and 100:1. We observed a 100% degradation of atrazine to hydroxyatrazine within 48 h at a ratio of DIMBOA to atrazine of 100:1. DIMBOA had the largest effect on atrazine, while it was about three times less effective on DEA and DIA. Corn (Zea mays L. cv. LG 2185) was exposed to 10 mg L–1 of either atrazine, DEA, or DIA for 11 d in a growth chamber experiment. Up to 4.3 µmol L–1 d–1 of hydroxyatrazine were formed in the nutrient solutions by plants exposed to atrazine, while the formation of hydroxylated metabolites from plants exposed to DEA and DIA was smaller and also delayed. The formation of hydroxylated metabolites increased in the solution with plant age in all atrazine, DEA, and DIA treatments. HMBOA (3,4-dihydro-2-hydroxy-7-methoxy-2H-1,4-benzoxazin-3-one), the lactam precursor of DIMBOA, and a tentatively identified derivative of MBOA (2,3-dihydro-6-methoxy-benzoxazol-2-one) were detected in the corn root exudates. Mass balance calculations revealed that up to 30% of the disappearance of atrazine and DEA, and up to 10% of DIA removal from the solution medium in our study could be explained by the formation of hydroxylated metabolites in the solution itself. Our results show that higher plants such as corn have the potential to promote the hydrolysis of triazine residues in soils by exudation of benzoxazinones.

Abbreviations: BPC, base peak chromatogram • ESI, electrospray ionization • HPLC, high performance liquid chromatography • MS, mass spectrometry • tr, retention time


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION AND CONCLUSIONS
 REFERENCES
 
ATRAZINE IS WIDELY USED to control annual grasses and broadleaf weeds primarily in corn and sorghum. The half-life of atrazine with respect to transformation into nonphytotoxic or less toxic metabolites is generally reported to range from 60 d to more than 1 yr. Complete mineralization is limited to generally less than 40% of the applied amount according to estimates of Assaf and Turco (1994). In soils, microbial transformation of atrazine primarily includes N-dealkylation to desethylatrazine, to desisopropylatrazine, to diaminochlorotriazine [2-chloro-4,6-diamino-s-triazine; DACT], or to a combination of these metabolites (Boundy-Mills et al., 1997). According to Panshin et al. (2000), DEA and hydroxyatrazine [2-hydroxy-4-(ethylamino)-6-(isopropylamino)-s-triazine] are the most prevalent metabolites of atrazine in soils.

Atrazine, DEA, and DIA pose a risk to surface waters and ground water on a worldwide level. Significant amounts of these compounds slowly leach from soils into the subsurface zone beneath or are transported by runoff or erosion to rivers and other surface waters (Goolsby et al., 1994; Spalding et al., 1994). Triazine-based compounds have been identified as a threat to ground water quality in many agricultural areas where they have been applied (Businelli et al., 2000). Field-monitoring studies have shown the presence of atrazine and its dealkylated metabolites DEA and DIA in many surface waters (Berg et al., 1995; Goolsby et al., 1994; Müller et al., 1997; Thurman and Meyer, 1996). Recently, endocrine disrupting effects of triazine herbicides have been described by Sanderson et al. (2000), Bisson and Hontela (2002), and Hayes et al. (2002). The Office of Pesticide Programs (OPP) (USEPA, 2002, 2003) has concluded that the toxicity of atrazine, simazine, propazine, DEA, DIA, and diaminochlorotriazine (DACT) arises from the same basic mechanism.

