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Influence of biotic and abiotic factors on dissipating metalaxyl in soil
Chemosphere 45 (2001) 941±947
www.elsevier.com/locate/chemosphere
In¯uence of biotic and abiotic factors on dissipating metalaxyl in soil Premasis Sukul 1, Michael Spiteller
*
Institute of Environmental Research, University of Dortmund, D-44221 Dortmund, Germany Received 13 September 2000; accepted 4 December 2000
Abstract Under laboratory condition, dissipation of metalaxyl in sterile and non-sterile soils, its sorption behaviour and fate in presence of light have been studied. The half-life value of metalaxyl was found in the range of 36±73 d in non-sterile soil. 5.3±14.7% dissipation was observed due to abiotic factors other than light. Metalaxyl was found photostable in soil showing half-life of 188±502 h under simulated sunlight. In adsorption study, a non-linear relationship between concentration of metalaxyl and its adsorption into soils was observed. Estimated kOC value increased as organic carbon content decreased. Adsorption and desorption kD values ranged between 53.5 and 151.1. Ó 2001 Elsevier Science Ltd. All rights reserved. Keywords: Biodegradation; Metalaxyl; Photodegradation; Soil; Sorption
1. Introduction Metalaxyl [N-(2,6-dimethylphenyl)-N-(methoxyacetyl) alanine methyl ester] is a systemic acylanilide fungicide. It oers a great promise for control of downy mildews and diseases caused by Pythium spp. and Phytophthora spp. (Urech et al., 1977). Metalaxyl formulations include granulars, wettable powders, dusts, emulsi®able concentrates, ¯owable concentrates, crystalline and liquid ready-to-use products. Application may be foliar, or soil incorporation, surface spraying (broadcast or band), drenching, sprinkler or drip irrigation, soil mix or seed treatment. Metalaxyl registered products either contain metalaxyl as the active ingredient or may be combined with a variety of other active
*
Corresponding author. Tel.: +49-231-7554081. E-mail address: [email protected] (M. Spiteller). 1 Present address: Department of Agricultural Biochemistry, F/Ag., Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, Nadia 741252, India.
ingredients like captan, carboxin, pentachloronitrobenzene, mancozeb, or thiabendazole (Sukul and Spiteller, 2000). Upon application in soil it is exposed to soil microorganisms, light and other physical chemical factors. The degradation of metalaxyl in soil had been reported mainly as biodegradation (Bailey and Coey, 1985; Droby and Coey, 1991; Huaguo et al., 1995). The compound had been found to persist for nearly 4 weeks after application to pearl millet and lettuce (Cho, 1981; Singh et al., 1986). Earlier studies (Burkhard, 1979; Yao et al., 1989; Sukul et al., 1992; Moza et al., 1994) demonstrated that metalaxyl undergoes photodegradation at varying rates in aqueous solution. Microbial transformation of metalaxyl in pure cultures was investigated (Zheng et al., 1989). Persistence behaviour of metalaxyl residues in dierent crops like tomatoes, rape seedlings, potato, etc., under dierent conditions were studied (Cabras et al., 1985; Stone et al., 1987; Dunsing et al., 1988; Milgroom et al., 1988). In this research, behaviour of metalaxyl was investigated in soils varying in their physical and chemical properties. Role of dierent biotic and abiotic factors in attenuating metalaxyl residues in soil was studied.
0045-6535/01/$ - see front matter Ó 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 5 - 6 5 3 5 ( 0 1 ) 0 0 0 1 0 - 8
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P. Sukul, M. Spiteller / Chemosphere 45 (2001) 941±947
Conventionally, the study on the role of microbes and of sunlight is the regular feature in determining the fate and behaviour of the pesticides. The present study would also take a look at the overall contribution of abiotic factors other than photodegradation towards dissipation of metalaxyl in soil. 2. Materials and methods 2.1. Soils Four soils representing dierent physical and chemical properties and geographical origin were included in the present study. Soil samples collected to a depth of 0±15 cm were kept in a greenhouse at 18 4°C in summer and winter at 60±80% of maximum water holding capacity. Shortly before the study was started, soils were sieved through a 2 mm sieve. Soil properties are given in Table 1. 2.2. Chemicals Metalaxyl, analytical standard grade (99%), was supplied by Riedel-de Haen, Germany. All the solvents used were of A.R. grade. 2.3. Metalaxyl application and incubation in soil To study degradation kinetics of metalaxyl in soil under sterilised and non-sterilised conditions, laboratory experiments were conducted with the recommended dose, three applications of 200 g a.i./ha. The selected rate of application of metalaxyl for this study was based on the maximum single use rate (600 g a.i./ha). Based on the conversion using the parameters soil depth (5 cm) and soil density (1.5 g/cm3 ), this corresponds to a weighed amount of 40 lg metalaxyl/50 g soil (dry weight). This calculated amount of metalaxyl was ap-
