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Anaerobic microbial degradation of the herbicide propanil
Soil Bid. Biochem. Vol. 17, No. 6, pp. 815-818, 1985 Printed in Great Britain. All rights reserved
Copyright 0
0038~0717/85 $3.00 + 0.00 1985 Pergamcn Press Ltd
ANAEROBIC MICROBIAL DEGRADATION OF THE HERBICIDE PROPANIL CHARLES A. PETTIGREW, M. J. B. PAYNWR and N. D. CAMPER Department of Microbiology and Department of Plant Pathology and Physiology, Clemson University, Clemson, SC 29631, U.S.A. (Aeeepted
31 March 1985)
Summary-The fate of propanil (3,4-dichloropropionanilide) in an anaerobic soil environment was studied. Two mineral salts media, one amended with O.OSo/,yeast extract and 0.05”/, tryptone, and both with 33 pg propanil ml-‘, were inoculated with IO”/, (v/v) soil to establish enrichment cultures. Cultures were incubated at 30°C under an atmosphe~ of 95% N, and 5% C02. There was a complete loss of propanil in 15 days in soil enrichment cultures. One degradation product was detected and identified by HPLC and TLC co-chromatography as 3,4-dichloroaniline. Propanil was also degraded in a soil-free medium containing 0.05% yeast extract and 0.05% tryptone and inoculated with supematant from a soil enrichment culture. After 100 days, propanil concentration decreased by 81%; 3,4_dichloroaniline and meta-chloroaniline were produced. No decrease in propanil concentration was detected in soil-free cultures without yeast extract and tryptone. No labeled CO2 or volatile products were detected after 80 days in soil-free enrichment cultures containing W-ring-labeled propanil.
INTRODUCTION
fate of the herbicide propanil (3&dichloropropionaniiide) has been studied extensively under aerobic conditions (Still and Herret, 1976). It is predominantly used in rice (Oryza sutiva) culture. Since rice paddies are periodically flooded providing a potential anaerobic environment, information about the anaerobic degradation of propanil and the organisms involved is of interest. Bartha and Pramer (1967) investigated propanil degradation in aerobic soil and showed an initial hydrolysis of propanii to 3,4-dichloroaniline (DCA) and the subsequent appearance of 3,~,4,~-tetrachloroa~obenzene (TCAB). It is believed that DCA is produced by a microbial acylamidase, but the method of TCAB production is unknown. Degradation of DCA and propanil in the presence of 4-~hloroaniline has been reported (Zeyer and Kearney, 1982). Steppe e? al. (1984) observed rapid degradation of propanil in anaerobic cultures inoculated with pond sediment; one of the two products detected was meta-chloropropionanilide. Reductive dehalogenation has been reported for diuron (Attaway et aI., 1982) and several halobenzoates (Horowitz et al., 1983). Our objectives were to: establish enrichment cultures with soil inoculum in the presence of propanil under anaerobic conditions; determine the fate of propanil in these enrichment cultures; and identify the resulting degradation products.
The
chloroaniline from Eastman Kodak, Rochester, New York. Propanil stock solutions (33 @gml-‘) were prepared in HPLC grade methanol, filter sterilized (0,2O~m, Gelman) and stored under 5% CO,:95% N,. Final methanol concentration in culture media did not exceed 0.33% (v/v). Meta- and para-chloropropionanilide were synthesized separately by treating 12.75 g of the appropriate chloroaniline, dissolved in 100 ml dry ethyl ether, with 4.6.5g propionyl chloride dissolved in 50 ml dry ethyl ether for 15-min; the mixture was cooled in an ice bath (4°C) and continuously stirred. Addition of 50 ml ice water (4°C) allowed separation; the aqueous layer was extracted twice with 50 ml dry ethyl ether. All ether extracts were combined and dried in an N, atmotiphere. iMefa- and parachloropropionanilide were recrystallized twice from a 3: I ethanoI-water solution after treatment with 100 mg activated charcoal. Both compounds showed 98% purity by HPLC. Soil samples Soil samples were obtained from a non-flooded rice field that had not been treated with propanil. Vegetation was removed and a top 30cm soil sample was collected. Samples were sealed in plastic containers and stored at 5°C. The soil was a clay-loam (41.9% silt, 37.9% clay, ZO.O*~ sand, 5.0% total organic matter, pH 4.6). Culture conditions
MATERIALS AND METHODS
Chemicals Technical grade propanil and “C-ring-labeled propanil (specific activity, 0.31 Bq mg”‘) were obtained from Rohm and Haas, Spring House, Pennsylvania; 3,4-dichloroaniline from Aldrich (Milwaukee, Wisconsin); paru-chloroaniline was purchased from Fischer, Fairlawn, New Jersey; and meta-
~n~chment cultures were grown in 250-ml roundbottom flasks sealed with n-butyl rubber stoppers. Each flask contained ZOOml mineral salts solution which contained (g 1-l): KH,P04, 3.0; K,HPO,, 3.0; NH&l, 0.2; NaCl, 1.0 and (mg 1-l) MnCl,, 31; H,BO,, 5.7; NaMoO,, 3; ZnSO,, 3.7; CoCl,, 5.50; MgCl,, 180; CaCl,, 135. Some cultures were supplemented with 0.05% yeast extract (Difco) and 0.05% tryptone (Difco) as noted. Media were then
816
CHARLES A. PETTIGREW et al.