Certain higher plants show a remarkably versatile metabolism that protects them from the potentially phytotoxic reactions with xenobiotics such as atrazine. Xenobiotic metabolism usually starts with an oxidation or hydrolysis step that serves to produce a functional group that is suitable for subsequent conjugation to an endogenous moiety such as glutathione, glucose, or an amino acid. In grasses, benzoxazinones appear to play an important role in this metabolism. Several authors have postulated that the major benzoxazinone of corn, DIMBOA, is involved in the natural defense of plants against bacteria, fungi, and insects (Bohidar et al., 1986; Figueroa et al., 1999; Leszczynski et al., 1995; Niemeyer, 1988a; Wilkes et al., 1999), in iron transport (Pethõ, 1992a, 1992b; Tipton and Buell, 1970) and in the detoxification of s-triazine herbicides by hydroxylation within the plants (Hamilton and Moreland, 1962; Raveton et al., 1997a; Tipton et al., 1971). Benzoxazinones are also known to catalyze the hydrolysis of the organophosphate pesticide diazinon (Ioannou et al., 1980), and have been found in several grasses, for example, Sorghum spp. (Malan et al., 1984), Triticum spp., including ancient wheats (Niemeyer, 1988a, 1988b), and Zea spp., including the Mexican variety teosinte (Chang and Brewbaker, 1975), and even in a few dicot families, for example, Acanthaceae and Scrophulariaceae (Friebe et al., 1998). It has been shown that the degradation of atrazine to hydroxyatrazine was promoted in the rhizosphere of corn (Alvey and Crowley, 1996). Moreover, it has also been shown that corn (Argandona and Corcuera, 1985; Friebe et al., 1998; Pethõ, 1992a), wheat (Pethõ, 1992b; Wu et al., 2001), and rye (Pethõ, 1992b) exude DIMBOA. Up to 40 µg L–1 of DIMBOA were observed in wheat root exudates of wheat seedlings grown in an agar medium (Wu et al., 2001).

The objectives of this study were to investigate the effect of isolated and purified DIMBOA on the degradation of atrazine, and its toxic breakdown products, DEA and DIA, in the absence of plants (i.e., using batch experiments), and to determine the effect of corn root exudates on these compounds using hydroponic experiments.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION AND CONCLUSIONS
 REFERENCES
 
Materials
Triazine compounds analyzed in this study are listed in Table 1 (Fig. 1A) . Table 2 (Fig. 1B) shows different benzoxazinone derivatives present in Poaceae plants.


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  		Table 1. Investigated triazine compounds and their abbreviations.

 


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  		Fig. 1. Chemical structures of (A) investigated triazine compounds, (B) benzoxazinones, and (C) benzoxazolinones.

 

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  		Table 2. Benzoxazinone derivatives in Poaceae and their abbreviations.

 
Atrazine (ATZ), desethylatrazine (DEA), desisopropylatrazine (DIA), hydroxyatrazine (ATZOH), hydroxydesethylatrazine (DEAOH), hydroxydesisopropylatrazine (DIAOH), and simazine (SIM) were obtained from Dr. Ehrenstorfer GmbH (Augsburg, Germany).

DIMBOA was isolated from corn seedlings using the procedure described by Hartenstein et al. (1992). Corn (Zea mays cv. LG 2185) was grown in quartz sand trays at 25°C in darkness. After 14 d the aboveground shoots were separated from the roots to induce the enzymatic release of the benzoxazinone aglycones. Cells were broken by freezing at –20°C to facilitate the extraction. A portion of 220 g frozen shoots was extracted with 0.5 L ethyl acetate in a blender. The organic suspension was then extracted with 100 mL of saturated NaHCO3 solution. The alkaline aqueous phase was acidified to pH 2 with concentrated HCl, once again extracted with ethyl acetate, and evaporated to dryness in a rotary evaporator (Büchi, Postfach, Switzerland). The yellow residue was washed with 5 mL cold diethyl ether, and recrystallized from ethyl acetate yielding 200 mg of purified DIMBOA. MBOA (2,3-dihydro-6-methoxy-benzoxazol-2-one) was purchased from Fluka (Buchs, Switzerland) and HMBOA (3,4-dihydro-2-hydroxy-7-methoxy-2H-1,4-benzoxazin-3-one) was kindly provided by Dr. Dieter Sicker, University of Leipzig (Germany).

Except for MES, obtained from Fluka, the other chemicals used in this study to prepare the nutrient solutions were obtained from Merck (Darmstadt, Germany). All solutions were prepared with high-purity 18 M{Omega} cm–1 water (Millipore, Bedford, MA) and were sterile filtered (0.2 µm) before use.

