Environ, Sci. Technoi. 1993, 27, 1943-1946
Rapid Hydrolysis of Atrazine to Hydroxyatrazine by Soil Bacteria Raphl T. Mandelbaum,*·* Lawrence P. Wackett,*·* and Deborah L. Allan* Department of Biochemistry and Institute for Advanced Studies in Biological Process Technology and Department of Soil Science, University of Minnesota, St. Paul, Minnesota 55108
Introduction Atrazine [ 2-chloro-4- (ethylamino)-6-(isopropy lamino) 1,3,5-triazine] is a widely used s-triazine herbicide. Approximately 800 million pounds was used in the United States between 1980 and 1990 (1). Numerous studies on the environmental fate of atrazine have shown that it is transformed slowly (2,3). Atrazine degradation can occur via biotic and abiotic processes. N-Dealkylation, dechlorination, and ring cleavage are the major degradative processes for atrazine. It is widely accepted that the atrazine dechlorination reaction in soils is a soil-catalyzed chemical process (Figure 1), while N-dealkylation reactions are biologically mediated (3-12). While s-triazine compounds with less bulky sidechain substituents undergo bacterially mediated dechlorination (13), atrazine was not transformed to hydroxyatrazine in this or other studies of bacterial atrazine degradation (3,14). Only a slow dechlorination of atrazine by soil fungi has been reported (7,15). Other data are interpreted to support nonbiological mechanisms of atrazine hydrolysis. Soils reported sterilized by sodium azide or heat retained the capacity to form hydroxyatrazine, presumably by organic matter catalysis (4, 16-19). These chemical transformations are strongly pH dependent with both acid and alkaline conditions promoting hydrolysis of atrazine (10, 20). The transformation of atrazine to hydroxyatrazine is of environmental significance. The latter compound is not effective as a herbicide (21). Several studies have shown that hydroxyatrazine rapidly becomes unavailable for extraction from soil, due to either biodegradation, bound residue formation, or both
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Environ. Sci. Technol. 1993.27:1943-1946. Downloaded from pubs.acs.org by EASTERN KENTUCKY UNIV on 08/23/18. For personal use only.
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(8, 22, 23).
In this study, we report the rapid transformation of atrazine to hydroxyatrazine at neutral pH by a soil bacterial mixed culture LFB6. This culture has been obtained from soil on the basis of its ability to utilize atrazine as a sole source of nitrogen (24). Addition of bacteria to atrazinecontaining artificial growth media or soils yielded hydroxyatrazine. The transformation was hydrolytic, as demonstrated by 180-labeling experiments. The observed rates are extremely fast, which suggests that small populations of soil bacteria may produce significant quantities of hydroxyatrazine. Materials and Methods The isolation of mixed culture LFB6 is described elsewhere (24). Culture LFB6 was grown in 500-mL Erlenmeyer flasks containing 300 mL of atrazine medium (24) for 3 days (ODgoo > 1) without shaking. The culture was then harvested by centrifugation (6000g, 20 min) and washed twice with 0.1 N sodium phosphate buffer (pH excess nutrients and residual atrazine 7.0) to remove t Department of Biochemistry and Institute for Advanced Studies in Biological Process Technology. Department of Soil Science. 1
0013-936X/93/0927-1943$04.00/0
©
1993 American Chemical Society
Chemical
c-nh-c2hb
h7c3-hn-c
Hydroxyatrazine
Cl
N'
Biological Dealkylation
h2n-c^
;-C,
C-NH2
Desethyldeslsopropylatrazlne
Previously reported atrazine degradation pathways in soli. Figure The microbial dealkylation of atrazine to form desethyldesisopropylatrazine may involve more than one microorganism. 1.