sterilized (12l”C, 22min) after addition of 0.0001% resazurin (final concn). Immediately after sterilization the media were purged with O,-free 5% CO,:95% N,. After the media had cooled cysteine HCl (0.5x, final concn), prepared anaerobically by the Hungate (1950) method, and filter sterilized propanil (33 pg ml-‘, final concn) were added. Each flask was inoculated with 20 g (wet wt) of soil. Controls for this experiment were prepared identically except that the soil was added before sterilization. Each experiment was performed in triplicate and compared to the corresponding control. Cultures were held at 30°C in a water bath. Excess gas (e.g. methane) produced was removed weekly using a sterile, 5-ml syringe (21 gauge needle), which had been flushed previously with O,-free 5% CO,:95% N1. Maintenance of a reduced anaerobic environment in all cultures was monitored. Visual analysis was facilitated by the presence of resazurin dye in the cultures. Electrochemical analysis used a platinum input electrode with a calomel reference electrode on a Fischer Acumet Eh/pH meter. Eh and pH were measured by flushing sealed 15-ml beakers, seated with stopper and electrodes, with O,-free 100% N, for 5 min before injection of samples. Soil-free enrichment cultures were prepared identically except inocula consisted of culture supernatant from soil inoculated cultures. The experiment consisted of three flasks and was repeated twice. Studies with “C-propanil
Culture conditions for the i4C-ring-labeled propanil degradation experiment were identical to the conditions mentioned above except “C-ring-labeled propanil (53.75 ngml-‘, final concn) was added. Na,S (0.05% final concn), which had been prepared anaerobically by the Hungate (1950) method, was added to aid media reduction under an N, atmosphere. The distribution of radioactivity was determined by purging O,-free 100% N, for 1 h through the cultures and subsequently through 75 ml 0.5 N H,S04 (to trap volatilized organic compounds) and 75 ml 0.5 N NaOH (to trap CO*). The amount of radioactivity in cultures and both traps was measured by combining 1 ml of sample and 15 ml of ReadySolv scintillation fluid (Beckman). Radioactivity was determined in a Beckman LS-100 C scintillation counter.
(60:40), at a flow rate of 2mlmin’. Recovery of propanil by extraction from soil-inoculated cultures at time zero was 87 + 6.4%. Extraction efficiency from standard propanil solutions devoid of soil was greater than 93 + 3.1%. Therefore, the proportion not recovered from soil containing media at time zero was attributed to adsorption. The organic phase contained the majority of propanil. Quantitation was based on peak area versus concentration of known standards. Determination of degradation products
Reference and synthesized compounds were recrystallized before use. Meta-chloropropionanilide (m.p. 85.5-86Y) and para-chloropropionanilide (m.p. 137.5-139°C) were identified by comparison with published melting point data (Good, 1961). Degradation products were identified by HPLC and TLC co-chromatography with known standards.