Degradation Experiment
The effect of DIMBOA on the degradation of atrazine, DEA, and DIA was monitored in high-purity water (18 M{Omega} cm–1). Pesticide solutions were prepared from methanolic stock solutions. Water was autoclaved before adding the pesticides. The final concentrations of pesticides were fixed at 50 µM. Experiments were performed in the presence and absence of 50 µM, 1 mM, or 5 mM DIMBOA resulting in ratios of DIMBOA to pesticides of 1:1, 20:1, and 100:1. All material was autoclaved before use and the prepared solutions were sterile filtered (0.2 µm) to prevent microbial contamination. The reaction mixtures were placed on a horizontal rotary shaker for 48 h at 125 rpm. After this time no more significant changes were observed in the pesticide concentration of the mixtures. Additionally, DIMBOA is known to decompose to MBOA in aqueous solution with a half-life of about 5.5 h at pH 6.8 and about 50 h at pH 5.0 and 28°C (Grambow et al., 1986), and MBOA does not induce the hydroxylation of atrazine (Raveton et al., 1997a). All samples were analyzed using high performance liquid chromatography (HPLC)–UV, and HPLC–mass spectrometry (MS) measurements were performed to confirm the identity of the formed metabolites.

Nutrient Solution Experiments
Corn was grown initially in a sterile quartz sand tray for 10 d. The seedlings were carefully washed in high-purity water before they were transferred to 100-mL brown glass bottles containing 0.1 strength Hoagland solution as described by Krämer et al. (1996). The nutrient solution pH was adjusted to 6 with NaOH in all treatments. Brown glass was used to protect the plant roots against light. For additional protection the bottles were wrapped in dark foil. To adapt the corn seedlings to the growth medium, they were grown for 5 d in nutrient solution before the start of the actual experiment. Nutrient solutions were permanently aerated with 0.2-µm filtered air. The experiments were performed for 11 d in a growth chamber on a 16 h (25°C)–8 h (15°C) day–night cycle. Treatments included 10 mg L–1 of either atrazine, DEA, or DIA, along with control treatments (pure nutrient solution, and nutrient solution containing of 1% methanol, the same amount of methanol that was added in the pesticide treatments). All glassware was autoclaved and all solutions were sterile filtered (0.2 µm) before use. Treatment solutions were replaced every 2 to 3 d by freshly prepared solutions to limit microbial interferences; the replaced solutions were again sterile filtered (0.2 µm) and stored at –20°C until analysis. Before freezing, subsamples of these solutions were combined and concentrated at 40°C in a rotary evaporator (Büchi) before being analyzed for the existence of benzoxazinones in the plant root exudates using HPLC–MS.

After harvest the shoots and roots were separated, washed with deionized water, and dried at 40°C. The oven-dried plant material was ground in a titanium mill. Root and shoot samples were microwave-extracted in 5 mL ethanol at 70°C, with simazine added as recovery standard. Plant extracts were filtered (0.45 µm) and diluted to 10 mL with ethanol. The plant extracts were analyzed for atrazine, DEA, DIA, and their hydroxylated metabolites by liquid chromatography coupled with tandem mass spectrometry (HPLC–MS–MS).

HPLC–UV and HPLC–MS Analysis
A Jasco (Easton, MD) high-performance liquid chromatographic system (PU-980) equipped with a UV spectrophotometric detector (UV 970) set at 220 nm and a 851-AS autosampler was used for the nutrient solution analyses. Samples of 100 µL were injected for the analysis of atrazine, DEA, DIA, and the hydroxylated metabolites. The HPLC separations were performed on a ODS(30) column (Ultracarb 5, 150 x 4.6 mm; Phenomenex, Torrance, CA) using gradient elution at a flow rate of 1 mL min–1. Initially, an isocratic system was used for 5 min with 15% acetonitrile (HPLC grade) and 85% buffer (KH2PO4 0.001 mol L–1 in H2O, pH 7.0). Then a linear gradient was applied increasing acetonitrile to 30% within 5 min, followed by a linear gradient further increasing acetonitrile to 80% within the next 15 min. After a postrun time of 5 min, initial conditions were reestablished within 3 min, and the system reequilibrated for another 5 min. The identity of the compounds was confirmed in two samples using HPLC–MS–MS.