plied with 400 ll of water (@ 500 l/ha) to 50 g soil in individual Erlenmeyer ¯asks (150 ml) plugged with cotton pad. Prior to application of metalaxyl, bulk soil samples were mixed for 1 h using a tumbling mixer (30 2 rpm). The moisture content was adjusted to 60% of maximum water holding capacity and checked gravimetrically every 2 weeks. One batch of soil sample was sterilised by autoclaving at 15 psi and 121°C for 20 min on three consecutive days. The experimental set-up for sterilised soils was performed aseptically. No eect of autoclaving on metalaxyl stability has been reported (Singh and Tripathi, 1982). However, metalaxyl stability in soil system under autoclaving had also been checked in the present study before incubating soil samples. To discard eect of light sterilised and non-sterilised soil samples were kept under dark. For photodegradation study 8 lg metalaxyl applied on soil surface (10 g dry weight basis) was kept in 100 ml reaction vessel made of quartz glass. The calculated amount of metalaxyl was applied with 80 ll of water (soil surface area 15.90 cm2 , soil layer 0.5 cm). To avoid microbial eect soils were sterilised with 1% HgCl2 . The reaction vessel was covered with a quartz plate and exposed to light of xenon lamp at 550 W=mm2 (Suntest, Heraeus equipped with coated quartz ®lters with a cuto at <285 nm) with 12 h of light/12 h of dark condition per day. The temperature of the soil was kept at 25 1°C through circulation of cold water. All experiments were performed in duplicate. Soil samples were taken for analysis at appropriate time intervals. Sterile soil samples treated with the pesticide at the same application rate as irradiated soil samples and maintained in darkness were used as experimental control. For adsorption±desorption studies replicates (3 10 g of air-dried soil) were treated with 0.01 M CaCl2 (20 ml) solution containing dierent concentrations (0.5, 1, 5, 10, 25 and 50 lg/ml) of metalaxyl in stoppered te¯on centrifuge tubes. These samples were turned end-overend at 29 rpm for 48 h on an overhead shaker.
Table 1 Physical±chemical characteristics of the test soils Soil
H ofchen
Passo Fundo
Laacher Hof
Verro Beach
Texture (%)
Clay Silt Sand
10.2 81.3 8.5
44.4 24.4 31.2
12.0 51.1 36.9
1.1 2.0 96.9
pH
H2 O CaCl2
7.8 7.2
5.8 5.1
8.1 7.3
6.3 5.9
Org.C (%)
2.6
1.6
0.9
0.2
Total N (%)
0.2
0.2
0.1
±
C.E.C (moles/kg)
15.0
13.1
±
±
Origin
Burscheid, Germany
Passo Fundo, Brazil
Monheim, Germany
Florida, USA
P. Sukul, M. Spiteller / Chemosphere 45 (2001) 941±947
943
2.4. Extraction
3. Results and discussion
Metalaxyl treated soil samples were extracted with methanol (3 50 ml) on an electric shaker (1 h), each followed by ultrasonic vibration (Branson Soni®er, Branson) for 5 min. The extracts were centrifuged (Beckman J2-HS, Beckman, USA) for 20 min (10,000 rpm) and the combined extracts were concentrated to dryness by a rotary evaporator. Finally, the residue was dissolved in 5 ml methanol and analysed by HPLC. In case of photodegradation study, soil samples collected after periodical intervals were transferred to centrifuge tubes and extracted with 3 20 ml methanol followed by ultrasonic vibration for 5 min each time. For sorption study after 48 h of shaking the contents were centrifuged at 3000 rpm for 10 min. The supernatants were decanted, volumes were measured and directly analysed by HPLC. Desorption study was carried out by adding extra amount of freshly prepared 0.01 M CaCl2 solution to the soil residue in the centrifuge tubes so that total amount of 0.01 M CaCl2 solution remained exactly 20 ml. The centrifuge tubes were again kept under overhead shaker for 48 h, decanted and desorbed solutions were analysed by HPLC. Blanks (without soil) were run through the same procedure. The concentration of pesticide adsorbed to the soil was calculated as
Dissipation of metalaxyl has been studied in sterilised and non-sterilised soils under laboratory condition and results are presented in Figs. 1 and 2. In non-sterilised soils metalaxyl dissipation was found to be higher than that in sterilised soils. Obviously, this suggests the possible microbial role in degrading metalaxyl. However, dissipation was also observed in sterilised soils, which might be due to the result of chemical and other abiotic factors other than photodegradation as the soils were kept under dark during the entire experimental period. Comparative role of microbes and abiotic factors other than light in dissipating metalaxyl in soil is presented in Table 2. After
CB
CS VS =M;
where CB and CS are the blank and supernatant solution concentration, VS is the solution volume, and M is the soil mass. Adsorption isotherm were ®tted to the Freundlich model in log form log
Fig. 1. Dissipation of metalaxyl in sterilised soils.
x 1 log kD log Ceq ; m n
where x/m is the concentration of pesticide adsorbed to the soil (lg/g), Ceq is the equilibrium concentration of pesticide in solution (lg/ml), log kD (intercept on Y-axis) and 1/n (slope) are the adsorption parameters calculated from the linear regression. 2.5. HPLC Metalaxyl active ingredient was estimated by HPLC using a Shimadzu LC 10 AT, equipped with a Macherey±Nagel (250/8/4) C18 column and a UV detector operated at 205 nm. For the ®rst 15 min the mobile phase (1 ml/min) was composed of 10% acetonitrile and 90% water adjusted to pH 4.0 with 0.15 M H3 PO4 . For the next 5 min the acetonitrile content was increased to 90%.
