Pestic. Sci. 1988, 22, 297-305

Effects of Microbial Inhibitors on the Degradation Rates of Metamitron, Metazachlor and Metribuzin in Soil

Richard Allen and Allan Walker

Institute of Horticultural Research, Wellesbourne, Warwick CV35 9EF, UK

(Revised manuscript received 22 September 1987; accepted 29 September 1987)

ABSTRACT

Rates of carbon dioxide evolution and degradation rates of metamitron, metazachlor and metribuzin were measured in two soils in the presence of three microbial inhibitors. The nonselective microbial inhibitor sodium azide reduced both carbon dioxide evolution and the rate of loss of all three herbicides in both soils, although the reduction in degradation rate of metamitron was small. The antibacterial antibiotic novobiocin enhanced carbon dioxide evolution @om both soils but had variable effects on the rates of herbicide degradation. It inhibited degradation of metazachlor and metribuzin, and in one of the soils its effects on metazachlor degradation were similar to those of sodium azide. Novobiocin inhibited degradation of metamitron to a small extent in one soil only. The antijiingal antibiotic cycloheximide also enhanced carbon dioxide evolution @om both soils. In general, its effects on herbicide degradation were similar to those of novobiocin, although the extent of inhibition was usually less pronounced. The results are discussed in terms of the relative involvement of microorganisms in degradation of the three herbicides.

1 INTRODUCTION

It is generally accepted that microorganisms are involved in the degradation of most soil-applied herbicides. There have been some recent attempts to relate rates of degradation in different soils,' or in a single soil in which pretreatment' or incubation conditions3s4 were varied, to parameters such as total microbial biomass

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298 R. Allen, A . Walker

or microbial respiration. Although the results have indicated some broad relationships with these microbial characteristics, different relationships have been observed for different herbicides. For example, Allen and Walker' reported that the rates of degradation of metazachlor and metribuzin in a range of mineral soils could be correlated with soil microbial activity, estimated as the rate of carbon dioxide evolution, but metamitron degradation in the same soils was not correlated with this microbial parameter. In experiments with a single soil but with various pretreatments, Walker and Brown2 found that atrazine degradation rates were highly significantly correlated with soil respiration, metamitron degradation rates were correlated with microbial biomass, but propyzamide degradation rates were not related to either of these. As discussed previously,' it is likely that different groups of microorganisms will be involved in the degradation of different compounds. It is therefore not surprising that parameters such as respiration or total biomass, which are coarse measurements of soil microbial characteristics, do not always correlate with degradation rates. Added to this is the possibility that some herbicides may be degraded by a combination of both microbial and nonmicrobial chemical transformation, hence complicating the situation yet further.

The present experiments were made to examine further the possible contribution of soil microorganisms to degradation of the herbicides metamitron, metazachlor and metribuzin. Rates of degradation were measured in the presence of antibacterial and antifungal antibiotics and the total microbial inhibitor sodium azide.

2 EXPERIMENTAL METHODS

2.1 Soils and herbicides

Two soils were used in these experiments. Their properties are listed in Table 1. Details of the methods used to derive these data were given previously.' The herbicides used were commercial wettable powder formulations of metamitron and metribuzin (700 g a.i. kg-') and a suspension concentrate formulation of

TABLE 1 Soil Properties

Soil Property Soil 1 Soil 2

Soil texture Mineral fraction: clay < 2 pm (%)

silt 2-50 pm (%) sand 5&2000 pm (%)

Organic carbon (%) PH Moisture content at applied pressure of 33 kPa (%) Freundlich adsorption constants (kf): metamitron

metazachlor metribuzin

Clay 1o:m 29 34 37

1.7 7.3

1.3 1.5 0.62

23

Sandy loam 17 8

75 2.4 6.4

2.9 2.7 0.93

20

Ej'cts of microbial inhibitors on herbicide degradation in soil 299

metazachlor (500 g litre-'), together with analytical grade samples of the three compounds. A sample of 14C-phenyl-labelled metamitron (specific activity 318 kBq mg- ') was used in some of the laboratory experiments.

