Absorption and Degradation of Metalaxyl in Mustard Plant(Brassica juncea)

Naresh Mehta, G. S. Saharan, and T. S. Kathpal*Department of Plant Pathology and *Department of Chemistry and Biochemistry, CCS Haryana Agricultural University, Hisar—125004, India

Received March 11, 1996

Absorption and degradation of metalaxyl were studied in mus-tard (Brassica juncea) plants after application as a seed dresser, afoliar spray, and a combination of both under subtropical condi-tions in India. Results indicated that absorption of metalaxyl in-creased up to 30 days when it was applied as a seed dresser;thereafter, it started declining and was not detectable after 60days of sowing. The maximum residues (average, 9.03 ppm) ofmetalaxyl were found after 1 day of spraying. The dissipation ofmetalaxyl after initial deposits on mustard plants was almost com-plete after 15 days of spraying. The safe waiting period of meta-laxyl was calculated to be 62 and 8 days for seed dresser and foliarapplication, respectively. The seeds raised through treatments un-der study were completely free from any detectable amount ofmetalaxyl residues. © 1997 Academic Press

INTRODUCTION

Metalaxyl (methyl-N-(2-methoxy acetyl)N-(2,6-xylyl) DL-alaninate), a systemic fungicide, is commercially available forthe control of diseases caused by theOomycetesgroup of fungion several crops (Urechet al., 1977; Sharom and Edgington,1982; Businelliet al., 1984; Reddyet al., 1990). A mixedformulation of metalaxyl and mancozeb (Ridomil MZ-72) hasbeen recommended to prevent repeated application of dithio-carbamate and to avoid evolution of new races of the pathogen(Businelli et al., 1984). Much work has been done on metal-axyl uptake, translocation, mode of action, residues, fungalresistance, and its effect on various host plants (Lyr, 1987).However, the information with regards to its absorption, per-sistence, and residual toxicity inBrassicasis scanty, especiallyunder subtropical conditions in India, where it is being usedagainst downy mildew and white rust diseases. The presentinvestigations were, therefore, undertaken to generate informa-tion on absorption and dissipation of metalaxyl in mustardunder subtropical agroclimatic conditions in India.

MATERIALS AND METHODS

Fungicide. Metalaxyl (Apron SD-35) was used for seedtreatment (at 2 g a.i. kg−1 seed) and Ridomil MZ-72 (metalaxyl8% and mancozeb 64%) was used as foliar application (at0.25%). The formulated and technical grade standards of meta-

laxyl were obtained courtesy of M/S Hindustan Ciba GeigyLimited, Bombay, India.

Plant. The mustard (Brassica juncea(L.) Czern and Coss.)cv. RH-30 seeds were sown in a randomized block design withthree replications in a 7 × 2.4-2 m plot with a row to rowdistance of 30 cm and a plant to plant distance of 10 cm duringthe 1991–1992 crop season. All cultural practices were fol-lowed according to the recommendations in the area.

Treatments. The various treatments were (a) seed treat-ment with Apron SD-35 at a rate of 2 g a.i. kg−1 seed (T1); (b)two foliar sprays with Ridomil MZ-72 at a rate of 0.25% at 40and 70 days after sowing (T2); and (c) seed treatment withApron SD-35 at a rate of 2 g a.i. kg−1 seed followed by twofoliar sprays of Ridomil MZ-72 at a rate of 0.25% at 60 and 80days after sowing (T3). Precautions were taken to avoid anydrifting of the chemicals, by applying them in the morninghours when the wind speed was less than 2 km/hr and by takingspecial care at the time of seed sowing. The control plots weremaintained away from the treated ones but in the same virginland.

Sampling. In case of seed treatment (T1), the plant sampleswere collected after 7, 15, 30, 40, 60, and 90 days of sowingwhereas in case of foliar application (T2), sampling was doneafter 1, 5, 10, 15, and 30 days of each spray. Sampling in thecase of T3 was done at 7, 15, 30, 40, and 60 days after seedtreatment and 1, 5, 10, and 15 days after each spray. Seedsamples from all the treatments were collected at harvest. Bulkplant samples of 500 g obtained from each replicate were col-lected in polythene bags and immediately brought to the labo-ratory for processing. Plant (25 g) and grain (20 g) samplesfrom each replicate were taken from the bulk and macerated ina blender before processing for extraction. Samples from con-trol plots were processed in the same manner.