Fig. 2. Dissipation of metalaxyl in non-sterilised soils.
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P. Sukul, M. Spiteller / Chemosphere 45 (2001) 941±947
Table 2 Comparative role of microbes and abiotic factors on dissipating metalaxyl in soil % degradation of metalaxyl after 60 days of application
Soil
H ofchen Passo Fundo Laacher Hof Vero Beach
Non-sterilised (+ microbes + abiotic factors other than light)
Sterilised () microbes + abiotic factors other than light)
Degradation due to microbes
51.5 69.2 61.4 41.1
14.7 11.9 7.4 5.3
36.8 57.3 54.0 35.8
60 days of application 5.3±14.7% attenuation of metalaxyl was observed due to abiotic factors other than light, while 35.8±57.3% dissipation was due to the in¯uence of microbial activity. When log residues were plotted against incubation period, a straight line was found suggesting a ®rst-order reaction in the dissipation behaviour of metalaxyl in both sterilised and non-sterilised soils. The half-life values were in the range of 36±73 and 232±602 d in nonsterilised and sterilised soils, respectively (Table 3). This conforms the earlier reports (EPA, 1994; Kookana et al., 1995). Persistence of metalaxyl in non-sterilised soils was found in the order of Passo Fundo < Laacher Hof < H ofchen < Vero Beach and H ofchen < Passo Fundo < Laacher Hof < Vero Beach in sterilised soils. Dierence in persistence of metalaxyl in dierent soils under different conditions might be due to dierent physicochemical properties and biological activities of the test soils. In Vero Beach soil under both sterilised and nonsterilised conditions attenuation of metalaxyl was least. This is due to its sandy nature and presence of low amount of clay and organic carbon. Adsorption which might be the prime factor of metalaxyl dissipation under sterilised condition is expected to be negligible in this soil. In non-sterilised Vero Beach soil low dissipation as compared to other soils may be explained due to its low organic carbon content and in turn less biological activity. The present investigation is well in accordance with the earlier ®ndings (Droby and Coey, 1991; Huaguo et al., 1995) stating the importance of microbial activity in degrading metalaxyl. Photodegradation of metalaxyl on the soil surface was studied using arti®cial sunlight under laboratory condition (Fig. 3). When log residues were plotted
Fig. 3. Photodegradation of metalaxyl in four dierent soils.
against time, a linear relationship was found stating a ®rst-order kinetic reaction (Table 4). The half-life values were found in the range 188±502 h of irradiation in four soils of dierent physicochemical properties. Soil clay content showed a linear relationship (r2 0,92) with photopersistence of metalaxyl in soils (Fig. 4). It may be due to the fact that metalaxyl enters into the intra-lattice structure of clay components and remains unexposed to light. Metalaxyl also appeared to be preferentially adsorbed on soil mineral surface (Sukop and Cogger, 1992). Metalaxyl on oven-dried soil photodecomposes slowly (Murthy et al., 1998). Less than
Table 3 Regression equation, correlation coecient and half-life values of metalaxyl under two conditions Soil H ofchen Passo Fundo Laacher Hof Vero Beach
Non-sterilised soil
Sterilised soil
Regression equation
r2
t1=2 (d)
Regression equation
r2
t1=2 (d)
Y Y Y Y
0.9693 0.9400 0.9435 0.9549
59.0 36.3 39.1 73.4
Y Y Y Y
0.9813 0.9999 0.9607 0.9302
231.6 301.0 501.7 602.1
1:57 1:56 1:63 1:60
0:0051X 0:0083X 0:0077X 0:0041X
1:61 1:61 1:60 1:61
0:0013X 0:001X 0:0006X 0:0005X
P. Sukul, M. Spiteller / Chemosphere 45 (2001) 941±947
2% conversion of metalaxyl on oven-dried soil was found when it was subjected to arti®cial sunlight for 72 h. In another report also metalaxyl was found to be more persistent in dry soil than wet soil in presence of Table 4 Regression equation, correlation coecient and half-life values of metalaxyl under Suntest Soil
Regression equation
r2
t1=2 (h)
H ofchen Laacher Hof Passo Fundo Vero Beach
Y Y Y Y
0.9207 0.9154 0.9395 0.9387
334.5 273.7 501.7 188.1
1:61 1:61 1:60 1:58
0:0009X 0:0011X 0:0006X 0:0016X
Fig. 4. Correlation between soil clay content and persistence of metalaxyl in soil under simulated sunlight.
Fig. 5. Equilibriation time for metalaxyl adsorption in dierent soils.
