Pesric. Sci. 1987, 21, 233-240

Enhanced Degradation of Iprodione and Vinclozolin in Soil: A Simple Colorimetric Test for Identification of

Rapid-degrading Soils

Allan Walker

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

(Revised manuscript received 14 April 1987; accepted 21 April 1987)

ABSTRACT

Measurements by gas-liquid and high performance liquid chromato- graphy confirmed that 3,5-dichloroaniline was an important degradation product o f iprodione in soil, and demonstrated that it could be extracted in relatively high concentrations when iprodione degradation was rapid. A colorimetric test based on the production of a diazo colour complex was shown to differentiate between amounts of the aniline in the range f rom 2 to 15 pg. The test was used to examine 3,5-dichloroaniline production f rom iprodione incubated at 50 mg kg-1 in 33 soils with previously-measured iprodione degrading properties. A positive colour reaction in acetone extracts made after 3 days was obtained with those soils in which the time to 90% degradation of iprodione was less than 6 days. A positive reaction after 7 days was obtained when this time was less than 14 days, and a positive reaction after 10 days was associated with a time to 90% degrada- tion of less than 22 days. Preliminary studies with vinclozolin indicated that the test could also be used to identify soils which rapidly degrade this fungicide.

1 INTRODUCTION

Previous experiments have shown that the rates of degradation of iprodione and vinclozolin are more rapid in soils which have been treated previously with the fungicides than in previously-untreated In a study involving 33 soils from commercial fields, for example, the time to 90% degradation of iprodione varied from less than 5 days in soils with an extensive pre-treatment history to over 90 days in two of the previously untreated soils.3 There was some evidence to suggest

233

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234 A. Walker

that performance of the fungicide against white rot disease (Sclerotiurn cepivorurn) of onions was poor in the rapid-degrading soils. From an advisory point of view, it would be useful if a simple test were available to characterise the degrading potential of different soils, so that those in which the fungicides are less likely to be effective could be identified. Such a test has been developed pre- viously for characterising carbofuran-degrading soils.4 It involves incubation of a small quantity of soil in an aqueous buffer solution containing carbofuran for 7 days, followed by examination of the solution for the presence of the phenolic hydrolysis product of carbofuran using a colorimetric procedure. Comparison with the colour produced by known standards provides a measure of the degrad- ing ability of the soil.

Experiments with [14C]-labelled iprodione and vinclozolin indicated that one of the main degradation products of both fungicides is 3,5-di~hloroaniline.~ Anilines undergo diazotisation reactions with sodium nitrite in the presence of hydro- chloric acid, and the diazo compounds can be coupled with substituted naphthyl derivatives to produce coloured complexe~.~ Colorimetric methods based on these reactions have been established for the quantitative measurement of residues of substituted urea herbicides in soil and plant The present experiments were made to examine the behaviour of 3,5-dichloroaniline in soil, to confirm its presence following degradation of iprodione and vinclozolin, and to determine whether a simple colour test could be used to characterise the degrad- ing potential of different soils.

2 EXPERIMENTAL METHODS

2.1 Soils and fungicides

Some of the properties of the 33 soils used are given in Table 1. The soils are listed in decreasing order of their ability to degrade iprodione, as indicated by the times to 90% degradation (DT,) of the fungicide. Full details of the. degradation measurements and further properties of the soils were reported previ~usly.~ The fungicides used were commercial wettable powder formulations of iprodione (‘Rovral’, 500 g kg-1 w.P.) and vinclozolin (‘Ronilan’, 500 g kg-l w.P.). Pure 33 - dichloroaniline was obtained from Fluorochem Ltd, Glossop, Derbyshire.