In order to estimate the amount of metalaxyl which could beremoved by simple washing, Apron-treated seed kept for 1–2weeks was washed under tap water for 5–6 min and dried inshade. Thereafter, the seed samples were analyzed for metal-axyl content.

Extraction and cleanup.Samples were collected at differ-ent intervals subject to metalaxyl residues, and extraction was

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done with acetone, according to the technique of Stoneet al.(1987) with some modifications. The entire extract was re-duced to 4–5 ml on a water bath after adding two to three dropsof liquid paraffin as an arrestant. The reduced amount wastransferred to a separating funnel and shaken with 15 ml hex-ane. The aqueous phase was drained out and the process wasrepeated twice. The hexane layers were combined and cleanedby following improved techniques of Kathpal and Dewan(1975). To hexane extract, acetone was added to make the ratioof hexane to acetone 4:1. Activated charcoal (0.5 g) was addedto the mixture and the contents were shaken for 30–60 sec. Thesupernate was filtered through a bed of anhydrous sodiumsulfate. The charcoal was washed twice with 15–20 ml hex-ane:acetone (4:1) mixture, each time stirring gently and allow-ing the carbon to settle. Combined filtrates were directly usedfor residue estimation by the GLC method.

A subsample (20 g) of seed from ground bulk samples col-lected from various treatments was kept overnight in acetoni-trile. The entire mixture was shaken for 1 hr for completeextraction. The content was filtered through Whatman filterpaper no. 1 and diluted four to five times with 4% aqueoussodium sulfate. The contents were transferred to a separatingfunnel and the residue was partitioned to hexane. The hexaneextract was cleaned by a method similar to that followed for themustard plant extract. The extract was reduced to 1 ml prior toinjection in the gas chromatograph.

Analysis. The plant and seed samples after cleanup wereanalyzed for metalaxyl residues by a gas–liquid chromatograph(HP 5890 A) equipped with a nitrogen phosphorus detector andmegabore column (10 m × 0.53 mm i.d. × 2.65mm film thick-ness) of methyl silicon gum (Ov-1) procurred from Hewlett–Packard (U.S.A.). The gas flows of nitrogen, hydrogen, and airwere maintained at 20, 3.5 ± 1, and 120 ml min−1, respectively.

The temperatures maintained were 200, 250, and 260°C for thecolumn, injection port, and detector, respectively. The chro-matogram obtained with standard metalaxyl is presented inFig. 1D with a retention time of 2.26 min.

Recovery experiment.To determine the efficiency and ac-curacy of the analytical method, a recovery experiment wascarried out with plant material fortified with technical metal-axyl. The samples were fortified at 1-, 2-, and 3-ppm levels andprocessed as above. The percentage recovery was calculated asusual and is presented in Table 1. The overall average recoverywas about 82%, and residues reported were calculated applyingcorrection factors. The detection limits of the metalaxyl werecalculated to be 0.2 ppm.

Calculations. The calculations of residue in test samples,residue half-life (RL-50), safe waiting period (SWP), and per-centage dissipation were made per the standard formulae ofDraper and Smith (1966) and Snedecor and Cochran (1968).The least significant difference and standard deviation (SD)were calculated using standard statistical methods. The meteo-rological observations are given in Fig. 2 as weekly averages,which include maximum and minimum atmospheric tempera-tures, morning and evening relative humidity, sunshine, andtotal rainfall. The observations were taken for the period (Oc-tober 1991–March 1992) from the meteorological research sta-tion of HAU, Hisar.

RESULTS

The magnitudes of residues of metalaxyl in different treat-ments are presented in Tables 2–7. The extent of persistenceand absorption of metalaxyl in plant samples when applied asa seed dresser (T1) are presented in Table 2. One-week-oldseedlings contained metalaxyl residues varying from 1.58 to

FIG. 1. Gas chromatogram. (A) Extract (1ml) of mustard plants containing Apron SD-35 at 2 g a.i. kg−1 seed after 40 days of sowing; (B) extract (1-ml)of mustard plants treated with Ridomil MZ-72 at 0.25%, 5 days after application; (C) extract (1ml) of mustard field treated with Ridomil MZ-72 at 0.25%, 1day after spraying; and (D) standard containing 3 mg metalaxyl.