metabolites. The pellet was resuspended in buffer to yield 1.5 mg of protein/mL. Soil inoculation experiments were conducted in 20-mL screw cap glass vials containing 3 g of air-dried soil (1.5% moisture) sieved through a 20-mesh screen. The soil was moistened with 1 mL of deionized water and preincubated for 3 days at 30 °C in the dark. Atrazine (460 /umol/mL) was suspended in methanol and sonicated for 30 sat 80% output of a Biosonic sonicator (Bronwill, Rochester, NY) to help solubilize the crystalline atrazine and reduce suspended particle size. The short sonication process did not cause any decomposition of the atrazine as determined by high-pressure liquid chromatography (HPLC) analysis. The atrazine suspension (6 µL) was thoroughly mixed into the preincubated soil and allowed to equilibrate at 4 °C in the dark for an additional period of 3 days. The experiment was initiated by adding 2 mL of culture LFB6 (0.75 mg of cell protein/mL) to the preincubated soil. The slurry was thoroughly stirred with a sterile spatula, the vials were capped, and the mixture was incubated on a reciprocal shaker (50 strokes/min) at 30 °C. Slurry samples removed before the end of the experiment were centrifuged to remove the soil. The supernatant was passed through a 0.2-¿um filter and frozen at -70 °C until analysis. For the labeling experiment with H2180,1 mL of 97.3 % H2180 (MSD Isotopes, St. Louis, MO) was combined with 1 mL of the atrazine suspension (prepared as previously described), and the solution was equilibrated at 4 °C overnight. Culture LFB6 (1 mL; 1.5 mg of cell protein/ mL) was centrifuged, and the pellet was dried for 10 min under a slow stream of air to further reduce its water content. The experiment was started by adding the atrazine suspended in H2180 to the air-dried pellet. The test tube was vigorously shaken for 30 s and then incubated 1 h at 30 °C. The reaction mixture was divided into 10 aliquots of 100 µL each and immediately frozen at -70 °C. Each 100µ aliquot was analyzed by HPLC. The eluting hydroxyatrazine peaks were pooled, the solvent volume was reduced using a rotary evaporator (Buchi, Switzerland) operated at 45 °C, and the residue was resuspended in 0.1 mL of methanol. Direct-insertion mass spectrometry was Environ. Sci. Technoi., Vol. 27, No. 9, 1993
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performed in a glycerol matrix with a Kratos mass spectrometer (Kratos) operated in the fast atom bombardment mode with xenon. Crude protein extracts from a 3-day-old LFB6 culture were prepared by sonicating a 3-mL cell suspension (OD6oo = 5.0) in 0.1 N sodium phosphate buffer (pH 7.0) for 45 s on ice at 50% intensity using a Biosonic sonicator. Broken cells were removed by centrifugation, and the supernatant was filtered through a 0.2-µ filter. Protein content of the resulting crude extract was assayed with a bicinchoninic acid assay (Pierce Chemical Co, Rockford, IL) and adjusted to 0.5 mg/mL using 0.1 N phosphate buffer (pH 7.0). The crude extract was incubated with 30 ppm atrazine at 30 °C. Metabolites were analyzed by HPLC. Soil metabolites were determined as previously described (24), and crude extract metabolites were analyzed using a 300 X 4.6 mm, 5-µ Cs column (Phase Separation Inc. Norwalk, CT). Atrazine (99.6%) was purchased from Chem Service Chemical Co. (WestChester, PA). 14C-uniformly ring-labeled atrazine (7.8 mCi/mmol; 99.6% radiochemical purity) was purchased from Sigma Chemical Co. (St. Louis, MO). Authentic samples of desisopropylatrazine, desethylatrazine, hydroxyatrazine, hydroxydesisopropylatrazine, and hydroxydidesalkylatrazine were a gift from Ciba Geigy Corp. (Greensboro, NC). Individual 100 ppm stock solutions of authentic atrazine and metabolite standards were prepared in methanol-aqueous 0.1 N H3PO4 and stored at 4 °C. Results and Discussion