2.2 Degradation of metazachlor and metribuzin

Fresh samples of Soils 1 and 2 (Table 1) were sieved (2 mm mesh), and duplicate amounts (1.2 kg) were stored overnight while further amounts (30 g) were dried at 110°C to determine soil moisture content. This was found to be between 60 and 65 % of the moisture content at an applied pressure of 33 kPa for both soils. A suspension of the commercial formulation of either metazachlor or metribuzin in water (10 ml) was incorporated by sieving into one sample of each soil to give an initial concentration of 4.0 mg herbicide kg- ' dry soil. Each soil sample was then subdivided into four amounts (300 g). For each soil/herbicide combination, the four subsamples were treated with aqueous suspensions of novobiocin or cycloheximide (300 mg in 20 ml water) or of sodium azide (240 mg in 20 ml water), or with water (20ml). Further water was added to all of the treatments to give the required moisture contents (Table 1). Each subsample was sieved to ensure thorough mixing, and divided between two Kilner jars (500 ml capacity). A vial containing 0.15 M aqueous sodium hydroxide solution (15 ml) was placed in the centre of each jar, and the jars were sealed. All of the containers were incubated at 20°C. Single subsamples of soil (25 g) were taken from each jar after 0,5,12,18 and 26 days and at these times the amounts of carbon dioxide evolved by the soils were determined as described before.' The total amount of carbon dioxide evolved never exceeded 1.75 x moles. Assuming a respiratory quotient of one, less than 42 cm3 of oxygen would be consumed, thus reducing the concentration of oxygen from approximately 20 to 12% of the total volume of air in the Kilner jars. Such changes in the partial pressure of oxygen without similar increases in carbon dioxide concentrations are unlikely to affect the growth of obligate aerobes or facultative anaerobes over short time period^.^ The decrease in weight of soil after each sampling was noted, and the calculations of microbial respiration per unit weight of soil adjusted accordingly. Residual herbicide concentrations were measured as before.' Mean extraction efficiencies from spiked samples were 9 0 1 ( 5.0) % and 94.8 ( f 2.8) % for metazachlor and metribuzin, respectively.

2.3 Degradation of metamitron

The results from preliminary experiments demonstrated that the presence of the antibiotics in soil interfered with the determination of metamitron residues by high- performance liquid chromatography (h.p.1.c.). Experiments with this compound were therefore made using l4C-labe1led herbicide. The soils were prepared as described above, the labelled herbicide was added in methanol (5 ml) to the sieved soil, and the solvent was allowed to evaporate. An aqueous suspension of the commercial formulation of metamitron was then incorporated by sieving as before to give a nominal initial concentration of 4 mg kg-' dry soil with a concentration of radioactivity of 77 kBq kg- dry soil. The experimental procedure was continued as described for metazachlor and metribuzin.

300 R . Allen, A . Walker

Metamitron was extracted from the soil by shaking with methanol-water as before.' The mean extraction efficiency from spiked samples was 95.6 (k 2.8) %. The samples were centrifuged, and subsamples of the clear supernatant (20 ml) were evaporated to remove the methanol on a vacuum rotary evaporator. Sodium sulphate (50 ml, 50 g litre-' in water) was added and the herbicide was partitioned into ethyl acetate (20 ml). The ethyl acetate layer was removed, dried by shaking with anhydrous sodium sulphate, and duplicate aliquots ( 2 ml) were pipetted into scintillation vials. Toluene-based scintillator (Cocktail T, BDH Chemicals Ltd; 10ml) was added and the samples were counted using a Rackbeta liquid scintillation counter (Pharmacia-LKB Ltd, Milton Keynes, UK). Efficiency correction was achieved using the external standard channels ratio method. Residual concentrations in the samples without inhibitors, determined from these radioactivity measurements, were compared with concentrations of parent herbicide measured on the same soil extracts by h.p.1.c.' for all time points. Radioassay gave residual concentrations equivalent to 102.3 ( f 4.3)% and 93.2 ( f 4.2) % of those measured by h.p.1.c. for Soils 1 and 2, respectively. This indicates that any degradation products extracted by methanol did not partition into ethyl acetate.

Engelhardt et aL6 described a thin-layer chromatography (t.1.c.) method for the separation of metamitron from its degradation products using the solvent system ethyl acetate +chloroform +acetic acid (12+ 8 + 1 by volume). The ethyl acetate extracts from the samples of Soil 1 without inhibitors, which remained following preparation of samples for counting, were evaporated to dryness, and the extracted material was redissolved in ethyl acetate (0.1 ml). These solutions were spotted on to silica gel t.1.c. plates and developed for 10 cm above the baseline using the solvent mixture described above. The plates were dried and examined using a Birchover Instruments radiochromatogram spark chamber to locate the zones of radioactivity. Only one spot was found with an R, value identical with that of the original labelled metarnitron, thus confirming that the radioactivity in the soil extracts which partitioned into ethyl acetate comprised only parent herbicide.

2.4 Statistical analyses

Respiration data and residual concentrations of each herbicide were analysed for each time point by a one-way analysis of variance. Soil respiration was averaged across the herbicide treatments, hence each data point was a mean of six measurements (duplicate samples from each soil/herbicide combination; error d.f. = 20). Each measurement of residual herbicide concentration was a mean of two values (error d.f. = 4).