945
natural sunlight (Saha and Sukul, 1997). Comparatively more photodecomposition was found in the present investigation due to the presence of moisture. In our experiment, soils were kept at 60% of their maximum water holding capacity. The present observation suggests photostability of metalaxyl, in general, which is consistent with other ®ndings (Singh and Tripathi, 1982; Sukul et al., 1992; Saha and Sukul, 1997). Metalaxyl is resistant to sunlight due to its kmax at 196 nm in aqueous solution and no absorption above 290 nm. Therefore, photolysis can only occur by indirect photolysis via photosensitisers which in presence of water and after a sequence of reactions lead to the formation of OH radicals. The latter can attack the substrate and lead to
Fig. 6. Adsorption isotherm of metalaxyl in three dierent soils.
Fig. 7. Adsorption isotherm of metalaxyl following a desorption step in three dierent soils.
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P. Sukul, M. Spiteller / Chemosphere 45 (2001) 941±947
Table 5 Constants and correlation coecients of the Freundlich equations for adsorption±desorption of metalaxyl in soils Soil/Process
Regression equation
r2
kD
H ofchen Adsorption Desorption
Y 0:73X 0:34 Y 1:45X 0:33
0.9883 0.9861
1.4 1.4
53.8 53.5
Laacher Hof Adsorption Desorption
Y 0:81X Y 0:64X
0:31 0:16
0.9818 0.9936
1.4 1.2
151.1 130.0
Passo Fundo Adsorption Desorption
Y 0:54X 0:16 Y 1:94X 0:13
0.9873 0.9915
1.2 1.1
73.1 71.2
hydroxylation of the molecule and subsequently to further degradation. For adsorption±desorption studies the incubation time for metalaxyl was set to 48 h to establish an equilibrium (Fig. 5). When the equilibrium concentration (C) was plotted against the amount of metalaxyl adsorbed (x) in microgram per unit amount of soil (m) in grams, both in logarithmic form, an excellent ®t of data by the Freundlich equation log x=m log kD 1=n log C was achieved with a coecient of determination 0.98±0.99 for both of adsorption and desorption process (Figs. 6 and 7). The kD for adsorption and desorption processes indicated that the adsorption of metalaxyl was in the order of H ofchen > Laacher Hof > Passo Fundo (Table 5). In adsorption study the values of 1/n were less than unity for all three soils indicating a non-linear relationship between concentration of metalaxyl and its adsorption into these soils. The variable slopes of the sorption isotherms obtained for dierent soil systems studied reveal that metalaxyl sorption on soil is a complex phenomenon involving dierent types of adsorption sites with dierent surface energies. kOC kOC kD 100=% organic carbon) were calculated for each soil, using kD and organic carbon data. The result shows that the kOC values estimated from the individual soil material tend to increase as organic carbon content decreases. The desorption kD values were nearly same to those for adsorption. This indicates involvement of same spectrum of forces in adsorption of metalaxyl during adsorption and desorption steps.
Acknowledgements This study was funded as part of a research grant to the ®rst author from the Alexander von Humboldt Foundation, Germany. The authors deeply appreciate the help rendered by Mr. J orn Sickerling and Mr. J urgen Scheen, University of Dortmund during preparation of the manuscript.
kOC
References Bailey, A.M., Coey, M.D., 1985. Biodegradation of metalaxyl in avocado soils. Phytopathology 74, 135±137. Burkhard, N., 1979. Photolysis of CGA-48988 (Ridomil) in aqueous solution under arti®cial sunlight conditions. Project Report, Agrochemicals Division, Ciba-Geigy, Basel, Switzerland. Cabras, P., Meloni, M., Pivisi, M.F., Cabitza, F., 1985. Behaviour of acylanilide and dicarboxymidic fungicide residues on green house tomatoes. J. Agric. Food Chem. 33, 86±89. Cho, J.J., 1981. Phytophthora rot of banksia, its host range and chemical control. Plant Dis. 65, 83±88. Droby, S., Coey, M.D., 1991. Biodegradation process and the nature of metabolism of metalaxyl in soil. Ann. Appl. Biol. 118, 543±553. Dunsing, M., Grote, D., Buesi, L., 1988. Research into the persistence and residue dynamics of metalaxyl in tomatoes in hydroponic culture with recirculating nutrient solution (NFT). Nachrichtenbl. Dtsch. P¯anzenschutzdienst Berlin 42, 173±176. EPA, 1994. Reregistration eligibility decision (RED): Metalaxyl. EPA 738-R-94-017, US Environmental Protection Agency, Washington, DC, pp. 1±49. Huaguo, W., Peng, G., Qi, M., 1995. Study on the residues and degradation of 14 C-metalaxyl in soil. Acta Agric. Univ. Pekin. 21, 395±401. Kookana, R.S., Di, H.J., Aylmore, L.A.G, 1995. A ®eld study of leaching and degradation of nine pesticides in a sandy soil. Aust. J. Soil Res. 33, 1019±1030. Milgroom, M.G., McCulloch, C.E., Fry, W.E., 1988. Distribution and temporal dynamics of metalaxyl in potato foliage. Phytopathology 78, 555±559. Moza, P.N., Sukul, P., Hustert, K., Kettrup, A., 1994. Photooxidation of metalaxyl in aqueous solution in the presence of hydrogen peroxide and titanium dioxide. Chemosphere 28, 341±347. Murthy, N.B.K, Hustert, K., Moza, P.N., Kettrup, A., 1998. Photodegradation of selected fungicides on soil. Fresenius Environ. Bull. 7, 112±117. Saha, T., Sukul, P., 1997. Metalaxyl ± its persistence and metabolism in soil. Toxicol. Environ. Chem. 58, 251±258. Singh, U.S., Tripathi, R.K., 1982. Physicochemical and biological properties of metalaxyl; octanol number, absorption
P. Sukul, M. Spiteller / Chemosphere 45 (2001) 941±947 spectrum and eect of dierent physicochemical factors on stability of metalaxyl. Indian J. Mycol. Plant Pathol. 12, 287±294. Singh, U.S., Tripathi, R.K., Kumar, J., Dwivedi, T.S., 1986. Uptake, translocation, distribution and persistence of metalaxyl in pearl millet (Pennisetum americanum (L) Lecke.). J. Phytopathol. 117, 112±115. Stone, J.R., Verma, P.R., Dueck, J., Westcott, N.D., 1987. Bioactivity of the fungicide metalaxyl in rape plants after seed treatment and soil drench applications. Can. J. Plant Pathol. 9, 260±264. Sukop, M., Cogger, C.G., 1992. Adsorption of carbofuran, metalaxyl and simazine: kOC evaluation and relation to soil transport. J. Environ. Sci. Health B 27, 565±590. Sukul, P., Moza, P.N., Hustert, K., Kettrup, A., 1992. Photochemistry of metalaxyl. J. Agric. Food Chem. 40, 2488±2492.