Benzoxazinones and reaction products in the degradation experiments and in corn root exudates were analyzed using an HP 1100 HPLC system (Hewlett-Packard, Palo Alto, CA) fitted with a diode array detector and directly connected to a Bruker Esquire LC electrospray ionization (ESI) ion trap mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) working in the positive ion mode. Five-microliter aliquots of the sample solutions were injected for the analysis. The HPLC separations were performed on an Uptisphere C18 HDO column (HP3HD#20QS, 3 µm; 200 x 2.1 mm; Interchim, Montluçon, France) using a gradient elution flow of 0.2 mL min–1 at 30°C. The mobile phase consisted of 0.1% HCOOH (formic acid) in H2O for Solvent A, and 0.1% HCOOH in acetonitrile for Solvent B. A linear gradient was used from 5 to 80% of Solvent B within 25 min.

The ESI–MS conditions were as follows: nitrogen (N2) was used as nebulizer gas (40 psi) and as drying gas (9 L min–1 N2 at 300°C). The capillary voltage was set at 4500 V, the capillary exit at 65 V, and the skimmer at 20 V; spectra were re-corded at normal resolution (0.6u full width at half-peak height), under ion charge control (ICC) conditions (10000) in the mass range from m/z 50 to 700 and 30 V trap drive value.

Atrazine, DEA, DIA, and their hydroxy metabolites in corn shoots and roots were analyzed using a Hewlett-Packard 1100 liquid chromatograph equipped with an online vacuum degassing device (DG4; Henggeler Analytic Instruments, Riehen, Switzerland), a binary gradient pump, an autosampler, an UV/VIS detector set at 220 nm, and a column oven coupled to an API 4000 mass spectrometer (Applied Biosystems, Rotkreuz, Switzerland). The HPLC separations were performed on a ODS(30) column (Ultracarb 5, 150 x 1 mm; Phenomenex) similar to that described for the nutrient solution analysis except that ammonium acetate (pH 7.0) was used instead of the phosphate buffer and the gradient elution flow was 0.1 mL min–1 instead of 1.0 mL min–1. The triple quadrupole mass spectrometer with electrospray interface (Applied Biosystems, Foster City, CA) and nitrogen as drying gas was operated in the positive ion mode. Highest fragmentation was achieved with the spray voltage set at 4500 V, the source temperature at 390°C, cone voltage at 60.0 V, entrance potential at 60.0 V, and the collision energy at 30.0 V.

Statistical Analysis
Analysis of variance (ANOVA) was performed using SYSTAT (version 10). If the F value indicated significant differences (P < 0.05), post hoc pair wise comparisons were performed using Bonferroni adjustment of probabilities. The SYSTAT t test comparison was used to compare the means of two data sets.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION AND CONCLUSIONS
 REFERENCES
 
Degradation Experiment
Figure 2 shows that DIMBOA had the largest effect on the degradation of atrazine, while it was about three times less effective on DEA and DIA degradation. Adding DIMBOA to atrazine at a ratio of 100:1 resulted in 100% degradation of atrazine to hydroxyatrazine. Only very small amounts of atrazine, DEA, and DIA were degraded to their corresponding hydroxylated metabolites (hydroxyatrazine, ATZOH; desethylhydroxyatrazine, DEAOH; desisopropylhydroxyatrazine, DIAOH) at a DIMBOA to atrazine, DEA, or DIA ratio of 1:1. Adding more DIMBOA to the reaction mixtures did not translate into an equivalent increase in the catalytic effect of DIMBOA. DIMBOA was more efficient at low than at high concentrations. No degradation or transformation of atrazine, DEA, or DIA was observed in the absence of DIMBOA during the experimental time.



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  		Fig. 2. Effect of DIMBOA on the degradation of atrazine, desethylatrazine (DEA), and desisopropylatrazine (DIA) in H2O for the DIMBOA to pesticides ratios of 1:1, 20:1, and 100:1.