3 RESULTS AND DISCUSSION

3.1 Soil respiration

Results from the measurements of carbon dioxide evolution made during the 26-day incubation period are summarised in Fig. 1. The values presented are means across herbicide treatments. The results of previous studies indicated that soil respiration

Eff;cts of' microbial inhibitors on herbicide degradation in soil 301

= I 1 I (a)

I I I 10 20 30

I

T I I

0 10 20 30 Days

Fig. 1. Carbon dioxide evolution from samples of (a) Soil 1 and (b) Soil 2 treated with (m) cycloheximide, (0) novobiocin, (A) sodium azide, or (0) untreated. I : 1.s.d. (P<O.O5; d.$ = 20).

rates were unaffected by any of the three herbicides at concentrations similar to those used in the present experiments.' The three inhibitors had different effects on respiration rates, although their relative effects were similar in both soils. The rate of carbon dioxide evolution from the untreated samples decreased with time as reported and discussed previously.' The rate of evolution from samples of soil treated with sodium azide, an aerobic respiration inhibitor, was slower than that from the control soil. During the period from 5 to 26 days, the average rates of carbon dioxide evolution from the azide-treated samples of Soils 1 and 2 were reduced to approximately 30 % and 40 % of the rate from the appropriate untreated control. These results demonstrate that although sodium azide inhibited respiration, the inhibition and hence soil sterilisation was incomplete.

The rate of carbon dioxide evolution was greatest in the presence of the antifungal antibiotic, cycloheximide. After 26 days, the total amount of carbon dioxide evolved from cycloheximide-treated samples was approximately three times that from untreated samples of both soil types. This rapid evolution of carbon dioxide may have been due, in part, to degradation of the antibiotic (present at an initial concentration of 640 mg C kg- dry soil), and in part, to increased activity of the

3 02 R . Allen, A . Walker

residual microbial population (presumably mainly bacteria) as it utilised the extra substrate made available from the microorganisms susceptible to cycloheximide. The sigmoid curves obtained are of similar shape to those reported previously for carbon dioxide evolution from soil treated with penicillin, chloromycetin and terram ycin.'

The rates of carbon dioxide evolution from the soils treated with the antibacterial antibiotic novobiocin decreased with time in both soils (Fig. 1). However, the cumulative amount of carbon dioxide produced in 26 days, relative to the untreated controls, was increased by 70% and 90% for Soils 1 and 2, respectively. The initial rapid increase in carbon dioxide evolution relative to the untreated controls may have resulted, in part, from respiratory uncoupling by the antibiotics, although the information available is not sufficient to confirm this.

3.2 Herbicide degradation

The influence of the microbial inhibitors on degradation of the three herbicides is illustrated in Fig. 2. The rate of loss of all three herbicides in both soils was most rapid in the untreated control samples. In general, sodium azide reduced the rates of loss although it did not prevent degradation completely. It severely reduced the rate of loss of metazachlor and metribuzin in both soils. The rate of metamitron degradation, however, was less affected. Although continued slow degradation in the azide treatments may have resulted from the incomplete sterilisation of the soils, it is possible that nonbiologicai chemical degradation could also be involved. There is evidence for nonbiological degradation of both metamitrod and metribuzin.' In the present experiments, 30 to 35% of the initial amount of metamitron was degraded during 26 days in the presence of sodium azide, which agrees well with the results of Engelhardt et ~ 1 . ~ who reported 50% degradation in an abiotic aqueous solution in 14 days.

The effects of the two selective inhibitors on degradation varied between herbicides and soils (Fig. 2). The antibacterial antibiotic, novobiocin, reduced the rate of degradation of metazachlor in both soils. In Soil 2, the residues after 5, 12 and 18 days were similar to those measured in samples sterilised with sodium azide, but in Soil 1, the inhibitory effect of novobiocin was not as great as that of sodium azide. Metazachlor degradation was also inhibited by the antifungal agent cycloheximide in Soil 2, but not to the same extent as with novobiocin. Cycloheximide did not inhibit degradation in Soil 1. There are a number of possible reasons for these differences in inhibitory action of the antibiotics between the two soils including the presence of different spectra of inhibitor-sensitive and -resistant microorganisms resulting from differences in soil properties, and direct effects of soil properties on the activity of the inhibitors. Previous studies have shown that adsorption by clay minerals can inactivate a number of antibiotics." In the present experiments, both of the antibiotics tested were most active in inhibiting metazachlor degradation in the soil with lower clay content (Soil 2). The relatively rapid loss of activity of cycloheximide is consistent with the results of previous experiments" which demonstrated complete degradation of this antibiotic in soil within 11 days. The similar effects of sodium azide and novobiocin on the early degradation rate of metazachlor indicate that the spectrum of microorganisms