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Sukul, P., Spiteller, M., 2000. Metalaxyl: persistence, degradation, metabolism and analytical methods. Rev. Environ. Contam. Toxicol. 164, 1±26. Urech, P.A., Schwinn, F., Staub, T., 1977. CGA 48988, a novel fungicide for the control of late blight, downy mildew, and related soil borne diseases. In: Proceedings of the British Crop Protection Conference, Brighton, 21±24 November, 1977, Boots, Nottingham, vol. 2, pp. 623. Yao, J.R., Liu, S.Y., Freyer, A.J., Minard, R.D., Bollag, J.M., 1989. Photodecomposition of metalaxyl in an aqueous solution. J. Agric. Food Chem. 37, 1518±1523. Zheng, Z., Shu-Yen, L., Freyer, A.J., Bollag, J.M., 1989. Transformation of metalaxyl by the fungus Sycephalastrum racemosum. Appl. Environ. Microbiol. 55, 66±71.
www.elsevier.com/locate/chemosphere
In¯uence of biotic and abiotic factors on dissipating metalaxyl in soil Premasis Sukul 1, Michael Spiteller
*
Institute of Environmental Research, University of Dortmund, D-44221 Dortmund, Germany Received 13 September 2000; accepted 4 December 2000
Abstract Under laboratory condition, dissipation of metalaxyl in sterile and non-sterile soils, its sorption behaviour and fate in presence of light have been studied. The half-life value of metalaxyl was found in the range of 36±73 d in non-sterile soil. 5.3±14.7% dissipation was observed due to abiotic factors other than light. Metalaxyl was found photostable in soil showing half-life of 188±502 h under simulated sunlight. In adsorption study, a non-linear relationship between concentration of metalaxyl and its adsorption into soils was observed. Estimated kOC value increased as organic carbon content decreased. Adsorption and desorption kD values ranged between 53.5 and 151.1. Ó 2001 Elsevier Science Ltd. All rights reserved. Keywords: Biodegradation; Metalaxyl; Photodegradation; Soil; Sorption
1. Introduction Metalaxyl [N-(2,6-dimethylphenyl)-N-(methoxyacetyl) alanine methyl ester] is a systemic acylanilide fungicide. It oers a great promise for control of downy mildews and diseases caused by Pythium spp. and Phytophthora spp. (Urech et al., 1977). Metalaxyl formulations include granulars, wettable powders, dusts, emulsi®able concentrates, ¯owable concentrates, crystalline and liquid ready-to-use products. Application may be foliar, or soil incorporation, surface spraying (broadcast or band), drenching, sprinkler or drip irrigation, soil mix or seed treatment. Metalaxyl registered products either contain metalaxyl as the active ingredient or may be combined with a variety of other active
*
Corresponding author. Tel.: +49-231-7554081. E-mail address: [email protected] (M. Spiteller). 1 Present address: Department of Agricultural Biochemistry, F/Ag., Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, Nadia 741252, India.
ingredients like captan, carboxin, pentachloronitrobenzene, mancozeb, or thiabendazole (Sukul and Spiteller, 2000). Upon application in soil it is exposed to soil microorganisms, light and other physical chemical factors. The degradation of metalaxyl in soil had been reported mainly as biodegradation (Bailey and Coey, 1985; Droby and Coey, 1991; Huaguo et al., 1995). The compound had been found to persist for nearly 4 weeks after application to pearl millet and lettuce (Cho, 1981; Singh et al., 1986). Earlier studies (Burkhard, 1979; Yao et al., 1989; Sukul et al., 1992; Moza et al., 1994) demonstrated that metalaxyl undergoes photodegradation at varying rates in aqueous solution. Microbial transformation of metalaxyl in pure cultures was investigated (Zheng et al., 1989). Persistence behaviour of metalaxyl residues in dierent crops like tomatoes, rape seedlings, potato, etc., under dierent conditions were studied (Cabras et al., 1985; Stone et al., 1987; Dunsing et al., 1988; Milgroom et al., 1988). In this research, behaviour of metalaxyl was investigated in soils varying in their physical and chemical properties. Role of dierent biotic and abiotic factors in attenuating metalaxyl residues in soil was studied.
