Environ. Sci. Technol. 1993, 27, 1943-1946
Rapid Hydrolysis of Atrazine to Hydroxyatrazine by Soil Bacteria Raphl T. Mandelbaum,tl* Lawrence P. Wackett,'gt 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-(isopropylamino)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 l), while N-dealkylationreactions 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 , 1 4 ) . Only a slow dechlorination of atrazine by soil fungi has been reported (7,251. Other data are interpreted to support nonbiologicalmechanisms 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 (8, 22, 23). In this study, we report the rapid transformation of atrazine to hydroxyatrazine at neutral pH by a soil bacterial mixed culture LFBG. 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 I80-labelingexperiments. 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 LFBG is described elsewhere (24). Culture LFBG was grown in 500-mL Erlenmeyer flasks containing 300 mL of atrazine medium (24)for 3 days (OD600 > 1)without shaking. The culture was then harvested by centrifugation (6000g, 20 min) and washed twice with 0.1 N sodium phosphate buffer (pH 7.0) to remove excess nutrients and residual atrazine t Department of Biochemistry and Institute for Advanced Studies in Biological Process Technology. t Department of Soil Science. 0013-936X/93/0927-1943$04.00/0
0 1993 American Chemical Society
Atrazine
\ \
Biologlcal Dealkylat ion
el
f\j HzN-C\~,C-NHZ
Desethyldesisopropyiatrazlne
Figure 1. Previously reported atrazine degradation pathways in soil.
The microbial dealkylation of atrazine to form desethyldesisopropylatrazine may involve more than one microorganism.
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 1mL of deionized water and preincubated for 3 days at 30 "C in the dark. Atrazine (460 pmol/mL) was suspended in methanol and sonicated for 30 s at 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 pL) 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 LFBG (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 O i l 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-pm filter and frozen at -70 "C until analysis. For the labeling experiment with H2180,1mL of 97.3% Hd80 (MSD Isotopes, St. Louis, MO) was combined with 1mL of the atrazine suspension (prepared as previously described), and the solution was equilibrated at 4 "C overnight. Culture LFBG (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 HZ180 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 100p L each and immediately frozen at -70 "C. Each 1OOpL 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. Technol., 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 LFBG culture were prepared by sonicating a 3-mL cell suspension (OD600 = 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-pm 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 ( 2 4 ) , and crude extract metabolites were analyzed using a 300 X 4.6 mm, 5-pm Cg column (Phase Separation Inc. Norwalk, CT). Atrazine (99.6 9% was purchased from Chem Service Chemical Co. (West Chester, PA). 14C-uniformlyring-labeled atrazine (7.8mCi/mmol; 99.6 73 radiochemical purity) was purchased from Sigma Chemical Go. (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 at,razine and metabolite standards were prepared in methanol-aqueous 0.1 N H3P04and stored at 4 OC.
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 ( 2 4 ) ,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 LFBG in liquid media was shown to liberate the atrazine ring carbon atoms as C02 ( 2 4 ) . 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 LFBG far exceeded those previously reported for native soils or bacterial cultures. Resting cell suspensions of culture LFBG 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 1044
Envlron. Sci. Tschnol., Vol. 27, No. 9, 1993
Standard mix
Webster Soil
L
L
0
Silica Sand
Y
0
'
F
63-
F
.
0 1
.
.
A 2
II
e
G
.
2 3 4
. . . . . . . , 5 6 7 8 9 101112
Time (min) Figure 2. High-pressure liquid chromatographyanalysis of soil inoculated with culture LFBG. Key: A, desethyldesisopropylhydroxyatrazine;B, desisopropylhydroxyatrazine; C, desethylhydroxyatrazine;D, desisopropylatrazine;E, desethylatrazine;F, hydroxyatrazine;G,atrazine. 1, extract from uninoculatedcontrol; 2, 1 h after inoculationwith culture LFBG; 3, 24 h after inoculation with culture LFBG.