RESULTS
All samples, from enrichment cultures and soil inoculated cultures, had Eh values that were less than - 150 mV throughout the incubation. Enrichment cultures exhibited a pH range of from 5.9 to 6.5 and soil inoculated cultures a pH range of from 5.7 to 6.4. Gas was produced in all inoculated cultures, with no gas production noted in the corresponding sterile controls. The propanil-containing media (with or without organic nutrients) were inoculated with soil. After 15 days, propanil was no longer detected in any soilinoculated cultures, except the sterile controls (Figs 1 and 2). A degradation product (DCA) occurred after 1 week in soil inoculated cultures (Figs 1 and 2). The
0.6
r
-
Extraction of cultures and analysis for propanil and products
Samples (15 ml) of cultures were removed anaerobically with sterile 25-ml syringes (18 gauge needles) which had been purged with 5% C02:95% N,. Aliquots (5 ml) were then mixed with 5 ml HPLC grade ethyl acetate for 1 min. The organic phase was decanted, and the aqueous phase extracted in the previous manner. Organic extracts were combined, air dried, and the residue dissolved in 5 ml HPLC grade methanol. A 10.0 pl aliquot of the 5 ml methanol solution was analyzed on a Varian 5000 liquid chromatography equipped with a CDS-l 11 microprocessor, 9176 recorder, and u.v.-50 detector. A micropack, MCH-10 (30 cm x 4 mm) column with matching precolumn was used. The solvent system was CH,OH:H,O
---
0
7
14
Control Active
21
Incubation
culture
28
35
42
time (days)
Fig. 1. Anaerobic degradation of propanil and 3,4_dichloroaniline (DCA) mineral salts medium using soil as inoculum. Single values represent the mean of triplicate experiments. Bars represent + 1 SD.
817
Degradation of propanil
culture extracts. DCA was not detected until 42 days. The concentration of MCA did not decrease by 100 days. Enrichment cultures with and without soil were established in the presence of i4C-ring-labeled propanil (data not presented). After 80 days no appreciable radioactivity was detected in either the acidic or basic traps. The amount of radioactivity in the culture extracts did not decrease during the incubation, except in cultures containing soil (indicating adsorption).
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DISCUSSION
Control Active
culture
20 \ 0
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IO 0 0
7
14
Incubation
21
28 time
35
42
(days)
degradation of propanil and Fig. 2. Anaerobic 3,~dichloroaniline (DCA) formation in mineral salts, 0.05% yeast extract, and 0.05% tryptone medium using soil as inoculum. Single values represent the mean of triplicate experiments. Bars represent 5 1 SD.
amount of DCA produced in cultures supplemented with organic nutrients was three times greater than in the unsupplemented cultures. The product (and the DCA standard) had a retention time of 6.8min compared to 10.6min for propanil. DCA did not persist; none was present after 28 days. Propanil concentration in the sterilized control decreased to a constant extractable level after I week. DCA did not appear in any sterile controls. Inoculum {essentially soil-free) from the supernatant of soil enrichment cultures was used to establish new enrichment cultures. These cultures without organic supplements (yeast extract and tryptone) showed no significnat loss of propanil (Fig. 3). Although appreciable propanil loss was not observed, a degradation product with a retention time of 6.8 min was detected at 100 days and identified as DCA (Fig. 3). There was no significant change in propanil concentration in corresponding sterile controls. Soil-free enrichment cultures consisting of mineral salts medium with 0.05% yeast extract and 0.05% tryptone amendments exhibited 81% loss of propanil by 100 days (Fig. 4). No reduction in concentration of propanil was detected in corresponding sterile controls. Two degradation products were detected in the yeast extract and tryptone amended cultures (Fig. 4). One product had a retention time of 6.8 min, as found in soil enrichments and was DCA. The second product had a retention time of 3.9 min and was identified by HPLC and TLC co-chromatography as meta-chloroaniline (MCA). MCA was detected only after 28 days, when DCA was no longer present in
The degradation of propanil in anaerobic environments was biologically mediated; there was no degradation in sterile controls. However, it was shown that the same degradation occurred in enrichment cultures containing no soil. This implies that a soil mediated physical or chemical event was not responsible for degradation. The decrease in propanil concentration in sterile controls containing soil may be caused by several factors including adsorption. Based on results obtained by Chisaka and Kearney (1970), soil adsorption would account for less than 15% of the decrease. Flasks were agitated prior to sampling; thus, sampling artifacts were not involved (also verified by small quantitative variations). The soil contained 5% organic matter. If sterilization did not drastically alter the humic structure of the soil, significant adsorption could occur (Bartha, 1971). The disappearance of DCA from soil inoculated cultures may have involved adsorption, but further
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Fig. 3. Anaerobic degradation of propanil and 3,4_dichloroaniline (DCA) formation in mineral salts using supematant from soil enrichment cultures as inoculum. Single values represent the mean of triplicate experiments. Bars represent f 1 SD.