 
The results of the HPLC–MS measurements of the aqueous reaction mixture DIMBOA + atrazine at the 1:1 ratio are shown in Fig. 3 . The base peak chromatogram (BPC) (Fig. 3A) showed four signals with various intensities. The formation of hydroxyatrazine (retention time, tr = 9.7 min, [M + H]+ = m/z 198, Fig. 3B) as a degradation product of atrazine (tr = 19.1 min, [M + H]+ = m/z 216, Fig. 3F) was confirmed by ESI–MS. There was no DIMBOA left at the end of the experiment, but its degradation product MBOA appeared (tr = 14.2 min, [M + H]+ = m/z 165, Fig. 3D). The signal at tr = 16.7 min corresponds to 1-chloro-3,5-diethylamino-2,4,6-triazine (DiEt-Atz, [M + H]+ = m/z 202, Fig. 3E). As the latter was also present in the purchased reference standard, it was not considered relevant for the reaction mechanism. The reaction mixture also contained an unidentified component with tr = 13.7 min and two signals in the mass spectrum ([M + H]+ = m/z 340 and 196, Fig. 3C).



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  		Fig. 3. High performance liquid chromatography (HPLC)–mass spectrometry (MS) investigation of the 1:1 DIMBOA + atrazine reaction mixture: (A) base peak chromatogram (BPC) and (B–F) electrospray ionization (ESI) mass spectra corresponding to the labeled signals at retention time (tr) 9.7, 13.7, 14.2, 16.7, and 19.1 min, respectively.

 
In the reaction mixtures of DIA and DEA with DIMBOA, we observed the formation of the respective hydroxylated metabolites, DEAOH, and DIAOH. No DIMBOA was detected in the reaction solutions at the end of the experiment, but MBOA and also the unidentified compound at tr = 13.7 min with m/z 340 and 196 (BPC and ESI–MS data for the DEA and DIA reaction mixtures not shown).

Nutrient Solution Experiment
Corn plants exposed to DEA and DIA responded with a significant decrease in root and shoot dry weight (Fig. 4A and 4B) , while there was no significant effect on biomass yield due to the atrazine treatment.



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  		Fig. 4. Treatment effects on the (A) root and (B) shoot biomass production (g plant–1) of corn (Zea mays L. cv. LG 2185). Columns with no common index are significantly different at P ≤ 0.05.

 
Concentrations of the triazines and their hydroxylated metabolites in plant roots were corrected for the recovery efficiency of the internal standard simazine, which was for root extracts 65 ± 13% (n = 10). In the roots, 15.5 µmol kg–1 of hydroxyatrazine were observed in plants exposed to atrazine, while 127 µmol kg–1 of DEAOH and 126 µmol kg–1 of DIAOH were observed in plants exposed to DEA or DIA, respectively. In the shoots, hydroxylated metabolites were also observed, but could not clearly be quantified due to the low recovery of the internal standard simazine in shoot extracts. The concentrations of atrazine, DEA, and DIA were in both, roots and shoots, below the detection limit.

Figure 5 shows the formation of hydroxylated metabolites in the nutrient solutions for the different triazine treatments. Up to 4.3 µmol L–1 d–1 of hydroxyatrazine were observed for plants exposed to atrazine, while the formation of hydroxylated metabolites from plants exposed to DEA or DIA was smaller and also delayed. The formation of hydroxylated metabolites increased with plant age in all atrazine, DEA, and DIA treatments. Only the formation of hydroxyatrazine slightly decreased toward the end of the experiment in the atrazine treatment. This decrease, however, was not statistically significant.



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  		Fig. 5. Formation of hydroxylated metabolites in the nutrient solutions per day (µmol L–1 d–1), where plants were exposed to 10 mg L–1 of either atrazine, desethylatrazine (DEA), or desisopropylatrazine (DIA). Atrazine treatment: hydroxyatrazine (ATZOH); DEA treatment: hydroxydesethylatrazine (DEAOH); DIA treatment: hydroxydesisopropylatrazine (DIAOH).