0045-6535/01/$ - see front matter Ó 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 5 - 6 5 3 5 ( 0 1 ) 0 0 0 1 0 - 8
942
P. Sukul, M. Spiteller / Chemosphere 45 (2001) 941±947
Conventionally, the study on the role of microbes and of sunlight is the regular feature in determining the fate and behaviour of the pesticides. The present study would also take a look at the overall contribution of abiotic factors other than photodegradation towards dissipation of metalaxyl in soil. 2. Materials and methods 2.1. Soils Four soils representing dierent physical and chemical properties and geographical origin were included in the present study. Soil samples collected to a depth of 0±15 cm were kept in a greenhouse at 18 4°C in summer and winter at 60±80% of maximum water holding capacity. Shortly before the study was started, soils were sieved through a 2 mm sieve. Soil properties are given in Table 1. 2.2. Chemicals Metalaxyl, analytical standard grade (99%), was supplied by Riedel-de Haen, Germany. All the solvents used were of A.R. grade. 2.3. Metalaxyl application and incubation in soil To study degradation kinetics of metalaxyl in soil under sterilised and non-sterilised conditions, laboratory experiments were conducted with the recommended dose, three applications of 200 g a.i./ha. The selected rate of application of metalaxyl for this study was based on the maximum single use rate (600 g a.i./ha). Based on the conversion using the parameters soil depth (5 cm) and soil density (1.5 g/cm3 ), this corresponds to a weighed amount of 40 lg metalaxyl/50 g soil (dry weight). This calculated amount of metalaxyl was ap-
plied with 400 ll of water (@ 500 l/ha) to 50 g soil in individual Erlenmeyer ¯asks (150 ml) plugged with cotton pad. Prior to application of metalaxyl, bulk soil samples were mixed for 1 h using a tumbling mixer (30 2 rpm). The moisture content was adjusted to 60% of maximum water holding capacity and checked gravimetrically every 2 weeks. One batch of soil sample was sterilised by autoclaving at 15 psi and 121°C for 20 min on three consecutive days. The experimental set-up for sterilised soils was performed aseptically. No eect of autoclaving on metalaxyl stability has been reported (Singh and Tripathi, 1982). However, metalaxyl stability in soil system under autoclaving had also been checked in the present study before incubating soil samples. To discard eect of light sterilised and non-sterilised soil samples were kept under dark. For photodegradation study 8 lg metalaxyl applied on soil surface (10 g dry weight basis) was kept in 100 ml reaction vessel made of quartz glass. The calculated amount of metalaxyl was applied with 80 ll of water (soil surface area 15.90 cm2 , soil layer 0.5 cm). To avoid microbial eect soils were sterilised with 1% HgCl2 . The reaction vessel was covered with a quartz plate and exposed to light of xenon lamp at 550 W=mm2 (Suntest, Heraeus equipped with coated quartz ®lters with a cuto at <285 nm) with 12 h of light/12 h of dark condition per day. The temperature of the soil was kept at 25 1°C through circulation of cold water. All experiments were performed in duplicate. Soil samples were taken for analysis at appropriate time intervals. Sterile soil samples treated with the pesticide at the same application rate as irradiated soil samples and maintained in darkness were used as experimental control. For adsorption±desorption studies replicates (3 10 g of air-dried soil) were treated with 0.01 M CaCl2 (20 ml) solution containing dierent concentrations (0.5, 1, 5, 10, 25 and 50 lg/ml) of metalaxyl in stoppered te¯on centrifuge tubes. These samples were turned end-overend at 29 rpm for 48 h on an overhead shaker.