CHARLFS
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A. PETTIGREWet al aerobic studies (Bartha and Pramer, 1967). However, dehalogenation associated with conversion of DCA to meta-chloroaniline has only been reported in anaerobic studies (Horowitz et al., 1983). The production of meta-chloroaniline from DCA is consistent with previous findings that showed preferential dehalogenation of the para-chlorine in 3,4-dichlorinated aromatic compounds (Attaway et al., 1982). Neither meta- nor puru-chloropropionanilide were detected as degradation products in this study. Steppe et al. (1984) observed rapid degradation of propanil in anaerobic cultures and reported the appearance of a product thought to be metu-chloropropionanilide. The lack of decrease in the concentration of DCA and MCA in the soil-free enrichment cultures suggests that there was no cleavage of the aromatic ring. Likewise, the lack of 14C0, and “C-volatile compound production indicated that ring cleavage was not detected. However, our system would not have detected the production of 14CH4.
DCA MCA
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70
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Incubation
time
I_ 96
(days)
Fig. 4. Anaerobic degradation of propanil and formation of 3,4-dichloroaniline (DCA) and 3-chloroaniline (MCA) in mineral salts, 0.05% yeast extract, and 0.05% tryptone medium using supernatant from soil enrichment cultures as inoculum. Single values represent the mean of triplicate experiments. Bars represent f I SD.
microbial degradation breakdown cannot be ruled out. The detection of DCA and MCA in soil-free cultures was probably aided by the lack of soil. The soil contained some required nutrients that aided microbial growth and therefore possibly degradation also. This is supported by the fact that enrichment cultures that lacked soil and nutritional amendments showed no significant propanil loss. The different conditions probably resulted in enrichment of different consortia or aided enzymatic reactions involved in propanil degradation. The identification of products as DCA and MCA supports a postulated sequence of propanil degradation to DCA and subsequently to MCA. The appearance of DCA after MCA in soil-free enrichment cultures may have been due to its adsorption to the microorganisms and subsequent release and conversion to MCA. Bartha (1971) showed that the bulk of immobilized residues were intact chloroanilines bound to organic matter. The initial production of DCA is similar to results obtained in
Acknowledgemenfs-Research supported by a grant from the National Agricultural Pesticide Impact Assessment Program. Journal Number 2357 of the South Carolina Agricultural Experiment Station.
REFERENCES
Attaway H. H., Camper N. D. and Paynter M. J. B. (1982) Anaerobic degradation of diuron by pond sediment. Pesticide Biochemistry and Physiology 17, 96-101. Bartha R. (1971) Fate of herbicide-derived chloroanilines in soil. Journal of Agricultural and Food Chemistry 19, 385-387. Bartha R. and Pramer D. (1967) Pesticide transformation to aniline and azo compounds in soil. Science 156, 1617-1618. Chisaka H. and Kearney P. C. (1970) Metabolism of propanil in soils. Journal of Agricultural and Food Chemisfry 18, 854-858. Good N. E. (1961) Inhibitors of the Hill reaction. Plant Physiology 36, 788-803. Horowitz A., Suflita J. M. and Tiedje J. M. (1983) Reductive dehalogenation of halobenzoates by anaerobic lake sediment microorganisms. Applied and Environmental Microbiology 45, 1459-1465. Hungate R. E. (1950) The anaerobic, mesophilic cellulolytic bacteria. Bacteriological Reviews 14, l-63. Steppe T. D., Camper N. D. and Paynter M. J. B. (1985) Anaerobic microbial degradation of selected 3,4-dihalogenated aromatic compounds. Pesticide Biochemistry and Physiology 23, 256-260. Still G. G. and Herrett R. A. (1976) Methylcarbamates, carbanilates, and acylanilides. In Herbicides: Chemistry, Degradation, and Mode of Action (P. C. Kearney and D. D. Kaufman, Eds), pp. 609-664. Dekker, New York. Zeyer J. and Kearney P. C. (1982) Microbial metabolism of propanil and 3,4-dichloroaniline, Pesticide Biochemistry and Physiology 17, 224-231.
