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Activities of dehydrogenase and protease in soil as influenced by monocrotophos, quinalphos, cypermethrin and fenvalerate
Agriculture, Ecosystems and Environment, 47 (1994) 319-326
319
Elsevier Science Publishers B.V., Amsterdam
Activities of dehydrogenase and protease in soil as influenced by monocrotophos, quinalphos, cypermethrin and fenvalerate V. Rangaswamy*, B.R. Reddy, K. Venkateswarlu Department of Microbiology, Sri Krishnadevaraya University, Anantapur 515003, India (Accepted 21 June 1993)
Abstract
Two organophosphorus insecticides, monocrotophos and quinalphos, and two synthetic pyrethroids, cypermethrin and fenvalerate, were assessed for their effects on the activities of dehydrogenase (in terms of triphenyl formazan formed from triphenyl tetrazolium chloride) and protease (as tyrosine formed from casein) in soil, collected from a fallow groundnut field. The influence of the selected insecticides on enzyme activities was dose-dependent; the activities increased with increasingconcentrations of the insecticides up to 2.5 kg ha- i. Higher rates (5-12.5 kg ha- 1) of the insecticides were either innocuous or toxic to the enzyme activities. The significant stimulation in the activity of dehydrogenase and protease, associated with 2.5 kg ha- 1, lasted up to 14 days and 30 days, respectively. The results of the present study clearly indicate that the insecticides, widely used in cultivation of groundnut, at field application rates enhance the activities ofdehydrogenase and protease in soil.
Introduction
Soil dehydrogenase, with different enzymes or enzyme systems, has a role in the initial stage of oxidation of soil organic matter (Tu, 1980). Proteases are involved in the initial hydrolysis, in soil, of the protein components of the organic nitrogen to simple amino acids. It has been shown that proteases in soil can hydrolyse not only added proteins (Kiss et al., 1975 ) but also native soil proteins and peptides (Dedeken and Voets, 1965 ). Groundnut (Arachis hypogeaeL. ), the most important oil seed crop of India, is being attacked by several insect pests, resulting in a marked reduction of yield (Ayyanna et al., 1982). Chemical control by spraying insecticides such as monocrotophos and quinalphos (organophosphates) (Patel and Vora, 1981 ), and cypermethrin and fenvalerate (synthetic pyrethroids ) (Das, 1988 ) has been the common practice in combating several major pests of groundnut. Any action altering the life functions of non-target soil microorganisms could *Corresponding author.
© 1994 Elsevier Science Publishers B.V. All rights reserved 0167-8809/94/$07.00
320
v. Rangaswamy et aL / Agriculture, Ecosystems and Environment 47 (1994) 319-326
indirectly affect soil enzyme activities (Tu, 1988). Although many studies are available on non-target effects of pesticides in soil (Adus, 1970; Bollen, 1961; Tu and Miles, 1976 ), knowledge of the action of pesticides on soil enzymes is scarce (Tu, 1980). However, very recently we reported the impact of monocrotophos, quinalphos, cypermethrin and fenvalerate on invertase and amylase activities in soil (Rangaswamy and Venkateswarlu, 1992). The present study was undertaken to further information on the influence of the above insecticides towards activities of dehydrogenase and protease in a vertisol. Materials and methods
Soil
A black vertisol, collected from a fallow groundnut field of Anantapur district (a semi-arid region in Andhra Pradesh, India), to a depth of 12 cm, was air-dried and sifted through a 2 m m sieve before use. The physico-chemical characteristics of the soil included pH 8.05; organic matter, 0.89%; total nitrogen, 0.053%, NaHCO3-extractable phosphorus, 4.75 #g g- ~ soil, and electrical conductivity, 0.348 m mhos. Insecticides
Technical samples of two organophosphorus insecticides, monocrotophos (dimethyl (E)- 1-methyl carbamoyl vinyl phosphate), and quinalpohos (o,odimethyl-o-quinoxaline-2-yl phosphorothioate), and two synthetic pyrethroids, cypermethrin (a-cyano-3-phenoxyphenyl-3-(2,2-dichlorovinyl)-2, 2dimethyl cyclopropane carboxylate), and fenvalerate (cyano- ( 3-phenoxyaphenyl)-methyl 4-chloro- ( l-methyl ethyl) benzene acetate ) were dissolved in acetone. Soil incubation
Aliquots (0.05 ml) from stock solutions of the insecticides, prepared in acetone, were applied with a 0.1 ml pipette to the surface of 10 g soil samples contained in test tubes (25mmX 200mm) as followed by Lethbridge and Burns (1976). The final concentrations (on w/w basis) of each insecticide included 10, 25, 50, 75, 100 and 125/lg g-i soil, which correspond to 1, 2.5, 5.0, 7.5, 10 and 12.5 kg ha -I (Anderson, 1978). The field application dose of the selected insecticides ranged from 2 to 2.5 kg h a - ~ (Anonymous, 1988 ). The soil samples receiving only 0.05 ml acetone served as controls. After complete evaporation of the solvent at room temperature, all the treatments including controls were maintained at 60% water-holding capacity (WHC), and