 
Figure 6A shows the HPLC–MS analysis of the corn root exudates in the concentrated nutrient solution sample. Besides the triazine compounds and their hydroxylated metabolites, several unidentified compounds were observed on the BPC (tr = 8.3 min, [M + H]+ = m/z 256; tr = 10.1 min, [M + H]+ = m/z 239 and 212; tr = 11.8 min, [M + H]+ = m/z 453; tr = 17.4 min, [M + H]+ = m/z 243; Fig. 6A). The unknown compound with the two signals m/z 340 and 196, which had also been found in the reaction mixtures of pure DIMBOA and pesticide (Fig. 3), was also detected in the corn root exudates. Additionally, the presence of 3,4-dihydro-2-hydroxy-7-methoxy-2H-1,4-benzoxazin-3-one (HMBOA) (tr = 11.6 m/z 178; MH+–H2O), the lactam precursor in the plant biosynthesis pathway of DIMBOA, was present in the corn root exudates (extracted ion chromatogram, EIC; Fig. 6B). To prove the presence of HMBOA, we spiked the exudates sample by adding pure HMBOA. The comparison of the extracted ion chromatograms (Fig. 6B, 6C) and the ESI–MS spectra (Fig. 6D, 6E) of the unspiked with the spiked sample clearly show that HMBOA was present in the corn root exudates.



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  		Fig. 6. High performance liquid chromatography (HPLC)–mass spectrometry (MS) analysis of corn root exudates. (A) Base peak chromatogram (BPC) of root exudates; (B) extracted ion chromatogram (EIC) at m/z 178 (corresponding to HMBOA-H2O which occurs in the ion source); (C) EIC at m/z 178 of the exudates sample spiked with pure HMBOA; (D) electrospray ionization (ESI)–MS at 11.6 min of the exudates samples; (E) ESI–MS at 11.6 min of the exudates sample spiked with pure HMBOA.

 
Figure 7 shows the fractions of atrazine, DEA, and DIA that were removed from the nutrient solutions. Losses due to adsorption onto material or volatilization were negligible (data not shown). The largest part, about 70% for atrazine and DEA, and about 90% for DIA, in the removal of the three compounds was due to unaccounted metabolites. About 30% of the atrazine and DEA removal from the nutrient solution was due to the formation of hydroxylated metabolites in the solution medium itself, while for DIA that fraction was about 10% (Fig. 7). The hydroxy metabolites measured in the plant roots and shoots only explained 1 to 2% of the pesticide removal.



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  		Fig. 7. Mass balance of pesticide removal from nutrient solutions. The numbers at the top of the columns correspond to the total removal of atrazine, desethylatrazine (DEA), or desisopropylatrazine (DIA) (expressed in µmol) from the solution within the experimental time of 11 d.

 

   DISCUSSION AND CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION AND CONCLUSIONS
REFERENCES
 
Degradation Experiment
The observation that increasing DIMBOA concentrations enhanced the degradation of atrazine, DEA, and DIA has also been reported by Raveton et al. (1997a), who studied the effect of different purified benzoxazinones on the degradation of atrazine, and by Ioannou et al. (1980), who described a catalyzing effect of DIMBOA on diazinon degradation. But in contrast to Raveton et al. (1997a), we observed a 100% degradation of atrazine within 48 h already at a ratio of DIMBOA to atrazine of 100:1, while Raveton et al. (1997a) observed full degradation only at DIMBOA to atrazine ratios of more than 1000:1. According to Raveton et al. (1997a) concentrations of DIMBOA as high as 10 mM were required to obtain hydroxylation of atrazine, while we observed the hydroxylation of atrazine, DEA, and DIA already at concentrations that were about 200 times lower. The lower detection limit might be due to differences in the analytical methods. Raveton et al. (1997a) used radiolabeled compounds, separated them on thin layer chromatography, and measured the radioactivity of the obtained fractions by scintillation counting. We used HPLC and HPLC–MS with direct injection of filtered samples onto the HPLC column. All DIMBOA used in the chemical reaction mixtures was degraded to MBOA at the end of the experiment. DIMBOA is known to decompose in aqueous solution near neutral pH to MBOA, with a half-life of about 5.5 h at pH 6.8 and about 50 h at pH 5.0 at 28°C (Grambow et al., 1986).