Copyright 0
0038~0717/85 $3.00 + 0.00 1985 Pergamcn Press Ltd
ANAEROBIC MICROBIAL DEGRADATION OF THE HERBICIDE PROPANIL CHARLES A. PETTIGREW, M. J. B. PAYNWR and N. D. CAMPER Department of Microbiology and Department of Plant Pathology and Physiology, Clemson University, Clemson, SC 29631, U.S.A. (Aeeepted
31 March 1985)
Summary-The fate of propanil (3,4-dichloropropionanilide) in an anaerobic soil environment was studied. Two mineral salts media, one amended with O.OSo/,yeast extract and 0.05”/, tryptone, and both with 33 pg propanil ml-‘, were inoculated with IO”/, (v/v) soil to establish enrichment cultures. Cultures were incubated at 30°C under an atmosphe~ of 95% N, and 5% C02. There was a complete loss of propanil in 15 days in soil enrichment cultures. One degradation product was detected and identified by HPLC and TLC co-chromatography as 3,4-dichloroaniline. Propanil was also degraded in a soil-free medium containing 0.05% yeast extract and 0.05% tryptone and inoculated with supematant from a soil enrichment culture. After 100 days, propanil concentration decreased by 81%; 3,4_dichloroaniline and meta-chloroaniline were produced. No decrease in propanil concentration was detected in soil-free cultures without yeast extract and tryptone. No labeled CO2 or volatile products were detected after 80 days in soil-free enrichment cultures containing W-ring-labeled propanil.
INTRODUCTION
fate of the herbicide propanil (3&dichloropropionaniiide) has been studied extensively under aerobic conditions (Still and Herret, 1976). It is predominantly used in rice (Oryza sutiva) culture. Since rice paddies are periodically flooded providing a potential anaerobic environment, information about the anaerobic degradation of propanil and the organisms involved is of interest. Bartha and Pramer (1967) investigated propanil degradation in aerobic soil and showed an initial hydrolysis of propanii to 3,4-dichloroaniline (DCA) and the subsequent appearance of 3,~,4,~-tetrachloroa~obenzene (TCAB). It is believed that DCA is produced by a microbial acylamidase, but the method of TCAB production is unknown. Degradation of DCA and propanil in the presence of 4-~hloroaniline has been reported (Zeyer and Kearney, 1982). Steppe e? al. (1984) observed rapid degradation of propanil in anaerobic cultures inoculated with pond sediment; one of the two products detected was meta-chloropropionanilide. Reductive dehalogenation has been reported for diuron (Attaway et aI., 1982) and several halobenzoates (Horowitz et al., 1983). Our objectives were to: establish enrichment cultures with soil inoculum in the presence of propanil under anaerobic conditions; determine the fate of propanil in these enrichment cultures; and identify the resulting degradation products.
The
chloroaniline from Eastman Kodak, Rochester, New York. Propanil stock solutions (33 @gml-‘) were prepared in HPLC grade methanol, filter sterilized (0,2O~m, Gelman) and stored under 5% CO,:95% N,. Final methanol concentration in culture media did not exceed 0.33% (v/v). Meta- and para-chloropropionanilide were synthesized separately by treating 12.75 g of the appropriate chloroaniline, dissolved in 100 ml dry ethyl ether, with 4.6.5g propionyl chloride dissolved in 50 ml dry ethyl ether for 15-min; the mixture was cooled in an ice bath (4°C) and continuously stirred. Addition of 50 ml ice water (4°C) allowed separation; the aqueous layer was extracted twice with 50 ml dry ethyl ether. All ether extracts were combined and dried in an N, atmotiphere. iMefa- and parachloropropionanilide were recrystallized twice from a 3: I ethanoI-water solution after treatment with 100 mg activated charcoal. Both compounds showed 98% purity by HPLC. Soil samples Soil samples were obtained from a non-flooded rice field that had not been treated with propanil. Vegetation was removed and a top 30cm soil sample was collected. Samples were sealed in plastic containers and stored at 5°C. The soil was a clay-loam (41.9% silt, 37.9% clay, ZO.O*~ sand, 5.0% total organic matter, pH 4.6). Culture conditions
MATERIALS AND METHODS
Chemicals Technical grade propanil and “C-ring-labeled propanil (specific activity, 0.31 Bq mg”‘) were obtained from Rohm and Haas, Spring House, Pennsylvania; 3,4-dichloroaniline from Aldrich (Milwaukee, Wisconsin); paru-chloroaniline was purchased from Fischer, Fairlawn, New Jersey; and meta-
~n~chment cultures were grown in 250-ml roundbottom flasks sealed with n-butyl rubber stoppers. Each flask contained ZOOml mineral salts solution which contained (g 1-l): KH,P04, 3.0; K,HPO,, 3.0; NH&l, 0.2; NaCl, 1.0 and (mg 1-l) MnCl,, 31; H,BO,, 5.7; NaMoO,, 3; ZnSO,, 3.7; CoCl,, 5.50; MgCl,, 180; CaCl,, 135. Some cultures were supplemented with 0.05% yeast extract (Difco) and 0.05% tryptone (Difco) as noted. Media were then
816
CHARLES A. PETTIGREW et al.