Table 1 Physical±chemical characteristics of the test soils Soil
H ofchen
Passo Fundo
Laacher Hof
Verro Beach
Texture (%)
Clay Silt Sand
10.2 81.3 8.5
44.4 24.4 31.2
12.0 51.1 36.9
1.1 2.0 96.9
pH
H2 O CaCl2
7.8 7.2
5.8 5.1
8.1 7.3
6.3 5.9
Org.C (%)
2.6
1.6
0.9
0.2
Total N (%)
0.2
0.2
0.1
±
C.E.C (moles/kg)
15.0
13.1
±
±
Origin
Burscheid, Germany
Passo Fundo, Brazil
Monheim, Germany
Florida, USA
P. Sukul, M. Spiteller / Chemosphere 45 (2001) 941±947
943
2.4. Extraction
3. Results and discussion
Metalaxyl treated soil samples were extracted with methanol (3 50 ml) on an electric shaker (1 h), each followed by ultrasonic vibration (Branson Soni®er, Branson) for 5 min. The extracts were centrifuged (Beckman J2-HS, Beckman, USA) for 20 min (10,000 rpm) and the combined extracts were concentrated to dryness by a rotary evaporator. Finally, the residue was dissolved in 5 ml methanol and analysed by HPLC. In case of photodegradation study, soil samples collected after periodical intervals were transferred to centrifuge tubes and extracted with 3 20 ml methanol followed by ultrasonic vibration for 5 min each time. For sorption study after 48 h of shaking the contents were centrifuged at 3000 rpm for 10 min. The supernatants were decanted, volumes were measured and directly analysed by HPLC. Desorption study was carried out by adding extra amount of freshly prepared 0.01 M CaCl2 solution to the soil residue in the centrifuge tubes so that total amount of 0.01 M CaCl2 solution remained exactly 20 ml. The centrifuge tubes were again kept under overhead shaker for 48 h, decanted and desorbed solutions were analysed by HPLC. Blanks (without soil) were run through the same procedure. The concentration of pesticide adsorbed to the soil was calculated as
Dissipation of metalaxyl has been studied in sterilised and non-sterilised soils under laboratory condition and results are presented in Figs. 1 and 2. In non-sterilised soils metalaxyl dissipation was found to be higher than that in sterilised soils. Obviously, this suggests the possible microbial role in degrading metalaxyl. However, dissipation was also observed in sterilised soils, which might be due to the result of chemical and other abiotic factors other than photodegradation as the soils were kept under dark during the entire experimental period. Comparative role of microbes and abiotic factors other than light in dissipating metalaxyl in soil is presented in Table 2. After
CB
CS VS =M;
where CB and CS are the blank and supernatant solution concentration, VS is the solution volume, and M is the soil mass. Adsorption isotherm were ®tted to the Freundlich model in log form log
Fig. 1. Dissipation of metalaxyl in sterilised soils.
x 1 log kD log Ceq ; m n
where x/m is the concentration of pesticide adsorbed to the soil (lg/g), Ceq is the equilibrium concentration of pesticide in solution (lg/ml), log kD (intercept on Y-axis) and 1/n (slope) are the adsorption parameters calculated from the linear regression. 2.5. HPLC Metalaxyl active ingredient was estimated by HPLC using a Shimadzu LC 10 AT, equipped with a Macherey±Nagel (250/8/4) C18 column and a UV detector operated at 205 nm. For the ®rst 15 min the mobile phase (1 ml/min) was composed of 10% acetonitrile and 90% water adjusted to pH 4.0 with 0.15 M H3 PO4 . For the next 5 min the acetonitrile content was increased to 90%.
Fig. 2. Dissipation of metalaxyl in non-sterilised soils.
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Table 2 Comparative role of microbes and abiotic factors on dissipating metalaxyl in soil % degradation of metalaxyl after 60 days of application
Soil
H ofchen Passo Fundo Laacher Hof Vero Beach
Non-sterilised (+ microbes + abiotic factors other than light)
Sterilised () microbes + abiotic factors other than light)
Degradation due to microbes
51.5 69.2 61.4 41.1
14.7 11.9 7.4 5.3
36.8 57.3 54.0 35.8
60 days of application 5.3±14.7% attenuation of metalaxyl was observed due to abiotic factors other than light, while 35.8±57.3% dissipation was due to the in¯uence of microbial activity. When log residues were plotted against incubation period, a straight line was found suggesting a ®rst-order reaction in the dissipation behaviour of metalaxyl in both sterilised and non-sterilised soils. The half-life values were in the range of 36±73 and 232±602 d in nonsterilised and sterilised soils, respectively (Table 3). This conforms the earlier reports (EPA, 1994; Kookana et al., 1995). Persistence of metalaxyl in non-sterilised soils was found in the order of Passo Fundo < Laacher Hof < H ofchen < Vero Beach and H ofchen < Passo Fundo < Laacher Hof < Vero Beach in sterilised soils. Dierence in persistence of metalaxyl in dierent soils under different conditions might be due to dierent physicochemical properties and biological activities of the test soils. In Vero Beach soil under both sterilised and nonsterilised conditions attenuation of metalaxyl was least. This is due to its sandy nature and presence of low amount of clay and organic carbon. Adsorption which might be the prime factor of metalaxyl dissipation under sterilised condition is expected to be negligible in this soil. In non-sterilised Vero Beach soil low dissipation as compared to other soils may be explained due to its low organic carbon content and in turn less biological activity. The present investigation is well in accordance with the earlier ®ndings (Droby and Coey, 1991; Huaguo et al., 1995) stating the importance of microbial activity in degrading metalaxyl. Photodegradation of metalaxyl on the soil surface was studied using arti®cial sunlight under laboratory condition (Fig. 3). When log residues were plotted
Fig. 3. Photodegradation of metalaxyl in four dierent soils.