MEHTA, SAHARAN, AND KATHPAL120

2.02 ppm with an average of 1.81 ppm. As a result of theincrease in absorption/translocation, residue increased to 3.46ppm in the seedlings collected 15 days after sowing. Thesamples obtained 30 days after sowing further indicated ahigher level (9.09 ppm) of metalaxyl. These observations in-dicated that the absorption of metalaxyl increased with thepassage of time. However, the samples collected 40 days aftersowing exhibited an average of 5.82 ppm metalaxyl (Fig. 1A),indicating thereby a reduction in metalaxyl after 30 days ofsowing. Metalaxyl residue was found to be below the detect-

able limit in leaves, inflorescence, and siliquae collected 60days after sowing.

Samples collected from T2, 1 day after the first spray doneon 40-day-old plants, contained metalaxyl residues varyingfrom 7.94 to 10.08 ppm (average, 9.03 ppm) (Fig. 1C), whereassamples collected 5 days thereafter indicated an average of0.68 ppm of metalaxyl indicating 92.56% dissipation (Fig. 1B;

FIG. 2. Weather conditions during the experiment (1991–1992).

TABLE 1Recovery of Metalaxyl by the GLC Method in Plant Samples

Amountadded(ppm)

Amountrecovered

(ppm)Recoverya

(%)

Averagerecovery

(%)

1 0.69 69.22 1.79 89.83 2.56 85.3 81.4

a Average of three replicates.

TABLE 2Persistence of Metalaxyl in Foliage after Seed Treatment (T1)

TreatmentDays ofsampling

Averageresiduelevel(ppm)

Rangea

(ppm)Dissipation

(%)SD(±)

Apron SD-35(at 2 g a.i./kg seed)

7 1.81 1.58–2.02 0.00 0.2315 3.46 3.03–3.80 +191.46 0.3730 9.08 8.63–9.84 +242.42 0.5440 5.82 5.57–6.01 35.90 0.1860 0.00 — 100.00 —90 0.00 — 100.00 —

a Average of three replicates.

ABSORPTION AND DEGRADATION OF METALAXYL 121

Table 3). Only 0.28 ppm of metalaxyl residue was recoveredfrom the samples collected 10 days after the spray with 96.89%dissipation. The residue dissipated more or less completely bythe 15th day after spraying because samples collected thereaf-ter did not reveal the presence of a detectable amount of meta-laxyl.

Dissipation of metalaxyl residues in mustard foliage after thesecond spray, given 30 days after the first, exhibited a trendalmost similar to that observed after the first spray. Leaf andinflorescence samples collected 1 day after the second spraycontained an average amount of 10.37 ppm metalaxyl, its levelreduced to 0.54 ppm on the fifth day, and by the tenth day, itwas only 0.29 ppm. The corresponding dissipation was about95 and 97% (Table 3).

In the treatment (T3) where seed treatment with Apron SD-35 was followed by two foliar sprays with Ridomil MZ-72 at60 and 80 days after sowing, the foliage samples were analyzedafter 7, 15, 30, 40, and 60 days of sowing. Analyzed data givenin Table 4A clearly indicate that the trend of uptake and dis-sipation of metalaxyl in mustard foliage was almost identical tothat observed in T1. The residue uptake increased up to 30days, after which it decreased significantly. Sixty-day-oldplants did not exhibit a detectable amount of metalaxyl resi-

dues. The samples collected 1 day after the first spray con-tained average residues of 8.21 ppm which declined to 0.49 and0.26 ppm after 5 and 10 days of spraying respectively (Fig.1A). Samples collected 1 day after the second spray, given 20days after the first spray, revealed initial deposits varying from8.64 to 10.22 ppm (average, 9.45 ppm). Residues further de-clined to 0.69, 0.27, and 0.0 ppm after 5, 10, and 15 days ofspraying, respectively (Table 4B).