sterilized (12l”C, 22min) after addition of 0.0001% resazurin (final concn). Immediately after sterilization the media were purged with O,-free 5% CO,:95% N,. After the media had cooled cysteine HCl (0.5x, final concn), prepared anaerobically by the Hungate (1950) method, and filter sterilized propanil (33 pg ml-‘, final concn) were added. Each flask was inoculated with 20 g (wet wt) of soil. Controls for this experiment were prepared identically except that the soil was added before sterilization. Each experiment was performed in triplicate and compared to the corresponding control. Cultures were held at 30°C in a water bath. Excess gas (e.g. methane) produced was removed weekly using a sterile, 5-ml syringe (21 gauge needle), which had been flushed previously with O,-free 5% CO,:95% N1. Maintenance of a reduced anaerobic environment in all cultures was monitored. Visual analysis was facilitated by the presence of resazurin dye in the cultures. Electrochemical analysis used a platinum input electrode with a calomel reference electrode on a Fischer Acumet Eh/pH meter. Eh and pH were measured by flushing sealed 15-ml beakers, seated with stopper and electrodes, with O,-free 100% N, for 5 min before injection of samples. Soil-free enrichment cultures were prepared identically except inocula consisted of culture supernatant from soil inoculated cultures. The experiment consisted of three flasks and was repeated twice. Studies with “C-propanil
Culture conditions for the i4C-ring-labeled propanil degradation experiment were identical to the conditions mentioned above except “C-ring-labeled propanil (53.75 ngml-‘, final concn) was added. Na,S (0.05% final concn), which had been prepared anaerobically by the Hungate (1950) method, was added to aid media reduction under an N, atmosphere. The distribution of radioactivity was determined by purging O,-free 100% N, for 1 h through the cultures and subsequently through 75 ml 0.5 N H,S04 (to trap volatilized organic compounds) and 75 ml 0.5 N NaOH (to trap CO*). The amount of radioactivity in cultures and both traps was measured by combining 1 ml of sample and 15 ml of ReadySolv scintillation fluid (Beckman). Radioactivity was determined in a Beckman LS-100 C scintillation counter.
(60:40), at a flow rate of 2mlmin’. Recovery of propanil by extraction from soil-inoculated cultures at time zero was 87 + 6.4%. Extraction efficiency from standard propanil solutions devoid of soil was greater than 93 + 3.1%. Therefore, the proportion not recovered from soil containing media at time zero was attributed to adsorption. The organic phase contained the majority of propanil. Quantitation was based on peak area versus concentration of known standards. Determination of degradation products
Reference and synthesized compounds were recrystallized before use. Meta-chloropropionanilide (m.p. 85.5-86Y) and para-chloropropionanilide (m.p. 137.5-139°C) were identified by comparison with published melting point data (Good, 1961). Degradation products were identified by HPLC and TLC co-chromatography with known standards.