14.Rangaswamy et al. / Agriculture, Ecosystems and Environment 47 (1994) 319-326
321
incubated in the laboratory at 2 8 + 4 ° C . After 7 or 10 days of incubation, duplicate soil samples were withdrawn for the assay of dehydrogenase (Chendrayan et al., 1980 ) and protease (Speir and Ross, 1975 ). The soil samples, in another experiment, were treated with only 2.5 kg h a of the four insecticides. Soil samples with no insecticide treatment served as controls. Soil moisture was maintained at 60% WHC and incubated in the laboratory at 28 _+4°C. Moisture levels were restored to their initial values during incubation. Duplicate soil samples of each treatment including the controls were withdrawn after 7 days, 14 days, 21 days, 28 days and 35 days, and 10 days, 20 days, 30 days, and 40 days of incubation, for the assay of dehydrogenase and protease, respectively.
Assay of dehydrogenase Dehydrogenase activity in soils was determined following the method of Casida et al. (1964) by the reduction of 2, 3, 5 -triphenyl tetrazolium chloride ( T T C ) . Each soil sample ( 10 g) was treated with 0.1 g CaCO3 and incubated for 24 h at 37°C. The triphenyl formazan formed was extracted from the reaction mixture with methanol and assayed at 485 n m in a Spectronic-20D spectrophotometer.
Assay of protease Untreated and insecticide-treated soil samples (2 g) were incubated for 24 h at 30°C with 10 ml of 0.1 M tris (2-amino-2 (hydroxymethylmethyl)-propane-1 : 3-diol, pH, 7.5 ) containing sodium caseinate (2% w / v ) . Four milliliters of aqueous solution ( 17.5% w / v ) of trichloro acetic acid was then added and the mixture was centrifuged. A suitable aliquot of the supematant was treated with 3 ml of 1.4 M NaCO3 followed by the addition of 1.0 ml FolinCicalteu reagent (33.3% v / v ) . The blue colour was read after 30 min at 700 n m in a spectrophotometer. Tyrosine was used as a standard.
Statistical analysis The concentrations of the enzymes were calculated on a soil weight (ovendried ) basis. The insecticide treatments were contrasted with untreated controis and the significant differences ( P < 0.05 ) between values of each sampiing and each insecticide were performed using Duncan's new multiple range ( D M R ) test (Duncan, 1955).
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• Rangaswamy et al. / Agriculture, Ecosystems and Environment 47 (1994) 319-326
R e s u l t s and d i s c u s s i o n
Soil dehydrogenase, one of the indices of microbial activity, measured in terms of triphenyl formazan accumulated from TTC, has been employed in the present study, as this enzyme is likely to be affected by addition of pesticides (Cervelli et al., 1978 ). Concentrations of monocrotophos, quinalphos, cypermethrin, and fenvalerate that effected a significant stimulation in enzyme activity during a 7 day incubation in soil samples ranged from 1 to 7.5 kg h a - 1 (Table 1 ). The accumulation of formazan was more striking at the 2.5 kg h a - 1 level. Even 10 and 12.5 kg h a - 1 levels of the selected insecticides (except 12.5 kg ha-1 of monocrotophos) were either stimulatory or had no influence on dehydrogenase activity. Likewise, methylparathion at 15 kg h a - 1 was reported to stimulate soil dehydrogenase activity (Naumann, 1970 ). But, Table 1 Effect of different concentrations of the insecticides after 7 days of incubation on activities of dehydrogenase ~in black soil Insecticide Monocrotophos concentration (kg ha-1 )
Quinalphos
Cypermethrin
Fenvalerate
0 1.0 2.5 5.0 7.5 10.0 12.5
38" 66 b 158 ~ 120 d 64 b 43 a 30"
38" 76 b 210 c 158 d 114 e 71 b 25"
(100) (200) (552) (146)
38 "b 61 ¢ 201 d 149 e
(300)
97 f
(255)
(187) (66)
51 be 23 a
(134) (61)
38 bc 62 ~ 178 e 92 f 49 Cd 28 ab 18b
(100) (163) (468) (242) (128) (74) (47)
(100) (173) (414) (315) (168) (113) (78)
(100) (161) (529) (392)
l#g formazan per gram of soil formed after 24 h incubation with TTC. Figures in parentheses indicate relative production percentages. Means, in each column, followed by the same letter are not significantly different ( P < 0.05 ) from each other according to DMR test. Table 2 Effect of the insecticides at 2.5 kg h a - 1 on activities of dehydrogenasel in soil Insecticide
Soilincubation (days) 7
Control Monocrotophos Quinalphos Cypermeyhrin Fenvalerate
38 a 178 b 158 b 210 ¢ 201 c
14 (100) (468) (415) (552) (258)
1Refer to Table 1 for footnotes.