Effects of microbial inhibitors on herbicide degradation in soil

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304 R . Allen, A . Walker

responsible for degrading this herbicide may be composed mainly of soil bacteria. Studies involving use of antibiotics have demonstrated that bacteria are responsible for degradation of the insecticide isofenphos in soil." The rate of ['"C] carbon dioxide evolution from labelled insecticide incubated in soil treated with cycloheximide (antifungal antibiotic) was the same as that from labelled insecticide incubated in control soil, whereas ['"C] carbon dioxide evolution from the insecticide in soil treated with chloramphenicol (antibacterial antibiotic) was very low and similar to that from soil sterilised by autoclaving.

There were no significant differences between residues of metribuzin in samples of soil treated with novobiocin or cycloheximide although the residues were significantly greater than those measured in the control samples of both soils. This indicates that a much wider spectrum of microorganisms may degrade metribuzin compared with metazachlor, and that this spectrum is wide enough for neither antibiotic alone to cause a drastic reduction in the rate of loss. Sodium azide was a more effective inhibitor than either of the antibiotics, providing further evidence that a wide spectrum of microorganisms may be involved.

There were no significant differences between residues of metamitron measured in samples of Soil 1 treated with novobiocin or cycloheximide, and in Soil 2, the residues were not significantly different from those measured in the controls treated with herbicide alone, except with novobiocin after incubation for 18 and 26 days. Sodium azide also had relatively little effect on metamitron degradation compared with its effects on rates of loss of the other two compounds. This is consistent with the results of earlier studies' which demonstrated that the rates of metribuzin and metazachlor degradation in 12 soils were correlated with soil microbial respiration, whereas those of metamitron were not.

Although carbon dioxide evolution from the soil was enhanced by the presence of the two antibiotics (Fig. 1) this was not reflected in enhanced degradation of any of the three herbicides. This may be a further indication that some respiratory uncoupling occurred in the presence of the antibiotics, since this would result in significant increases in apparent respiration rate without a concomitant increase in energy available for other processes. Herbicide degradation was either inhibited or unaffected by the antibiotic treatments. When inhibition occurred, there is the possibility that this might have resulted from preferential degradation of the antibiotics by the surviving microorganisms rather than from a direct inhibition of the active herbicide degraders, but there is insufficient information to confirm whether this was so. More detailed examination of the microbial characteristics of the soil will be required before more definite conclusions can be reached.

4 CONCLUSIONS

The main conclusion from these experiments is that they indicate that different groups of soil microorganisms are involved in degradation of the three herbicides. The results are in general agreement with those reported previously' and provide further evidence for greater involvement of microorganisms in degradation of metazachlor and metribuzin than in degradation of metamitron.

Effects of microbial inhibitors on herbicide degradation in soil 305

ACKNOWLEDGEMENTS

These experiments formed part of a thesis submitted by R. Allen to the Depart- ment of Plant Sciences, Leeds University for the degree of PhD, and the authors thank Dr D. J. Pilbeam of that Department for helpful discussions. The work was financed by an AFRC postgraduate research studentship. The authors are grateful to Bayer AG for their gift of radiolabelled metamitron, and to Bayer (UK) Ltd and BASF (UK) Ltd for their interest in this work and for samples of analytical grade herbicides.

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of Soil-Applied Herbicides, pp. 63-71. 3. Anderson, J. P. E. Soil Biol. Biochem. 1981, 13, 155-161. 4. Anderson, J. P. E. Soil Biol. Biochem. 1984, 16,483490. 5. Hattori, T. Microbial Life in the Soil - an Introduction. Marcel Dekker, New York,

1973, 427 pp. 6. Engelhardt, G.; Ziegler, W.; Wallnofer, P. R.; Jarczyk, H. J.; Oehlmann, L. J . Agric.

Food Chem. 1982,30, 278-282. 7. Allen, R. PhD Thesis, University of Leeds, 1985. 8. Nissen, T. V. Nature (London) 1954, 174, 22C227. 9. Savage, K. E. Weed Sci. 1977, 25, 55-59.

10. Martin, N.; Gottlieb, D. Phytopath. 1952, 42, 294-296. 11. Gottlieb, D.; Siminoff, P.; Martin, M. M. Phytopath. 1952, 42, 493496. 12. Racke, K. D.; Coats, J. R. J . Agric. Food Chem. 1987, 35, 94-99.