RESULTS
All samples, from enrichment cultures and soil inoculated cultures, had Eh values that were less than - 150 mV throughout the incubation. Enrichment cultures exhibited a pH range of from 5.9 to 6.5 and soil inoculated cultures a pH range of from 5.7 to 6.4. Gas was produced in all inoculated cultures, with no gas production noted in the corresponding sterile controls. The propanil-containing media (with or without organic nutrients) were inoculated with soil. After 15 days, propanil was no longer detected in any soilinoculated cultures, except the sterile controls (Figs 1 and 2). A degradation product (DCA) occurred after 1 week in soil inoculated cultures (Figs 1 and 2). The
0.6
r
-
Extraction of cultures and analysis for propanil and products
Samples (15 ml) of cultures were removed anaerobically with sterile 25-ml syringes (18 gauge needles) which had been purged with 5% C02:95% N,. Aliquots (5 ml) were then mixed with 5 ml HPLC grade ethyl acetate for 1 min. The organic phase was decanted, and the aqueous phase extracted in the previous manner. Organic extracts were combined, air dried, and the residue dissolved in 5 ml HPLC grade methanol. A 10.0 pl aliquot of the 5 ml methanol solution was analyzed on a Varian 5000 liquid chromatography equipped with a CDS-l 11 microprocessor, 9176 recorder, and u.v.-50 detector. A micropack, MCH-10 (30 cm x 4 mm) column with matching precolumn was used. The solvent system was CH,OH:H,O
---
0
7
14
Control Active
21
Incubation
culture
28
35
42
time (days)
Fig. 1. Anaerobic degradation of propanil and 3,4_dichloroaniline (DCA) mineral salts medium using soil as inoculum. Single values represent the mean of triplicate experiments. Bars represent + 1 SD.
817
Degradation of propanil
culture extracts. DCA was not detected until 42 days. The concentration of MCA did not decrease by 100 days. Enrichment cultures with and without soil were established in the presence of i4C-ring-labeled propanil (data not presented). After 80 days no appreciable radioactivity was detected in either the acidic or basic traps. The amount of radioactivity in the culture extracts did not decrease during the incubation, except in cultures containing soil (indicating adsorption).
2.0
:1\
1.6 I
3
5 0
1.2
;\
1 \
L
0.8
‘,
2
\
\ \
0.4
0.0 35 30
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---
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’
_
--_a
DISCUSSION
Control Active
culture
20 \ 0
\
IO 0 0
7
14
Incubation
21
28 time
35
42
(days)
degradation of propanil and Fig. 2. Anaerobic 3,~dichloroaniline (DCA) formation in mineral salts, 0.05% yeast extract, and 0.05% tryptone medium using soil as inoculum. Single values represent the mean of triplicate experiments. Bars represent 5 1 SD.
amount of DCA produced in cultures supplemented with organic nutrients was three times greater than in the unsupplemented cultures. The product (and the DCA standard) had a retention time of 6.8min compared to 10.6min for propanil. DCA did not persist; none was present after 28 days. Propanil concentration in the sterilized control decreased to a constant extractable level after I week. DCA did not appear in any sterile controls. Inoculum {essentially soil-free) from the supernatant of soil enrichment cultures was used to establish new enrichment cultures. These cultures without organic supplements (yeast extract and tryptone) showed no significnat loss of propanil (Fig. 3). Although appreciable propanil loss was not observed, a degradation product with a retention time of 6.8 min was detected at 100 days and identified as DCA (Fig. 3). There was no significant change in propanil concentration in corresponding sterile controls. Soil-free enrichment cultures consisting of mineral salts medium with 0.05% yeast extract and 0.05% tryptone amendments exhibited 81% loss of propanil by 100 days (Fig. 4). No reduction in concentration of propanil was detected in corresponding sterile controls. Two degradation products were detected in the yeast extract and tryptone amended cultures (Fig. 4). One product had a retention time of 6.8 min, as found in soil enrichments and was DCA. The second product had a retention time of 3.9 min and was identified by HPLC and TLC co-chromatography as meta-chloroaniline (MCA). MCA was detected only after 28 days, when DCA was no longer present in
The degradation of propanil in anaerobic environments was biologically mediated; there was no degradation in sterile controls. However, it was shown that the same degradation occurred in enrichment cultures containing no soil. This implies that a soil mediated physical or chemical event was not responsible for degradation. The decrease in propanil concentration in sterile controls containing soil may be caused by several factors including adsorption. Based on results obtained by Chisaka and Kearney (1970), soil adsorption would account for less than 15% of the decrease. Flasks were agitated prior to sampling; thus, sampling artifacts were not involved (also verified by small quantitative variations). The soil contained 5% organic matter. If sterilization did not drastically alter the humic structure of the soil, significant adsorption could occur (Bartha, 1971). The disappearance of DCA from soil inoculated cultures may have involved adsorption, but further
1.4
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I.0
9 n
0.8
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Fig. 3. Anaerobic degradation of propanil and 3,4_dichloroaniline (DCA) formation in mineral salts using supematant from soil enrichment cultures as inoculum. Single values represent the mean of triplicate experiments. Bars represent f 1 SD.