Interestingly, there was also an unidentified compound at 13.7 min with m/z 340 and 196. Due to the similarity of the UV-spectra of this unidentified compound with MBOA and the similar retention time on the analytical column, we propose a MBOA derivative with an additional methoxy group either at Position 7 (R3), or at the nitrogen atom at Position 3 (R1) (Table 2, Fig. 1C). The 2,3-dihydro-6,7-dimethoxybenzoxazol-2-one (M2BOA) with its 3,4-dihydro-2,4-dihydroxy-7,8-dimethoxy-2H-1,4-benzoxazin-3-one (DIM2BOA-Glc) precursor in corn has been isolated and identified by Klun et al. (1970). A DMBOA (2,3-dihydro-3,4-dimethoxybenzoxazol-2-one), an MBOA derivative with the additional methoxy group at the nitrogen atom, would be a novel compound, which has not yet been described in literature so far. Unfortunately, it was not possible to clearly identify the structure of this compound due to its low concentration. In wheat plants, high concentrations of 3,4-dihydro-2-hydroxy-4,7-dimethoxy-2H-1,4-benzoxazin-3-one-ß-D-glucopyranoside (HDMBOA-Glc) have been observed (Grambow et al., 1986), and accumulation of HDMBOA-Glc was shown to be induced in wheat (Bücker and Grambow, 1990), wheat and rye (Oikawa et al., 2002), and corn leaves (Oikawa et al., 2001) by treatments that are known to elicit defense reactions in Poaceae plants. After enzymatic release from the glucosidic precursor, they form highly instable HDMBOA aglycones, which are rapidly degraded to MBOA (Grambow et al., 1986). Possibly, our isolated and purified DIMBOA standard contained traces of a MBOA derivative with an additional methoxy group. It may also have formed in the reaction mixtures of DIMBOA with the triazine pesticides.

The mechanism by which DIMBOA catalyzes the hydroxylation of atrazine, DEA, or DIA is not yet clear. It seems plausible that a heterolytic cleavage of the N–O bond of DIMBOA is involved, rendering a highly electrophilic compound susceptible to nucleophilic attack at different positions (Hashimoto and Shudo, 1996). The 7-methoxy group situated at the para-position to the hydroxamic acid group or a free hydroxy group at Position 2 may have facilitated the heterolytic cleavage of the N–O bond of the benzoxazinone as proposed by Hashimoto and Shudo (1996). The electrophilic reactivity depends on the leaving group properties of the R1 group (Table 2, Fig. 1B) in Position 4 (Grambow et al., 1986). However, our HPLC–MS analysis did not show the formation of chlorinated DIMBOA derivatives, which were postulated by Raveton et al. (1997a) as by-products of the reaction of DIMBOA with atrazine.

Nutrient Solution Experiment
While Wu et al. (2001) observed that different wheat accessions exuded between 50 and 200 nmol L–1 of DIMBOA, we did not detect DIMBOA in our corn root exudates. However, we clearly identified HMBOA, the lactam precursor in the biosynthesis pathway of DIMBOA (Fig. 6). In addition, we observed an unidentified compound with m/z 340 and 196, which might be an MBOA derivative, as it also appeared in the experiments with pure chemical reaction mixtures. Concentrations of DIMBOA-Glc and its 8-methoxylated analog (DIM2-BOA-Glc) decrease with plant age (Cambier et al., 2000). These compounds were no longer detectable in wheat leaves 3 wk after germination in a study by Bücker and Grambow (1990). Thus, a possible reason for the fact that we did not find DIMBOA in the root exudates of our corn seedlings may have been that our corn seedlings had already reached an age at which DIMBOA-Glc concentrations were no longer detectable when the samples were taken. Interestingly, Bücker and Grambow (1990) observed a drastic increase of the benzoxazinone glucoside HDMBOA-Glc in wheat leaves at the same time when the DIMBOA-Glc decreased in response to an inoculation with a pathogen. The induced accumulation of HDMBOA-Glc as plant response to certain stress elicitors has also been reported by Oikawa et al. (2001)(2002). This indicates that some stress factors may have induced an increased biosynthesis of defense compounds. The fact that the formation of hydroxylated metabolites increased with plant age in our hydroponic experiments may also indicate an increased plant defense reaction. In corn roots, the concentration of HDMBOA-Glc was found to be the main benzoxazinone glucoside 10 d after germination (Cambier et al., 2000). Friebe et al. (1998) observed much higher amounts of DIMBOA-Glc in corn root exudates than of the corresponding aglycones. According to Raveton et al. (1997a), DIMBOA monoglucosides are as effective in catalyzing the hydroxylation of atrazine as the DIMBOA aglycone. It is possible that benzoxazinone glucosides were present in our corn root exudates. The presence of the tentatively identified MBOA derivative would fit such an interpretation. We did not analyze the exudates for the presence of benzoxazinone glucosides, as they would have been hard to detect on the HPLC system used in our experiments due to the high polarity of these compounds and the resulting low retention time on the analytical column.