2.2 Studies with 3,s-dichloroaniline

Separate samples (500 g) of a sandy loam soil from Wharf Ground field at the Institute of Horticultural Research, Wellesbourne (soil 28, Table 1) were treated with 3,5-dichloroaniline at concentrations of 5 , 10, 15 and 20 mg kg-1 dry soil. The chemical was added in acetone ( 5 ml) and, when the solvent had evaporated, sufficient water was added to give a soil moisture content equivalent to an applied pressure of 33 kPa (1243% mass/mass). The soils were mixed thoroughly and incubated in polypropylene containers at 20°C. Immediately after preparation and then 3, 7, 10 and 15 days later, duplicate subsamples (50 g) of soil were removed from each treatment and extracted with acetone (50 ml) by shaking for 1 h on a wrist action shaker. The concentrations of 3,5-dichloroaniline in the

Colorimetric test for ideniificaiion of rapid-degrading soils 235

TABLE 1 Soil Properties and Responses in the Colorimetric Test

Soil number

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

Organic p H matter mJ) ________

2.49 3.13 3.84 3.50 7.47 3.41 3.74 3.69 2.55

10.09 3.69 3.67 3.69 3.49 3.33 3.43 3.46 3.45 3.50 3-28 3.50 6.66 3.61 3.41 3.66 3.39 3.82 2.49 2.43 2.86 2.12 3.94 1.49

6.2 7.2 7.0 7.3 6.6 7.2 6.0 7.6 7.6 6.6 6.9 7.3 6.9 7.2 7.4 7.1 7.3 7.2 7.8 7.5 7.4 7.2 7.3 7.8 7.6 7.5 7.6 6.2 6.2 7.3 6.9 5.7 6.4

Water content

(%)

12.8 13.6 11.4 22.7 19.8 19.6 10.6 18.9 20.7 24.3 20.7 19.5 19.5 21.8 20.0 19.7 20.2 19.6 19.5 20.0 19.8 32.8 20.7 18.5 19.8 19.2 20.0 12.8 24.7 23.8 12.2 21.9 9.9

3.8 4.8 4.9 5.1 5.1 5.2 6.0

10.3 10.4 11.1 12.4 12.6 14.5 15.8 17.1 17.6 18.4 19.1 22.0 22.6 22.8 24.6 28.3 33.1 33.1 35.5 37.9 54.5 57.0 57.6 58.5 92.0 93.0

Colorimetric responsea after: __ ~ ______ 3 days 7 d a y s __ * * *

* * * * * * * *

* * * *

* * * 0 + 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

*

10 days

* * * * * * * * * * * * * * * * *

* * * * *

* * *

* * * * + + + 0 + + 0 + 0 0 0 0 0 0 0 0 0 0 0 0

* * * * * * * * * * * * * * * * * * * * * * * * *

* * * * * * * * * * * * + + *

* * * + + 0 0 0 0 0 0 0 0 0 0 0 0

OSee Section 3.2 for interpretation of colorimetric response.

acetone extracts were measured by high performance liquid chromatography (h.p.1.c.) using the same methods and conditions as described previously for iprodione.3 The retention time in the solvent system used (acetonitrile+ water, 60+40 by volume) was 4.5 min. Residues were also measured directly in the acetone extracts by gas-liquid chromatography (g.1.c.) using a nitrogen (flame ionisation) detector. The column used (1.5 mx5 mm i.d.) was packed with 3% OV1 on Chromosorb WHP, the carrier gas flow rate was 40 ml min-' and the column temperature was 175°C. The retention time of 3,5-dichloroaniline was 1.8 min.

236 A. Walker

2.3 The colorimetric test

The colorimetric procedure used was a modification of that described by Dalton and Pease.6 Standard solutions of pure 3,5-dichloroaniline in acetone (2 ml) were pipetted into glass stoppered tubes (50 ml) to give amounts of the aniline in the range from 1 to 20pg. Similarly, acetone extracts (3 ml) of appropriate soil samples (Section 2.2) were also added to glass stoppered tubes. Hydrochloric acid (10 ml, IM) was added followed by sodium nitrite solution (1 ml, 20 g litre-' in water). The tubes were stoppered, shaken and allowed to stand for 15 min. Sulphamic acid solution (1 ml, 100 g litre-' in water) was added and the samples allowed to stand for 10 min with occasional shaking. Finally, a solution of N-1- naphthyl ethylenediamine dihydrochloride (2 ml, 20 g litre-' in water) was added, producing a magenta colour which varied in intensity according to the concentration of 3,5-dichloroaniline present originally. The intensity of the col- our was assessed visually after 15 min.