The purpose of this study was to determine whether a bacterial mixed culture, previously reported to mineralize atrazine in a liquid growth medium (24), could metabolize atrazine to hydroxyatrazine in soil. This was of interest since hydroxyatrazine formation has not previously been attributed to bacterial activity. Moreover, it is widely reported that the formation of hydroxyatrazine in soil is due to abiotic processes (3). In Webster clay loam and silica sand, each spiked with 100 ppm atrazine and inoculated with mixed bacterial culture LFB6, hydroxyatrazine was detected after 1 h (Figure 2). Hydroxyatrazine was rigorously identified by HPLC retention time, TLC Rf value, ultraviolet spectroscopy, and mass spectrometry. After 24 h, more than 80 % and 95 % of the atrazine in the clay loam soil and the sand samples, respectively, were degraded. Hydroxyatrazine was formed as a transient intermediate compound which was further degraded. Previously, culture LFB6 in liquid media was shown to liberate the atrazine ring carbon atoms as CO2 (24). Surprisingly, dealkylated metabolites such as desisopropylatrazine or desethylatrazine were not detected (at a detection level of 100 ppb) except for the uninoculated silica sand treatment in which a trace amount of desisopropylatrazine was formed. Previous reports of microbial atrazine degradation indicated dealkylation to be the initial metabolic step (3). Degradation rates of atrazine in soil by culture LFB6 far exceeded those previously reported for native soils or bacterial cultures. Resting cell suspensions of culture LFB6 degraded atrazine at a rate of 0.13 mmol per 100 mg of cell protein per h. Similar degradation rates have only been reported for chemical hydrolysis of atrazine at pH values above 13 or below 1 (4) or under the combined effect of pH 4 and a high concentration of humic acid in 1944
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Standard mix B D
OI
234
56
7
8
9101112
Time (min) Figure 2. High-pressure liquid chromatography analysis of soil inoculated with culture LFB6. Key: A, desethyldesisopropylhydroxyatrazlne; B, desisopropylhydroxyatrazine; C, desethylhydroxyatrazine; D, desisopropylatrazine; E, desethylatrazine; F, hydroxyatrazine; G, atrazine. 1, extract from uninoculated control; 2, 1 h after inoculation with culture LFB6; 3, 24 h after inoculation with culture LFB6. a muck soil (19). Thus, it was of interest to determine whether high rates of atrazine degradation could be catalyzed by bacterial enzymes at neutral pH. In Figure 3, a cell-free crude protein extract of culture LFB6, buffered at pH 7.0, rapidly transformed atrazine to hydroxyatrazine (Figure 3B). After 24 h, hydroxyatrazine was further degraded to a more polar metabolite with a retention time similar to those recorded for authentic samples of dealkylated hydroxyatrazine (Figure 3C). Atrazine degradation did not occur in the buffer alone (Figure 3A). A control of protein alone indicated that atrazine or hydroxyatrazine was not present in the protein preparation (Figure 3D). Crude extract boiled for 10 min lost its ability to degrade atrazine (data not shown). These experiments demonstrated that hydroxyatrazine formation occured at neutral pH and required heat-labile component(s) in cell-free protein extracts. Dealkylated s-triazines such as desethylsimazine were dechlorinated by a Pseudomonas sp (13) via a proposed hydrolytic mechanism. Similarly, culture LFB6 could dechlorinate atrazine under both aerobic and oxygenlimited conditions. Thus, we hypothesized a hydrolytic mechanism was operative. However, the apparent hydrolytic dechlorination of pentachlorophenol to tetrachloro-p-hydroquinone is now known to be catalyzed by a flavoprotein oxygenase (25). In this context, it was important to determine the source of oxygen in biologically derived hydroxyatrazine. We have determined that hydroxyatrazine formation by culture LFB6 is hydrolytic. Atrazine exposed for 1 h to nongrowing cells of culture LFB6 in H2180 yielded [ 180] hydroxyatrazine as demonstrated by fast atom bombardment mass spectroscopy (Figure 4). The major peak
-1-1-1-1-1-1-1-1-12.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
22.5
Time (min) Figure 3. High-pressure liquid chromatography analysis of atrazine in crude extract prepared from culture LFB6: (A) control of atrazine In buffer (pH 7.0) after 24 h; (B) atrazine In crude extract after 1 h; (C) disappearance of atrazine In crude extract after 24 h; (D) crude extract not amended with atrazine.
