The degradation of methazole in soil. II. Studies with methazole, methazole degradation products and diuron

Pesfic. Sci. 1978, 9, 333-341

The Degradation of Methazole in Soil. 11. Studies with Methazole, Methazole Degradation Products and Diuron

Allan Walker and Michael G. Roberts

National Vegetable Research Station, WelIesbourne, War wic k. CV35 9EF

(Manuscript received 7 December 1977)

[14C]-Labelled methazole, 1-(3,4-dichlorophenyl)-3-methylurea (DCPMU), 1-(3,4- dichloropheny1)urea (DCPU), and diuron were incubated in soil at 20°C and field capacity soil moisture content. Decomposition followed first-order kinetics ; half- lives for degradation of these four compounds were 2.4, 144, 30 and 108 days respectively. The amount of DCPMU and DCPU that could be extracted decreased with time and the decrease was accompanied by the generation of an equivalent amount of 14COz. This was not so in the studies with diuron and methazole, however, and the decrease in the concentrations of radioactivity extracted from soil treated with these compounds could not be entirely accounted for as carbon dioxide. It is concluded that the unextractable radiochemical that was present was DCPMU. Methazole appeared to be degraded through DCPMU to 3,4-dichloroaniline (DCA) with the production of only traces of DCPU.

1. Introduction

Previous studies have shown that the herbicide methazole 2-(3,4-dichlorophenyl)-4-methyl-1,2,4- oxadiazolidine-3,5-dione, is rapidly degraded in soil.1-3 The major degradation product is 1-(3,4- dichlorophenyl)-3-methylurea (DCPMU) which is appreciably more stable than the parent compound. In plants, DCPMU is further degraded to 1-(3,4-dichlorophenyl)urea (DCPU).4-6 When methazole is degraded in soil, there is a build-up of unextractable material?, 3 the nature of which has not been determined. In the present experiments, the degradation of methazole, DCPMU and DCPU in soil was examined over a period of 120 days. The herbicide diuron was also studied be- cause of the similarity of its degradation pathway to that of methazole.7- * The structures of these compounds are shown in Figure 1.

2. Experimental

2.1. Materials Soil containing 1.38% organic carbon, 29% clay and with a pH of 6.1 was taken from the surface 10 cm of Soakwaters, a field at the National Vegetable Research Station. The herbicides and other compounds used were [14C]-labelled methazole, both 2-(3,4-di~hloro[~~C]phenyl)-4-methyl-1,2,4- oxadiazolidine-3,5-dione and 2-(3,4-dichlorophenyl)-4-methyl-[3-~~C]-1,2,4-oxadiazolidine-3,5-dione and carbonyl-labelled DCPMU, DCPU and diuron. The specific activities of these compounds were 83, 29, 29, 0.94 and 4.1 mCi g-1 respectively and their radiochemical purity was greater than 99 %. Analytical grade samples of methazole, DCPMU, DCPU and diuron were also used.

2.2. Preparation of DCPMU The sample of DCPMU was prepared from methazole labelled in C-3 of the heterocyclic ring. A solution of the labelled methazole in methanol (5 ml, 1 .O pCi ml-1) was added to water (30 ml) in an evaporation flask (50ml). The methanol was evaporated on a vacuum rotary evaporator at

0031-613X/78/0800-0333 $02.00 0 1978 Society of Chemical Industry

333

334 A. Walker and M. G. Roberts

R HN- C -N R’R’

I

(1) (11) Figure 1. Structure of methazole (I), diuron (11; R1=R2=CH3) and their principal degradation products DCPMU

(11; Rl=CHa, RZ=H) and DCPU (11, R1=R2=H).

40°C and the aqueous solution which remained was left under vacuum on the evaporator at this temperature for about 45 min. The solution was cooled and extracted three times with ethyl acetate (50 ml). It was shown that about 20% of the radioactivity had been lost during the process, possibly by volatilisation, but that all of the activity in the aqueous phase partitioned into the ethyl acetate. The ethyl acetate extracts were combined and evaporated to a small volume (about 1 ml) and 10 pl of the solution was subjected to thin-layer chromatography as described previous1y.l The ethyl acetate solution was shown to contain only DCPMU.

2.3. Incubation studies Separate amounts (1 kg) of air-dry soil were treated with phenyl-labelled and carbonyl-labelled methazole, DCPMU, DCPU and diuron and prepared for incubation by the methods described previous1y.l The final concentration of radioactivity in each sample was nominally 4 pCi kg-l dry soil and the concentration of each compound was adjusted, where necessary, to 4 pg g-l with un- labelled pure compound. Subsamples (700 g) of each treatment were incubated in polythene bottles at room temperature (about 18-23°C) and field capacity soil moisture content (16.8 %). Duplicate amounts (30 g) from each treatment were removed immediately after preparation and at intervals during the subsequent 120 days and their content of methazole, DCPMU, DCPU and diuron determined by the extraction and thin-layer chromatographic methods described bef0re.l The RF value for diuron in the benzene + acetone solvent system (7 + 3, by volume) used for thin-layer chromatography was 0.75.