The mustard seeds from all the treatments were free fromdetectable amounts of metalaxyl residues (Table 5). Data in-dicated that metalaxyl could not be carried to seed even inthose plants where it was sprayed 30–40 days before harvest.The RL-50 and SWP of metalaxyl in mustard plants are givenin Table 6. In treatment T1, the RL-50 was calculated to be17.57 days and the SWP to be about 62 days. In case T2 theRL-50 was observed to be 2 days and the SWP to be 8 days.

Keeping in view the MRL values of 0.5 mg kg−1 (WHO,1991), the mustard leaves should not be used as a vegetablebefore 60 days of growth after seed treatment, as is the practicein India. Similarly, a safe waiting period of 8–9 days is sug-

TABLE 3Persistence of Metalaxyl in Foliage after Foliar Application (T2)

Treatment

Average residue levela (ppm)

Day

1 5 10 15 30

Foliar spray Ib 9.03 0.68 0.28 0.0 0.0Range 7.94–10.08 0.49–0.80 0.25–0.32 — —Dissipation (%) 0.00 92.46 96.89 100.0 100.0

Foliar spray II 10.37 0.54 0.29 0.0 0.0Range 9.57–11.10 0.51–0.57 0.29–0.30 — —Dissipation (%) 0.00 94.79 97.20 100.0 100.0

a Average of three replicates.b Foliar sprays: 40, 70 DAS at 0.25%.

TABLE 4APersistence of Metalaxyl in Foliage after Seed Treatment and

Foliar Sprays (T3)

TreatmentDays tosampling

Averageresiduelevel(ppm)

Rangea

(ppm)Dissipation

(%)SD(±)

Apron SD-35(at 2 g a.i./kg seed)

7 1.81 1.58–2.02 0.00 0.23015 3.46 3.03–3.80 +191.46 0.37030 9.08 8.63–9.84 +242.42 0.54040 5.82 5.57–6.01 35.90 0.18060 0.00 — 100.00 —

a Average of three replicates.

TABLE 4BPersistence of Metalaxyl in Foliage after Seed Treatment and

Foliar Sprays (T3)

Treatments

Average residue levelsa (ppm)

Day

1 5 10 15 30

Foliar spray Ib 8.21 0.49 0.26 0.0 0.0Range 7.89–8.76 0.47–0.52 0.23–0.29 — —Dissipation (%) 0.00 94.03 96.83 100.0 100.0

Foliar spray II 9.45 0.69 0.27 0.0 0.0Range 8.64–10.22 0.59–0.80 0.25–0.30 — —Dissipation (%) 0.00 92.69 97.14 100.0 100.0

SD ±, Spray I 0.382 0.28 0.034 — —SD ±, Spray II 0.673 0.121 0.028

a Average of three replicates.b Foliar sprays: 60 and 80 DAS at 0.25%.

TABLE 5Translocation of Metalaxyl Residues into Mustard Seed

Following Different Treatments at Harvest

Treatments

Averageresidue(ppm)

Seed treatmenta (Apron SD-35) (T1) NDb

Foliar sprayc (Ridomil MZ-72) (T2); 40, 70 DASd NDSeed treatment + foliar spray (T3); 60, 80 DAS ND

a At 2 g a.i. kg−1 seed.b ND, not detectable.c At 0.25%.d DAS, days after sowing.

MEHTA, SAHARAN, AND KATHPAL122

gested in the case of treatments where only spraying applica-tion are given. Washing of Apron-treated seed under laboratoryconditions reduces surface residues by about 60%. The initialdeposits of 22.47 ppm were reduced to 8.98 ppm after 5–6 minwashing (Table 7), thereby indicating that a major part of themetalaxyl was lost by washing, dilution, etc.