Alvey and Crowley (1996) suggested that an increased formation of hydroxyatrazine in the rhizosphere of corn exposed to atrazine may be due to atrazine uptake by the plant and excretion of hydroxyatrazine after hydrolysis within the plant. While we observed about 2 to 3 µmol L–1 of hydroxyatrazine in the corn roots, we found about 5 to 8 µmol L–1 of hydroxyatrazine in the surrounding nutrient solution medium resulting in a concentration gradient unlikely to drive the excretion of hydroxyatrazine from the root cells into the surrounding nutrient solution medium. Within the plants, DIMBOA glucosides are stored in the cell vacuoles. According to Raveton et al. (1997b), the hydroxylation of atrazine by DIMBOA is likely to take place in the plant cell vacuoles due to the fact that DIMBOA is highly active at slightly acidic pH, which is the case of vacuolar pH, and much less effective at pH 7, which is a pH close to that of the plant cell cytosol. Raveton et al. (1997b) also demonstrated that hydroxyatrazine accumulated in the plant cells, and was unable to diffuse freely into the medium. These observations do not fit the hypothesis of Alvey and Crowley (1996). For benzoxazinones, however, average concentrations of 3 to 5 mmol kg–1 fresh weight in the roots of corn plantlets were reported by Argandona and Corcuera (1985). These values are much higher than benzoxazinone concentrations of corn root exudates reported by Friebe et al. (1998). In this case the resulting concentration gradients would in fact be sufficient to drive substantial root excretion of benzoxazinones into the surrounding medium.

About 70% of atrazine and DEA, and about 90% of DIA removal from the nutrient solutions could not be accounted for by our analyses. As atrazine is readily taken up by plants, the unaccounted metabolites in our nutrient solution experiment may be explained by plant uptake and formation of metabolites not analyzed in this study. Besides hydroxylation of atrazine by benzoxazinones, corn also uses the formation of glutathione conjugates as detoxification pathway (Shimabukuro et al., 1970). Both detoxification pathways, the hydroxylation by benzoxazinones and the formation of glutathione conjugates, were shown to concurrently occur within corn plants by Cherifi et al. (2001). We did not analyze corn roots and shoots for the presence of glutathione conjugates. Additionally, the formation of non-extractable residues could also have contributed to the unaccounted for metabolites.

Up to 30% of the disappearance of atrazine and DEA, and up to 10% of DIA removal from the solution medium in our study could be explained by the formation of hydroxylated metabolites in the nutrient solution itself. This indicates that this pathway may indeed play an important role in the fate of atrazine and its toxic breakdown products, DEA and DIA, in the rhizosphere. Hydroxylated metabolites of triazines were frequently associated with a decline in bioavailability and therefore toxicity due to the formation of soil-bound residues as observed by Mersie and Seybold (1996) and Capriel et al. (1985). Our results show that higher plants such as corn have the potential to promote the hydrolysis of triazine residues in soils by exudation of benzoxazinones, thus reducing their toxicity and mobility. The ability of certain plants to exude high amounts of benzoxazinones into the rhizosphere holds promising potential with respect to environmental protection and sustainable management of agricultural land.


   ACKNOWLEDGMENTS
 
This study was financially supported by the Indo-Swiss Collaboration in Biotechnology (ISCB) of the Swiss Agency of Development and Cooperation (SDC), Project BR1, and the Swiss National Science Foundation, Fellowship Award, Project PA00A-101501. We greatly thank Dr. Dieter Sicker of the University Leipzig, Germany, for providing the HMBOA reference standard, and Dr. Christa Werner of the University Zurich, Switzerland, for making all the gray literature about benzoxazinones available to us she had been collecting for years. Special thanks also to René Saladin of the Institute of Terrestrial Ecology, ETH Zurich, Switzerland, for his help in the isolation of DIMBOA from corn tissues.


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 RESULTS
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