against time, a linear relationship was found stating a ®rst-order kinetic reaction (Table 4). The half-life values were found in the range 188±502 h of irradiation in four soils of dierent physicochemical properties. Soil clay content showed a linear relationship (r2 0,92) with photopersistence of metalaxyl in soils (Fig. 4). It may be due to the fact that metalaxyl enters into the intra-lattice structure of clay components and remains unexposed to light. Metalaxyl also appeared to be preferentially adsorbed on soil mineral surface (Sukop and Cogger, 1992). Metalaxyl on oven-dried soil photodecomposes slowly (Murthy et al., 1998). Less than
Table 3 Regression equation, correlation coecient and half-life values of metalaxyl under two conditions Soil H ofchen Passo Fundo Laacher Hof Vero Beach
Non-sterilised soil
Sterilised soil
Regression equation
r2
t1=2 (d)
Regression equation
r2
t1=2 (d)
Y Y Y Y
0.9693 0.9400 0.9435 0.9549
59.0 36.3 39.1 73.4
Y Y Y Y
0.9813 0.9999 0.9607 0.9302
231.6 301.0 501.7 602.1
1:57 1:56 1:63 1:60
0:0051X 0:0083X 0:0077X 0:0041X
1:61 1:61 1:60 1:61
0:0013X 0:001X 0:0006X 0:0005X
P. Sukul, M. Spiteller / Chemosphere 45 (2001) 941±947
2% conversion of metalaxyl on oven-dried soil was found when it was subjected to arti®cial sunlight for 72 h. In another report also metalaxyl was found to be more persistent in dry soil than wet soil in presence of Table 4 Regression equation, correlation coecient and half-life values of metalaxyl under Suntest Soil
Regression equation
r2
t1=2 (h)
H ofchen Laacher Hof Passo Fundo Vero Beach
Y Y Y Y
0.9207 0.9154 0.9395 0.9387
334.5 273.7 501.7 188.1
1:61 1:61 1:60 1:58
0:0009X 0:0011X 0:0006X 0:0016X
Fig. 4. Correlation between soil clay content and persistence of metalaxyl in soil under simulated sunlight.
Fig. 5. Equilibriation time for metalaxyl adsorption in dierent soils.
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natural sunlight (Saha and Sukul, 1997). Comparatively more photodecomposition was found in the present investigation due to the presence of moisture. In our experiment, soils were kept at 60% of their maximum water holding capacity. The present observation suggests photostability of metalaxyl, in general, which is consistent with other ®ndings (Singh and Tripathi, 1982; Sukul et al., 1992; Saha and Sukul, 1997). Metalaxyl is resistant to sunlight due to its kmax at 196 nm in aqueous solution and no absorption above 290 nm. Therefore, photolysis can only occur by indirect photolysis via photosensitisers which in presence of water and after a sequence of reactions lead to the formation of OH radicals. The latter can attack the substrate and lead to
Fig. 6. Adsorption isotherm of metalaxyl in three dierent soils.
Fig. 7. Adsorption isotherm of metalaxyl following a desorption step in three dierent soils.
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P. Sukul, M. Spiteller / Chemosphere 45 (2001) 941±947
Table 5 Constants and correlation coecients of the Freundlich equations for adsorption±desorption of metalaxyl in soils Soil/Process
Regression equation
r2
kD
H ofchen Adsorption Desorption
Y 0:73X 0:34 Y 1:45X 0:33
0.9883 0.9861
1.4 1.4
53.8 53.5
Laacher Hof Adsorption Desorption
Y 0:81X Y 0:64X
0:31 0:16
0.9818 0.9936
1.4 1.2
151.1 130.0
Passo Fundo Adsorption Desorption
Y 0:54X 0:16 Y 1:94X 0:13
0.9873 0.9915
1.2 1.1
73.1 71.2
hydroxylation of the molecule and subsequently to further degradation. For adsorption±desorption studies the incubation time for metalaxyl was set to 48 h to establish an equilibrium (Fig. 5). When the equilibrium concentration (C) was plotted against the amount of metalaxyl adsorbed (x) in microgram per unit amount of soil (m) in grams, both in logarithmic form, an excellent ®t of data by the Freundlich equation log x=m log kD 1=n log C was achieved with a coecient of determination 0.98±0.99 for both of adsorption and desorption process (Figs. 6 and 7). The kD for adsorption and desorption processes indicated that the adsorption of metalaxyl was in the order of H ofchen > Laacher Hof > Passo Fundo (Table 5). In adsorption study the values of 1/n were less than unity for all three soils indicating a non-linear relationship between concentration of metalaxyl and its adsorption into these soils. The variable slopes of the sorption isotherms obtained for dierent soil systems studied reveal that metalaxyl sorption on soil is a complex phenomenon involving dierent types of adsorption sites with dierent surface energies. kOC kOC kD 100=% organic carbon) were calculated for each soil, using kD and organic carbon data. The result shows that the kOC values estimated from the individual soil material tend to increase as organic carbon content decreases. The desorption kD values were nearly same to those for adsorption. This indicates involvement of same spectrum of forces in adsorption of metalaxyl during adsorption and desorption steps.
Acknowledgements This study was funded as part of a research grant to the ®rst author from the Alexander von Humboldt Foundation, Germany. The authors deeply appreciate the help rendered by Mr. J orn Sickerling and Mr. J urgen Scheen, University of Dortmund during preparation of the manuscript.
kOC
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