2.4. Collection of [Wl-labelled carbon dioxide Further samples of treated soil (100 g) were used for monitoring the production of 14C02 during the experiment. The apparatus for trapping the evolved 14C02 consisted of a pump which passed air through two gas washing bottles containing 1~ sodium hydroxide and a column of ‘Indicarb’ soda lime. The carbon dioxide-free air was saturated with water vapour and then passed through a manifold leading to a series of flasks (250 ml) containing the treated soils. The air from each flask was then passed through a gas washing bottle containing a mixture (40 ml) of ethanolamine+ 2-methoxyethanol(l+ 3, by volume). At intervals of about 7 days, the trapping solution was replaced and made up to 50 ml with methanol. Duplicate subsamples (2 ml) were placed in counting vials with scintillation fluid (5 ml) and counted as before.1

2.5. Wet oxidation of soils The apparatus used for wet oxidation of soil samples was based on that described by Fuchs and de Vries.9 The sample (about 5 g dry soil) was mixed with a similar weight of sodium dichromate in a Buchner flask and water (20 ml) was added. The apparatus was sealed and a stream of nitrogen was passed through. Concentrated sulphuric acid (20ml) was run into the flask from a separating funnel and the flask heated for 45 min. The evolved 14C02 was dried by passing through concentrated sulphuric acid and absorbed in a mixture (35 ml) of ethanolamine+ 2-methoxyethanol (1 + 3, by volume). The solutions were made up to 50 ml with methanol and counted as before.

Degradation of methazole. I1 335

3. Results and discussion

3.1. Incubation studies

The data in Figures 2 and 3 show that methazole was unstable in this soil, confirming the results of previous experiments.' After about 40 days, only trace amounts of the parent compound remained.

(Dovs)

Figure 2. Degradation of phenyl-labelled methazole in soil. A, methazole; 0, DCPMU; 0, 14C02; A, unextracted.

-0 ,.--. /.--o

I A-AI 0 20 40 60 80 I00 I20

(Days)

Figure 3. Degradation of carbonyl-labelled methazole in soil. A, methazole; 0, DCPMU; 0, W O Z ; A, unextracted.

A. Walker and M. G. Roberts 336

There was a rapid rise in the amount of DCPMU extracted from the soil which reached a maximum of approximately 65 % of the initial methazole concentration after 15 to 20 days. Subsequent loss of DCPMU occurred more slowly and at the end of the experiment, after 120 days, the amount of DCPMU extracted was equivalent to 45 % of the initial methazole concentration. This slow loss of DCPMU from the soil is further illustrated by the data in Figure 4. When DCPMU was incubated

I .o A $-. / ------r 4

@'@--@

I A ~ A - A J 0 20 40 60 80 I00 I20

( D a d

Figure 4. Degradation of DCPMU in soil. A, DCPMU; @, 14C02; A, unextracted; 0, increase in amount of unextracted material from time 0.

in the soil, over 50% of the amount initially extracted could still be extracted after 120 days. DCPU (Figure 5) was also more stable than the parent compound, and after 120 days about 5 % of the initial amount remained. Diuron (Figure 6) was of similar stability to DCPMU in this soil and about 50% remained at the end of the experiment.

Also shown in Figures 2 to 6 are the cumulative amounts of labelled carbon dioxide evolved during the fust 105 days of the experiment and the amounts of radioactivity which were not extracted from the soil at each sampling time. If the reduction in the amount extracted resulted from loss as labelled carbon dioxide, these two curves would be the same. With DCPU this is apparently so but with the other four compounds, the loss of extractability is greater than the loss a sWO2. A later product in the degradation of methazole, diuron and DCPMU in soil is likely to be3,4-dichloroaniline (DCA).' With phenyl-labelled methazole, formation of this compound should result in little loss of W02 and as DCA becomes incorporated into soil organic matter and cannot be readily extracted from the soil,lO a build-up of unextracted radioactivity would be expected. However, degradation of carbonyl-labelled methazole, DCPMU and diuron to DCA should be accompanied by loss of the labelled carbon atom, and the discrepancies between the amounts unextracted and generation of labelled carbon dioxide with these compounds suggests that DCA formation alone cannot explain the observed increases in the amounts of unextracted radioactivity.

The results in Figure 4 suggest that the unextracted material produced when methazole and diuron are degraded in soil may be associated with DCPMU. The initial recovery of DCPMU from soil was low; only 65-70% of the amount nominally applied. When the amounts of unextracted radioactivity are corrected for this low initial recovery (subtracting the initial amount of unextracted radioactivity from all subsequent values), there is close agreement with the cumulative loss of 14C02.