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 LFBG 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 LFBG is hydrolytic. Atrazine exposed for 1h to nongrowing cells of culture LFBG in Hz180 yielded [180]hydroxyatrazine as demonstrated by fast atom bombardment mass spectroscopy (Figure 4). The major peak
n
OH
n
E c
0
cv
3
2 c
0
!2 ? L
0
c
0
a, a,
t
n
D
2.5
7.5
5.0
10.0
12.5
15.0
17.5
20.0
22.5
Time (min) Flguro 3. High-pressure llquld chromatographyanalysts of atrazine in crude extract prepared from culture LFBB: (A) control of atrazine in buffer (pH 7.0) after 24 h; ( 6 )atrazlne in crude extract after 1 h; (C) dlsappearanceof atrazlne In crude extract after 24 h; (D) crude extract not amended with atrazine.
treatment consisting of authentic hydroxyatrazine solubilized in 97.3% Hz180 did not show any spontaneous exchange of l80 hydroxyl groups, even when the hydroxyatrazine was incubated with Hz180 for 24 h. The small peak at m/z 198in Figure 4B was due to some residual Hz160 carried over from bacterial cells grown in Hz1600containing medium. Mass spectra of authentic hydroxyatrazine (Figure 4A) yielded a hydroxyatrazine peak at m l 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 microbialenzymatic 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
I
1
10 0
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-0108-89from 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.
I
223
I
'2 20
'240
'260
'280
m/z Figure 4. Fast atom bombardment mass spectra of authentic hydroxyatrazine(A)and hydroxyatrazineformed fromatrazineby culture LFB6 in H21a0(6). The peaks at mlz 185 and 277 are from the glycerol (x2 1 and x3 1, respectively)and m/r 207 represent two molecules of glycerol sodlum. The starred peaks represent the parent ions.
+
+
+
at mlz 200 (199 + 1) indicated the incorporation of l 8 0 from H2180during atrazine dechlorination. A control
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(6) Obien, S. R.; Green, R. E. Weed Sci. 1969, 17, 509-514. (7) Kaufman, D. D.; Blake, J. Soil Biol. Biochem. 1970,2, 7380. (8) Skipper, H. D.; Volk, V. V. Weed Sci. 1972, 20, 344-347. (9) Muir, D. C.; Baker, B. E. Weed Res. 1978, 18, 111-120. (10) Fernandez-Quintanilla, C. M.; Cole, A,; Slife, F. W. Theory and Practice of the Use of Soil Applied Herbicides. Proc. E WRS Symp. 1981; pp 301-308. (11) Adams, C. D.; Randtke, S. J. Environ. Sci. Technol. 1992, 26, 2218-2227. (12) Sorenson, B. A. Ph.D Thesis, University of Minnesota, 1992. (13) Cook, A. M.; Hutter, R. J. Agric. Food Chem. 1984, 32, 581-585. (14) Behki, R. M.; Khan, S. U. J . Agric. Food Chem. 1986,34, 746-749. (15) Couch, R. W.; Gramlich, J. V.; Davis, D. E.; Funderburk, H.H. Proc. South. Weed Sci. SOC.1965, 18,623-631. (16) Harris, C. I. J . Agric. Food Chem. 1967, 15, 157-162. (17) Agnihorti, N. P.; Panday, S. Y.; Jain, H. K. J . Agric. 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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(20) Best, J. A,; Weber, J. B. Weed Sci. 1974, 22, 364-373. (21) Gysin, H.; Knusli, E. Adv. Pest Control Res. 1960,3, 289358. (22) Hance, R. J.; Chesters, G. Soil Biol. Biochem. 1969,1,309315. (23) Goswami, K. P.; Green, R. E. Environ. Sci. Technol. 1971, 5,426-429. (24) Mandelbaum, R. T.; Wackett, L. P.; Allan, D. I,. Appl. Environ. Microbiol. 1993, 59, 1695-1701. (25) Xun, L.; Topp, E.; Orser, C. S. J . Bacteriol. 1992,174,57455747. (26) Armstrong, D. E.; Chesters, G. Environ. Sci. Technol. 1968, 2, 683-689. (27) Zimdahl, R. L.; Freed, V. H.; Montgomery, M. L.; Furtick, W. R. Weed Res. 1970, 10, 18-26. (28) Garret, S. D. In Soil Fungi and Soil Fertility; Pergamon Press: London, 1981; pp 76-77. (29) Gray, P. H. H.; Wallace, R. H. Can. J . Microbiol. 1957, 3, 71.1-714.
Received for review May 6, 1993. Revised manuscript received June 15, 1993. Accepted June 16, 1993.