184 ~ 249 b 237 b 284 c 276 c
21 (100) (135) (128) (154) (150)
357" 426 b 393 ~b 438 c 393 ~b
28 (100) (119) (110) (122) (110)
215 ~ 215" 241 b 222 b 229 b
35 (100) (100) (112) (103) (107)
152" 149 a 155" 158" 152"
(100) (98) (102) (104) (100)
323
V. Rangaswamy et aL / Agriculture, Ecosystems and Environment 47 (1994) 319-326
Table 3 Effect of different concentrations of the insecticides after 10 days of incubation on activities of protease t in soil Insecticide concentration (kg ha-1 )
Monocrotophos
Quinalphos
Cypermethrin
Fenvalerate
0 1 2.5 5 7.5 10 12.5
403 ab 630 d 672 e 555 f 475 ¢ 439 be 383 ~
403 ~b 588 Cd 639 a 565 ¢ 450 b 389 "b 362"
403 ~ 520 b 608 ¢ 560 d 494 e 434 f 345 s
403 a 515 b 691 ¢ 612 d 547 e 540 f 371"
(100) (156) (166) (138) (118) (109) (95)
(100) (146) (159) (140) (112) (97) (90)
(100) (129) (151) (139) (123) (108) (88)
(100) (128) (172) (152) (136) (117) (92)
tag tyrosine per gram of soil formed after 2 h incubation at 30°C with 1% casein. Refer to Table 1, for other footnotes. Table 4 Effect of the insecticides at 2.5 kg h a - l on activity of protease t in soil Insecticide
Soil incubation (days) l0
Control Monocrotophos Quinalphos Cypermethrin Fenvalerate
403" 672 d 639 ¢ 608 b 691 d
20 (100) (166) (158) (150) (171)
520" 688 b 672 b 677 b 723 ¢
30 (100) (132) (129) (130) (139)
350" 420 b 434 c 399 b 446 c
40 (100) (120) (124) (114) (127)
277" 289" 293" 277" 280 a
(100) (104) (106) (100) (101)
'/lg tyrosine per gram of soil formed after 2 h incubation at 30 °C with casein. Refer to Table 1 for other footnotes.