Degradation of methazole. LI 337

(Days)

Figure 5. Degradation of DCPU in soil. A, DCPU; 0. 14COa; A, unextracted.

(Days)

Figure 6. Degradation of diuron in soil. A, diuron; 0, DCPMU; 0, 14C02; A, unextracted.

This further suggests the possibility that the amounts of DCPMU which are not extracted with methanol are also not readily degraded. Further tests were made to check that formation of unextractable compounds was a real effect. At 83 days, samples of soil from the treatment involving carbonyl-labelled methazole, DCPMU and DCPU were oxidised to determine the total amounts of


radioactivity in the soil. The amounts obtained were compared with the amounts of methanol- extractable radioactivity. The results (Table 1) show that oxidation of the soils treated with methazole or DCPMU released more radioactivity than did the normal methanol extraction. With DCPU, the amounts determined by oxidation and methanol were similar. This confirms that unextracted radioactivity is present in soils treated with methazole and DCPMU but not when treated with

Table 1. Recovery of radioactivity from methazole, DCPMU and DCPU-treated soil after 84 days incubation

Recovery (dis min-' g-l) "

Fraction Methazole DCPMU DCPU

Methanol extraction 4564 3131 1866 Wet oxidation 6387 4793 2106 Cumulative l4Co2 1827 1782 8038

Total recoverable* 8214 6575 10144 Extractable at time 0 8854 4786 8898

(1 Dis min-1 g-l=(disintegrations/minute) per gram dry soil. b Total residual activity at 84 days determined by wet oxidation

plus cumulative loss as l4Co2 to day 84.

DCPU. Also shown in Table 1 are the total amounts of radioactivity accounted for when the cumulative amounts of W02 evolved by the soil to day 84 are added to the total residual radioactivity at this time determined by wet combustion. These values are compared with the amounts of methanol- extractable radioactivity determined at time 0. These data suggest initial recoveries of 106, 73 and 89 % for methazole, DCPMU and DCPU respectively. Although variable, these results confirm the apparent low initial recovery of DCPMU from the soil.

The implication from the data in Table 1 is that inefficient extraction of DCPMU from the soil is responsible for the rapid rise in the amount of unextractable radioactivity when methazole is incubated in soil (Figures 2 and 3). A final experiment was made to determine the rate at which DCPMU becomes unextractable from the soil when added as an aqueous or methanolic solution. Solutions of labelled DCPMU in water and methanol were prepared and duplicate subsamples (2 ml) were counted. Separate amounts (5 ml) of these solutions were added to soil (30 g) in 100 ml conical flasks. The flasks were stoppered and shaken and the compound extracted with methanol (50 ml) in the normal way after 0, 1,2,4 and 24 h. Similar experiments with aqueous solutions only were made with methazole and DCPU. The results are shown in Table 2. Immediately after addition of DCPMU to the soil in water, the recovery was only 84%, and after 4 h, over 30% of the radioactivity could not be extracted into methanol. Given the inherent stability of DCPMU in soil

Table 2. Recovery of methazole, DCPMU and DCPU by methanol extraction

Recovery (% of applied)

DCPMU from Time (h) Methanol Water Methazole DCPU

0 83.5 84.2 99.1 85.3 1 76.6 75.9 2 71.5 74.2 96.0 90.0 4 67.4 75.2

24 65.1 74.1 80.7 88.5

- -

- -


(Figure 3), it seems unlikely that this loss results from degradation. Similar results were obtained when solutions of DCPMU in methanol were examined, further suggesting that degradation was not the cause, and also suggesting no reduction in extraction efficiency by aqueous methanol, Methazole was recovered initially from soil but after 24 h, almost 20% of the radioactivity could not be extracted. Since methazole is rapidly degraded in this soil, this degree of change could result from its degradation to DCPMU. With DCPU, the results were somewhat variable, but they suggest little change in the efficiency of extraction which averaged about 88 %. These results therefore further suggest that DCPMU is the main source of the unextracted radioactivity produced during methazole degradation in soil.

3.2. Pathway of degradation In previous studies of methazole degradation in soil, Carringer et a1.2 and Brockmans have proposed the orderly degradation route for methazole of opening of the heterocyclic ring with loss of C02 to form DCPMU, followed by demethylation to DCPU and subsequently hydrolysis to DCA. How- ever, only small amounts of DCPU were found in their treatments. In the present work, DCPU was not detected in either of the treatments involving methazole (Figures 2 and 3) and the results in Figure 4 show that DCPU was a minor degradation product of DCPMU. Unpublished data from the National Vegetable Research Station have shown that DCPU is produced when methazole is degraded in plants and that it can be determined by the methanol extraction and thin-layer chromatographic techniques described above. The results in Figure 5 also show that DCPU can be determined by the techniques used. These results also suggest that if produced, DCPU would be suffici- ently stable to build-up in significant concentrations. This is illustrated by the data in Figure 7. The positions for the curves shown in this figure were calculated assuming that methazole is degraded via DCPMU and DCPU to DCA by a series of first-order reactions. The half-lives used in the calculations were derived from the data in Figures 2-5 and were 2.4,144 and 29 days respectively for methazole, DCPMU and DCPU. To take account of the observed low recovery of DCPMU, it was assumed that only 70% would be extracted (the mean recovery shown by the data in Figure