treatment consisting of authentic hydroxyatrazine solubilized in 97.3% H2180 did not show any spontaneous exchange of 180 hydroxyl groups, even when the hydroxyatrazine was incubated with H2180 for 24 h. The small peak at m/z 198 in Figure 4B was due to some residual H2160 carried over from bacterial cells grown in H2160containing medium. Mass spectra of authentic hydroxyatrazine (Figure 4A) yielded a hydroxyatrazine peak at m/z 198 (197 +1). These findings suggest that microbial dechlorination of atrazine may occur in oxygen-limited environments such as groundwater and subsoil. Many authors cite the work of Armstrong et al. (4) in support of a chemical mechanism for soil hydroxyatrazine formation (3, 8, 9,11,12,18,19,26,27). In contrast, our work suggested that microbial degradation of atrazine to hydroxyatrazine may be significant in groundwater and soil. In this light, it is important to reevaluate some of the points supporting the conclusion that hydroxyatrazine in the environment is chemically formed: (a) Soil boiled for 15 min and then incubated for over 30 days enhanced the degradation of atrazine by more than 20-fold (4). It was concluded that the “sterilized” soil enhanced the degradation of atrazine via a chemical pathway. Numerous soil metabolism studies have shown that boiling for 15 min will not sterilize soils but will likely enrich for heat-resistant bacteria (28). (b) No microbial degradation of atrazine was detected following perfusion of “sterilized” soil with medium containing 0.3 g/L ammonium nitrate and 0.1 g/L sucrose as a carbon source (4). In our studies, such high levels of ammonium nitrate strongly inhibited atrazine biodegradation and sucrose could not serve as a carbon source for atrazine-degrading bacteria, (c) In a nonsterile soil, the correlation between high organic matter and hydroxyatrazine formation could have resulted from increased microbial enzymatic activity associated with high levels of organic matter (29). We demonstrated the hydrolytic dechlorination of atrazine by a bacterial culture in soil. The dechlorination is mediated by bacterial enzymes and not via chemical hydrolysis. We have obtained over 30 atrazine-degrading bacterial cultures out of 100 soil samples taken from three separate atrazine-contaminated sampling sites. Many of those cultures produced hydroxyatrazine from atrazine. This suggests that biological transformation of atrazine to hydroxyatrazine may be widespread in soils previously exposed to atrazine.
Acknowledgments
This work was supported by a grant from the Legislative Commission on Minnesota Resources to L.P.W. and D.L.A. and partially supported by postdoctoral fellowship Grant SI-0108-89 from BARD, The United States-Israel Binational Agricultural Research and Development Fund, to R.T.M. The assistance of Tom Krick with mass spectrometry is gratefully acknowledged.
Literature Cited Fast atom bombardment mass spectra of authentic hydroxyatrazine (A) and hydroxyatrazine formed from atrazine by culture LFB6 In H2180 (B). The peaks at m!z 185 and 277 are from the glycerol (x2 + 1 and *3+1, respectively) and mtz 207 represent two molecules of glycerol + sodium. The starred peaks represent the parent Ions.
Figure 4.
at m/z 200 (199 + 1) indicated the incorporation of 180
from H2180 during atrazine dechlorination. A control
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746-749. (15) Couch, R. W.; Gramlich, J. V.; Davis, D. E.; Funderburk, . H. Proc. South. Weed Sci. Soc. 1965, 18, 623-631. (16) Harris, C. I. J. Agrie. Food Chem. 1967, 15, 157-162. (17) Agnihorti, N. P.; Panday, S. Y.; Jain, . K. J. Agrie. Chem. 1976, 9, 15-22. (18) Nearpass, D. C. Soil Sci. Soc. Am. Proc. 1972, 36, 606-610. (19) Li, G. C.; Felbeck, G. T. Soil Sci. 1972, 114, 201-209.
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Received for review May 6, 1993. Revised manuscript received June 15, 1993. Accepted June 16, 1993.