2.4 The colorimetric test and iprodione degradation

A preliminary evaluation of the ability of the colorimetric test to distinguish between soils showing variations in iprodione degradation rate was made using soils 1 ,2 ,11,12,21 and 22 (Table 1). Twelve subsamples of each soil (50 g) were weighed into conical flasks (100 ml) and iprodione in water (4 ml) added to give four replicate samples of each soil containing 10,50 or 100 mg iprodione kg-1 dry soil. Sufficient water was added to give the required soil moisture content (Table 1). The soils were allowed to stand for 1 h and were then vigorously shaken to incorporate the fungicide. The flasks were loosely capped with aluminium foil and incubated at 20°C. After 3 and 7 days, duplicate samples of each treatment were extracted with acetone (50 ml) and the acetone extracts examined for the pre- sence of 3,5-dichloroaniline by h.p.l.c., g.1.c. and the colorimetric test as described before.

TABLE 2 Extractable Residues of 3,s-Dichloroaniline Over a 15-day Incubation Period

Initial Extractable 3,5-dichloroaniline (mg kg-') after (days): ~~ ~

0 3 7 I0 15 concentration (mg kg-') ___________-____ ~ ~ __

Residues measured by g.1.c. 5 4.61 1.54 0.74 0.46 0.29

10 8.62 4.14 2.32 1.48 0.82 15 14.0 7.31 4.92 3.11 1.84 20 17.7 8.93 6.70 4.15 2.17

Residues measured by h.p.1.c. 5 4.32 1.47 0.65 0.35 0.19

10 8.66 3.96 2.14 1.22 0.71 15 13.7 7.15 4.66 2.93 1.62 20 17.5 8.70 6.21 3.93 1.98

Colorimetric test for identification of rapid-degrading soils 231

2.5 Colorimetric evaluation of the degrading potential of 33 soils

Six subsamples (50 g) of the 33 soils listed in Table 1 were treated with 50 mg iprodione kg-l dry soil as before (Section 2.4) and incubated at 20°C. Further subsamples of soil 1 (Table 1) which had been treated previously in the field with 3 doses of vinclozolin ( 3 kg ha-l on 23 May, 3 July and 5 August 1986) were prepared for incubation in the same way but containing 50 mg vinclozolin kg-*. Untreated control samples of the same soil (soil 28, Table 1) were also incubated with added vinclozolin. After 3, 7 and 10 days, two samples from all of the treatments were extracted with acetone and the acetone extracts examined for the presence of 3,5-dichloroaniline using the colorimetric procedure. The intensity of colour produced was compared with that given by 2, 5 and 1Opg pure 3,5- dichloroaniline.

3 RESULTS AND DISCUSSION

3.1 Determination of 3,s-dichloroaniline residues

The residues of 3,5-dichloroaniline extracted from soil at intervals during the incubation period of 15 days are shown in Table 2. The mean initial recovery of the applied dose was 88(*2*3)% and 90(&3-3)% when measured by h.p.1.c. and g.1.c. respectively, and the residues measured by h.p.1.c. after 3 ,7 ,10 and 15 days were consistently less than those measured by g.1.c. Calculations using all of the data in Table 2 showed that, on average, the residues measured by h.p.1.c. were 91.2(+8.6)% of those measured by g.1.c. suggesting some losses during evapora- tion of the solvents or during so1vent:solvent partitioning. Subsequent tests indicated that the amount of 3,5-dichloroaniline measured in an acetone solution which had been evaporated to dryness and then redissolved in the same volume of acetone was only 94.7% of that in the original solution thus confirming small losses during solvent evaporation. The results in Table 2 indicate a relatively rapid change in extractability with increasing incubation time. On average, about 50% of the amounts recovered initially were still extracted after 3 days, and only 10% after 15 days. They also indicate somewhat more rapid rates of loss at lower initial concentrations. The residues measured by g.1.c. after 15 days were equivalent to 6.3, 9.5, 13.1 and 12.3% of those recovered at zero time for the treatments involving initial concentrations of 5 , 10, 15 and 20 mg kg-I respectively. Studies with other chloro-substituted anilines have also shown a rapid loss of extract- ability when incubated with soil, and it has been suggested that complex forma- tion with soil organic matter is an important route for loss.8 In studies with 4-chloroaniline, Bartha'reported that up to 77% of the applied dose was bound to soil after 20 days incubation, and Chisaka and Kearney'" demonstrated similar loss of extractability of 3,4-dichloroaniline. The results in Table 2 suggest that if significant amounts of 3,5-dichloroaniline are produced during degradation of appropriate dicarboximide fungicides in soil, they should be extractable with acetone, at least for short periods following their formation.