4.c

3.c

- - I 0

0 2.0 a -

I .o 'Yo2

DCPU

0 20 40 60 80 I00 I20 !Davs)

Figure 7. Calculated curves for methazole degradation assuming degradation through DCPMU and DCPU to DCA by a sequence of first-order reactions.


4, Table 1 and Table 2). When compared with the observed data for heterocyclic ring-labelled methazole (Figure 3), the results show that loss as 14C02 is underestimated and that DCPU is calculated to account for about 12% of the initial dose from day 40 onwards.

The results from the present experiments suggest an alternative route for methazole degradation. There was close agreement between the apparent build-up of unextracted radioactivity and loss of 14C02 (Figure 4) and the data therefore suggest that demethylation to DCPU is not involved in degradation. A similar pqthway of degradation has been proposed for linuron, 1-(3,4dichloro- phenyl)-3-methoxy-l-methylurea, where loss of herbicide from the soil is not associated with N- demethylation or demethoxylation, but with loss of the carbonyl group.11912 The way in which this proposed pathway for methazole degradation in soil can explain the observed results is shown in Figure 8 for phenyl-labelled methazole and in Figure 9 for carbonyl-labelled methazole. Half-lives

4.c

3.c

- 1 0

o1 7.0 3. 1

I .o

'A 1 A

IA [ A I I 0 20 40 60 80 I00 I20

(Duysl

Figure 8. Degradation of phenyl-labelled methazole in soil-calculated compared with observed. A. methazole: 0, DCPMU; A, unextracted.

assumed that only 70% of the DCPMU produced during degradation was extracted from the soil. With phenyl-labelled methazole, the unextracted residue included DCA whereas with carbonyl- labelled methazole, formation of this compound was assumed to result in release of W02. In both instances good agreement was obtained between observed and predicted degradation patterns.

4. Conclusions

Methazole is degraded rapidly in soil to produce DCPMU as the main degradation product. There is a build-up of unextracted material when methazole is degraded in soil and the results suggest that this is mainly DCPMU. It is proposed that methazole is degraded to DCA with DCPMU as the only intermediate and that demethylation of DCPMU to DCPU is a minor route.

Acknowledgements Part of this work formed the project carried out by M. G. Roberts of the Department of Biological Sciences, Bath University during Sandwich Course training at Wellesbourne. We are grateful to


0 2 0 40 60 80 100 '20

(Dcvs)

Figure 9. Degradation of carbonyl-labelled methazole in soil-calculated compared with observed. A, methazole; 0, DCPMU; 0, 14C02; A, unextracted.

Velsicol Chemical Ltd for a gift of samples of labelled methazole and analytical grade methazole, DCPMU and DCPU, to Du Pont (UK) Ltd for samples of labelled and analytical grade diuron and to Dr G. G. Briggs, Rothamsted Experimental Station, Harpenden for the sample of labelled DCPU.

References 1. 2. 3. 4. 5. 6. 7. 8. 9.

10. 11. 12.

Walker, A. Pestic. Sci. 1978, 9, 326. Carringer, R. D.; Rieck, C. E.; Harger, T. R. Proc. 28rh A. Meet. Sth Weed Sci. Sor. 1975, 292. Brockman, F. E.; Duke, W. B. Weed Sci. 1977,25, 304. Jones, D. W.; Foy, C. L. Pestic. Biochem. Physiol. 1972, 2, 8. Dorough, H. W.; Whitacre, D. M.; Cardona, R. A. J. agric. Fd Chem. 1973, 21, 797. Butts, E. R.; Foy, C. L. Pestic. Eiochem. Physiol. 1974, 4, 44. Dalton, R. L.; Evans, A. W.; Rhodes, R. C. Weeds 1966,14, 31. Smith, J. W.; Sheets, T. J. J. agric. Fd Chem. 1967, 15, 577. Fuchs, A. ; De Vries, F. W. Int. J . appl. Radiat. Isotopes 1972, 23, 361. Hsu, T. S.; Bartha, R. J. agric. Fd Chem. 1976, 24, 118. Borner, H. Z . Ppkrankr. PpParh. PflSchutz. 1965, 72, 516. Wallnofer, P. Weed Res. 1969, 9, 333.

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The degradation of methazole in soil. II. Studies with methazole, methazole degradation products and diuron