DISCUSSION

Analysis of mustard plants periodically collected from T1

revealed that metalaxyl absorption increased in foliage up to 30days and started reducing thereafter (Table 2). The averageamount of metalaxyl was roughly two to five times higher inthe samples collected between 15 and 30 days of sowing com-pared to those collected after 7 days of sowing. It has beenreported that absorption mobility, and translocation of anycompound into a plant system depend upon lipophilicity,which is measured as the octanol water coefficient. The octanolwater coefficient of metalaxyl, i.e., 1.56, being more than 1suggests that the fungicide is readily absorbed and translocatedin plants (Singh and Tripathi, 1982). The gradual increase inmetalaxyl residues in mustard plants observed in T1 must bedue to its absorption from the seed or soil surface. Maximumrecovery of metalaxyl in the foliage raised from treated seedwas 9.08 ppm after 30 days of sowing, indicating that a majorportion was lost either through diffusion from seed into soilduring germination or remained in the seed parts and wasabsorbed slowly (Singhet al., 1986). The current studies(Table 7) also indicated that about 60% of the fungicide fromthe treated seed is removed by washing, which remains in thesoil, giving longer protection to the seed in addition to trans-location and absorption of fungicide slowly in the growingseedlings. The absorption and translocation of metalaxyl havebeen observed by various workers in different crops. The pes-ticide dissipated more or less completely in 14 days in cowpea,30–45 days in pearl millet, and 45 days in maize (Tripathi andSingh, 1983; Reddyet al., 1990), thus, indicating that metal-axyl persisted in the plant parts for a longer period when ap-plied as a seed dresser.

The persistence of metalaxyl in plant parts ofB. junceawiththe foliar application of Ridomil MZ-72 (T2) indicated thatmaximum residue was recovered after 1 day of spraying. It was

significantly reduced with the passage of time and was notdetectable after 15 days of spraying. The presence of a higherconcentration of metalaxyl 1 day after spraying may be dueeither to a direct relationship between fungicide accumulationand transpiration of water loss or to its lipophilic activity asdemonstrated by various workers (Peterson and Edgington,1971; Singh and Tripathi, 1982). A higher amount of metalaxyl(6.3–9.5 ppm) has also been recorded in foliage when appliedas a foliar spray (Speck and Dirr, 1980; Businelliet al.,1984;Reddyet al., 1990). The rapid degradation/dissipation of fun-gicide in subsequent sampling after spraying may be due todilution of the toxicant due to plant growth coupled with fa-vorable climatic conditions, i.e., clear sunshine, temperature,and relative humidity prevailing during the postapplication pe-riod (Fig. 2).

The analysis of samples after seed treatment followed byfoliar application (T3) revealed a trend of residues similar tothat recorded in earlier samples. The factor responsible for thistype of trend has been discussed previously.

Seeds collected from plants at harvest from all three treat-ments were free from residues, thereby indicating that metal-axyl could not be carried over to the seed when applied onplant parts, probably due to its easily degradable naturecoupled with dilution due to plant growth. A similar trend hasbeen reported by Reddyet al. (1990) in pearl millet.

The critical observations of these results revealed that seed-lings, leaves, inflorescence, and siliquae readily absorbedmetalaxyl after its application and subsequently degraded. Itsresidue in plant parts remained at a level much higher than theprescribed maximum residue limit of 0.5 ppm (WHO, 1991) upto 40 days of sowing, thereby indicating that the foliage isunsafe for human consumption. However, in the subsequentsampling it reached within the safe limit. The fungicide de-graded quickly when it was applied as a foliar spray and plantswere in the safe limit after 10 days of spraying. As there wasno detectable residue in mustard seed at harvest, both the for-mulations of metalaxyl at the tested doses can be consideredsafe from the point of view of health hazards. Hence, the samecan be recommended for large-scale adoption by farmers.

TABLE 7Metalaxyl Content in the Seed after Treatment with

Apron SD-35

Treatment

Averagea

residue(ppm)

Range(ppm)

SD(±)

Seed treatment(without washing) 22.47 21.16–23.78 1.85

Seed treatment(with washing)b 8.98 8.27–9.69 1.00

a Average of three replicates.b Washed under tap water after 1–2 weeks of treatment and dried in the

shade.

TABLE 6Safe Period and Residue Half-Life Values of Metalaxyl

in Mustard

TreatmentRL-50a

(days)SWPb

(days)

Seed treatment 17.57 62.33Foliar spray I 2.08 8.69Foliar spray II 1.79 7.85

Note.MRL, 0.5 mg/kg (WHO, 1991).a RL-50, residue half-life.b SWP, safe waiting period.