238 A. Walker

3.2 Colorimetric test for 3,5-dichloroaniline

When the colorimetric procedure was used with a range of standard solutions containing pure 3,5-dichloroaniline, visual examination indicated no colour change with 1 pg chemical, and only a small but relatively insignificant pink colouration with 2 pg. On the other hand, 5 pg produced a distinct magenta colour which increased in intensity with 10 and 15 pg. Examination of the colours produced by 15 and 20 pg indicated no difference in colour intensity discernible to the naked eye, although dilution may well have differentiated between them. In all of the subsequent tests, colours were assessed relative to those given by 5 and 1Opg 3,5-dichloroaniline. The notation used to define the responses was 0 to indicate no colour change, + to indicate a small but insignificant pink colouration, and *, * * and * * * to indicate levels of magenta colour equivalent to <5,5-10 and >10 pg 3,5-dichloroaniline respectively. An assessment of the colours produced in subsamples (3 ml) of the acetone extracts from soils incubated with different amounts of 3,5-dichloroaniline for different periods is shown in Table 3. The results indicate that the test effectively distinguished between the different residual concentrations, and clearly characterised the decay of 33-

TABLE 3 Colorimetric Responses of 3,5-Dichloroaniline Residues

Extracted from Soil

Initial Colorimetric response after concentration incubation for (days):

3 7 10 15 (mg kg-’)

* * * + 0 0 + + *

* * * * * * * *

5 10 15 * ~.

* * * * * * * * * ** 20

TABLE 4 Residues of 3,5-Dichloroaniline and Colorimetric Responses Following Incubation of Iprodione at an Initial Concentration of

50 mg kg-I in Six Soils

Soil DTw 3,5-dichloroaniline Colour reaction number (days) residue (mg kg-‘) after (days):

after (days): - ______._

3 7 3 7 * * * * * * * * * * * *

3.8 7.3 18.7 1 2 4.8 9.2 16.4 *

* * 11 12.4 0.18 1.68 0 12 12.6 0.25 3.92 0 21 22.8 0-18 0.42 0 + 22 24.6 0.10 0-24 0 0

Colorimetric lest for identification of rapid-degrading soils 239

dichloroaniline in the soil (Table 3 compared with Table 2). The results further confirm that the intensity of the colour reaction was most effective in distinguish- ing between amounts of 3,5-dichloroaniline in the range from about 2 to 15 yg.

3.3 Preliminary evaluation of iprodione-degrading soils

The residues of 3,5-dichloroaniline recovered from the six soils incubated with iprodione at 50 mg kg-' (Table 4) show that large amounts were recovered after 3 and 7 days from the two rapid-degrading soils. The concentrations shown were measured by g.1.c.; those measured by h.p.1.c. were, as before, marginally smaller. In the two soils with an intermediate rate of degradation, the degradation product was present in relatively small, but significant concentrations after 7 days. Only trace amounts were found in soils 21 and 22 which were the slowest 'degraders' of those examined. Similar measurements made with the soils incu- bated with 10 mg iprodione kg-' gave significant levels of 3,5-dichloroaniline after 3 and 7 days in soils 1 and 2 only. Incubation with 100 mg iprodione kg-' resulted in concentrations approximately double those shown in Table 4.