CHARLFS
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A. PETTIGREWet al aerobic studies (Bartha and Pramer, 1967). However, dehalogenation associated with conversion of DCA to meta-chloroaniline has only been reported in anaerobic studies (Horowitz et al., 1983). The production of meta-chloroaniline from DCA is consistent with previous findings that showed preferential dehalogenation of the para-chlorine in 3,4-dichlorinated aromatic compounds (Attaway et al., 1982). Neither meta- nor puru-chloropropionanilide were detected as degradation products in this study. Steppe et al. (1984) observed rapid degradation of propanil in anaerobic cultures and reported the appearance of a product thought to be metu-chloropropionanilide. The lack of decrease in the concentration of DCA and MCA in the soil-free enrichment cultures suggests that there was no cleavage of the aromatic ring. Likewise, the lack of 14C0, and “C-volatile compound production indicated that ring cleavage was not detected. However, our system would not have detected the production of 14CH4.
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Fig. 4. Anaerobic degradation of propanil and formation of 3,4-dichloroaniline (DCA) and 3-chloroaniline (MCA) in mineral salts, 0.05% yeast extract, and 0.05% tryptone medium using supernatant from soil enrichment cultures as inoculum. Single values represent the mean of triplicate experiments. Bars represent f I SD.
microbial degradation breakdown cannot be ruled out. The detection of DCA and MCA in soil-free cultures was probably aided by the lack of soil. The soil contained some required nutrients that aided microbial growth and therefore possibly degradation also. This is supported by the fact that enrichment cultures that lacked soil and nutritional amendments showed no significant propanil loss. The different conditions probably resulted in enrichment of different consortia or aided enzymatic reactions involved in propanil degradation. The identification of products as DCA and MCA supports a postulated sequence of propanil degradation to DCA and subsequently to MCA. The appearance of DCA after MCA in soil-free enrichment cultures may have been due to its adsorption to the microorganisms and subsequent release and conversion to MCA. Bartha (1971) showed that the bulk of immobilized residues were intact chloroanilines bound to organic matter. The initial production of DCA is similar to results obtained in
Acknowledgemenfs-Research supported by a grant from the National Agricultural Pesticide Impact Assessment Program. Journal Number 2357 of the South Carolina Agricultural Experiment Station.
REFERENCES
Attaway H. H., Camper N. D. and Paynter M. J. B. (1982) Anaerobic degradation of diuron by pond sediment. Pesticide Biochemistry and Physiology 17, 96-101. Bartha R. (1971) Fate of herbicide-derived chloroanilines in soil. Journal of Agricultural and Food Chemistry 19, 385-387. Bartha R. and Pramer D. (1967) Pesticide transformation to aniline and azo compounds in soil. Science 156, 1617-1618. Chisaka H. and Kearney P. C. (1970) Metabolism of propanil in soils. Journal of Agricultural and Food Chemisfry 18, 854-858. Good N. E. (1961) Inhibitors of the Hill reaction. Plant Physiology 36, 788-803. Horowitz A., Suflita J. M. and Tiedje J. M. (1983) Reductive dehalogenation of halobenzoates by anaerobic lake sediment microorganisms. Applied and Environmental Microbiology 45, 1459-1465. Hungate R. E. (1950) The anaerobic, mesophilic cellulolytic bacteria. Bacteriological Reviews 14, l-63. Steppe T. D., Camper N. D. and Paynter M. J. B. (1985) Anaerobic microbial degradation of selected 3,4-dihalogenated aromatic compounds. Pesticide Biochemistry and Physiology 23, 256-260. Still G. G. and Herrett R. A. (1976) Methylcarbamates, carbanilates, and acylanilides. In Herbicides: Chemistry, Degradation, and Mode of Action (P. C. Kearney and D. D. Kaufman, Eds), pp. 609-664. Dekker, New York. Zeyer J. and Kearney P. C. (1982) Microbial metabolism of propanil and 3,4-dichloroaniline, Pesticide Biochemistry and Physiology 17, 224-231.