ABSORPTION AND DEGRADATION OF METALAXYL 123

CONCLUSIONS

(a) Metalaxyl absorption in mustard plants increased up to30 days of its application as a seed dresser. Thereafter, itstarted declining and was nondetectable after 60 days of itsapplication.

(b) The dissipation of metalaxyl after foliar application wasalmost complete after 15 days of application.

(c) The safe waiting period of metalaxyl was calculated tobe 62 and 8 days for seed dresser and foliar application, re-spectively.

(d) Metalaxyl (Apron SD-35) application at the rate of 2 ga.i. kg−1 seed as a seed dresser and Ridomil MZ-72 at the rateof 0.25% as a foliar application may be considered safe fromthe point of view of toxic residues under the tropical agrocli-matic conditions of Haryana.

ACKNOWLEDGMENTS

The senior author is highly thankful to Dr. (Mrs.) Beena Kumari (Asst.Chemist) and the Department of Chemistry and Biochemistry for providing thenecessary facilities for conducting the research. The technical sample providedby M/S Hindustan Ciba Geigy Limited, Bombay, and the financial help ren-dered by CSIR, New Delhi, are also fully acknowledged.

REFERENCES

Businelli, M., Marucchini, C., Patumi, M., Zazzerini, A., and Toree, G. C.(1984). Efficacy of metalaxyl for blue mould control and its persistence ontobacco.Pestic. Sci.15, 11–16.

Draper, N. R., and Smith, H. (1966).Applied Regression Analysis,pp. 7–13.Wiley, New York.

Kathpal, T. S., and Dewan, R. S. (1975). Improved clean-up technique for theestimation of endosulfan and aldrin residues.J. AOAC58, 1076–1080.

Lyr, H. (1987). Modern Selective Fungicides—Properties, Application,Mechanism of Action.Longman, Harlow/New York.

Peterson, C. A., and Edgington, L. V. (1971). Transport of benomyl in tovarious plant organs.Phytopathology61, 91–92.

Reddy, M. V. B., Shetty, H. S., and Reddy, M. S. (1990). Mobility, distributionand persistence of metalaxyl residues in pearl millet (Pennisetum america-num (L.) Leeke).Bull. Environ. Contam. Toxicol.45, 250–257.

Sharom, M. S., and Edgington, K. V. (1982). The absorption, mobility andpersistence of metalaxyl in soil and aqueous systems.Can. J. Plant Pathol.4, 334–340.

Singh, U. S., and Tripathi, R. K. (1982). Physiochemical and biological prop-erties of metalaxyl. InFirst National Symposium on Systemic Fungicides,June 17–18. Jabalpur, India.

Singh, U. S., Tripathi, R. K., Kumar, J., and Dwivedi, T. S. (1986). Uptake,translocation, distribution and persistence of metalaxyl in pearl millet (Pen-nisetum americanumL. Leeke).J. Phytopathol.117,112–135.

Snedecor, G. W., and Cochran, W. G. (1968).Statistical Methods,pp. 139–141. Oxford and IBH, New Delhi.

Speck, M., and Dirr, E. (1980). Gas chromatographic determination of meta-laxyl (Ridomil) residues in tobacco.J. Chromatogr.200,313–316.

Stone, J. R., Verma, P. R., Dueck, J., and Westcotto, N. D. (1987). Bioactivityof the fungicide metalaxyl in rape plants after seed treatment and soil drenchapplication.Can. J. Plant Pathol.9, 260–264.

Tripathi, R. K., and Singh, U. S. (1983). Metalaxyl: Physiochemical and bio-logical properties. InRecent Advances in Plant Pathology(H. Akhtar, K.Singh, B. P. Singh, and V. P. Agnihotri, Eds.), pp. 201–226. Print House,Lucknow.

Urech, P. A., Schwinn, F., and Staub, T. (1977). CGA 48988, a novel fungicidefor the control of late blight, downy mildew and related soil borne diseases.Proc. Br. Crop. Protect. Conf. Pests Dis.9, 623–631.

World Health Organization (WHO) (1991).Guide to Codex Maximum Limitsfor Pesticide Residues,Part 2. Codex Alimentarious Commission, FAO.

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