The results from the colour test involving the same soil extracts indicated that it was particularly effective in characterising very rapid degradation. The assess- ments made for the samples involving an iprodione concentration of 50 mg kg-' are given in Table 4. With this concentration incubated in the soil, the results also suggest that the test could differentiate between those soils in which 90% degrada- tion occurred in 10-12 days and those in which it occurred in 20-25 days. The colour test was not so successful when a lower iprodione concentration (10 mg kg-') was used since there was no response from the extracts of soils 11 and 12. There was also no advantage in using a higher initial concentration of fungicide (100 mg kg-*) since the colour assessments made did not differ from those in Table 4. 3.4 Use of the colour test with 33 soils

The results of the colour assessments made following incubation of samples of all 33 soils for 3 ,7 and 10 days with 50 mg kg-I iprodione are shown in Table 1. They confirm the conclusion made previously that the test is particularly effective in identification of rapid-degrading soils. All of the soils with DT, of <6 days gave a positive colour response after 3 days. Soil 10 gave an apparently anomalous result , since it also gave a positive reaction after 3 days although the DT,, determined previously was more than 10 days. The assessments made after 7 days incubation (Table 1) also confirm the suggestion made previously (Section 3.3) that the test can differentiate between soils in which the DT,, is less than or greater than 12-15 days. A further categorisation of soils was possible using the colour assessments made after 10 days' incubation; only those soils with a DT, less than 20-22 days gave a positive colour reaction at this time. There was, however, somewhat more variability in these results, with soils 18 and 19 in particular giving an unexpectedly strong colorimetric response. It is possible that some of the discrepancies between the DT, values and the colorimetric responses may be associated with the two- stage degradation of iprodione reported previ~us ly .~ The DT, refers to loss of parent iprodione which degrades to 3,5-dichloroaniline via an unidentified inter-

240 A . Wulker

mediate. Variations in the rate of degradation of this intermediate between soils may well account for some of the apparently anomalous observations.

The results of the tests made with extracts of the two soils incubated with vinclozolin demonstrated that the test may also be useful for characterising soils that degrade this fungicide rapidly. A strong positive colour reaction (* * *) was obtained after 3 ,7 and 10 days when vinclozolin was incubated in a sample of soil from an area in the field treated three times previously with the fungicide. There was no colour reaction following incubation of the fungicide for similar periods in previously untreated soil from the same site.

Tests with untreated controls of all 33 soils indicated no positive reactions in the colorimetric test, demonstrating that 3,5-dichloroaniline residues that might have been present as a result of previous fungicide applications did not contribute to the responses listed in Table 1.

These results therefore show that the ability of soils to degrade certain dicar- boximide fungicides can be assessed by a relatively rapid and simple colorimetric procedure. There is a need to evaluate the test in a wider range of soils, par- ticularly with known degradation rates of vinclozolin, and to calibrate it with respect to the biological performance of the fungicides. It has the potential to indicate soils in which enhanced degradation has been induced, and hence to identify those in which efficacy problems might be encountered.

REFERENCES

1. Walker, A.; Entwistle, A. K.: Dearnaley, N. J. British Crop Protection Coiincil Monograph No. 27, Soils and Crop Protection Chemicals 1984. 117-123.

2. Walker, A.; Brown. P. A.; Entwistle. A. R. Pestic. Sci. 1986, 17, 183-193. 3. Walker, A. Pestic, Sci. 1987, 21, 219-231. 4. Chapman, R. A.; Moy, P.; Henning, K. J. Environ. Sa. Health, 1985, B20,313-319. 5 . Finar, I . L. Organic Chemistry, Vol. 1, Fimdamental Principles, Longmans, London,

6. Dalton, R. L.; Pease, H. L. J . Ass. Off. Agric. Chem. 1962, 45, 377-381. 7. Bleidner. W. E. ; Baker, II M.; Leyitsky, M.; Lowen, W. K. J . Agric. Food Chcrn.

1954,2,476479. 8. Still, G. G. ; Herrett, R . A. In: Herbicides: Chemistry. Degradation, Mode of Action.

(Kearney. P. C.; Kaufman, D. D.. Eds), Marcel Dekker, New York, 1976, pp. 609- 664.

1963, p. 582-605.

9. Bartha, R . J . Agric. Food Chem. 1971, 19, 385-387. 10. Chisaka, H. ; Kearney, P. C . J. Agric. Food Chem. 1970. 18, 854858.