complete inhibition of the enzyme activity was noticed with 150-300 kg h a - 1 methylparathion at temperatures of 12-15 °C (Naumann, 1972 ). The extent of dehydrogenase activity of soil samples under the impact of the selected insecticides at 2.5 kg h a - 1 was also determined after incubating the insecticide-treated samples for 7, 14, 21, 28 and 35 days (Table 2). In general, the dehydrogenase activity was relatively less in the soils maintained under non-flooded conditions as reported by Chendrayan et al. (1980). This can be expected because dehydrogenase activity is significantly more pronounced in flooded soils as most dehydrogenases are of anaerobic origin (Chendrayan et al., 1980). There was a progressive increase in the accumulation of formazan with increasing period of incubation up to 21 days which gradually decreased further. In fact, application of insecticides to soils led to an initial striking increase in dehydrogenase activity.By the end of 5 weeks of soil incubation, insecticide application had virtually no influence on the enzyme activity. On the contrary, application ofturbufos, triazophos and trich-
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K Rangaswamy et al. / Agriculture, Ecosystems and Environment 47 (1994) 319-326
lornat at 5 and 10 #g g- 1 to an organic soil initially inhibited dehydrogenase activity followed by the stimulatory effect at the end of 2 weeks of incubation at 28°C (Tu,1981). Similarly, carbaryl at 10, 50 and 100 p.p.m, was known to inhibit dehydrogenase activity in three soils supplemented with glucose (Narayana Rao, 1988 ). However, significant increase in dehydrogenase activity was noticed with lower concentrations (0.5 and 5/tg g- ~) of five pyrethroid insecticides, permethrin (FMC 33297 ), FML 45498, Shell WL 41706, Shell WL 43467 and Shell WL 43775, after 3 weeks (Tu, 1980). The soil samples, treated with concentrations ranging from 1 to 12.5 kg h a - 1 of the insecticides, and incubated for 10 days, were supplemented with 1% casein in order to determine the non-target effects of these insecticides on protease activity, measured in terms of tyrosine formed after 2 h at 30°C. Application of quinalphos up to 5 kg h a - 1, or monocrotophos up to 7.5 kg h a - 1, and cypermethrin or fenvalerate up to 10 kg h a - ~greatly enhanced the activity of protease in soil (Table 3 ). The activity of protease was significantly more pronounced in soil samples that received 2.5 kg ha-~ of the four insecticides. Except the 12.5 kg h a - 1 level of cypermethrin, which was inhibitory, the higher concentrations (up to 12.5 kg h a - ~) of the insecticides were non-toxic to the soil enzyme. Similarly, population ofAzospirillum sp. in soil, the same soil used in the present study, and its nitrogen-fixing activity (Rangaswamy et al., 1989), and also ammonification and nitrification (Rangaswamy and Venkateswarlu, 1990) were either unaffected or enhanced by the application, even up to a level of 5 kg h a - 1, of monocrotophos or quinalphos. When the selected insecticides were applied at 2.5 kg ha-1 (the concentration which was seemingly stimulatory to the enzyme) to the soil samples, protease activity was enhanced significantly until 30 days of soil incubation (Table 4). However, incubation of insecticide-treated samples up to 40 days resulted in no stimulation of the enzyme activity. However, Endo et al. ( 1982 ) reported depression in protease activity of soils treated with 100 and 1000 p.p.m, of cartaphydrochloride under upland conditions. The results of the present study clearly indicate that the insecticides, widely used in cultivation of groundnut, at field application rates enhance the activities of dehydrogenase and protease in soil.
Acknowledgements One of us (VRS) is grateful to the University Grants Commission, New Delhi, India, for financial assistance.
References Adus, L.J., 1970. The action of herbicides and pesticides on the microflora (a review). Meded. Rijksfac. Landb. Gent., 35,465-492.
K Rangaswamy et aL/Agriculture, Ecosystemsand Environment 47 (1994) 319-326
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Anderson, J.R., 1978. Pesticide effects on non-target soil microorganisms. In: I. R. Hill and S.J.L. Wright (Editors), Pesticide Microbiology. Academic Press, London, pp. 313-533. Anonymous, 1988. Agricultural Diary. Department of Agriculture, Andhra Pradesh, India, pp. 42-44. Ayyanna, T., Arjuna Rao, P., Subbaratnam, G.V., Krishnamurthy Rao, B.H. and Narayana, K.L., 1982. Chemical control of Spodoptera litura Fabricus on groundnut crop. Pesticides, 16(8): 19-20. Bollen, W.B., 1961. Interactions between pesticides and soil microorganisms. Ann. Rev. Microbiol., 15: 69-92. Casida Jr., L.E., Klein, D.A. and Santoro, T., 1964. Soil dehydrogenase activity. Soil Sci., 98: 371-376. Cervelli, S., Nannipieri, P. and Sequi, P., 1978. Interactions between agrochemicals and soil enzymes. In: R.G. Burns (Editor), Soil Enzymes, Academic Press, London, pp. 251-293. Chendrayan, K., Adhya, T.K. and Sethunathan, N., 1980. Dehydrogenase and invertase activities of flooded soils. Soil Biol. Biochem., 12:271-273. Das, B.B., 1988. Efficacy of some synthetic pyrethroids and conventional insecticidal spray against groundnut pest complex. Pesticides, 22 ( 1 ): 10-12. Dedeken, M. and Voets, J.P., 1965. Recherches sur le metabolisme des acides amines dans le sol. 1. Le metabolisme de glycine, alanine, acide aspartique et acide glutaminique, Annales de 1 'Institut Pasteur, Paris 10g. Supplement 3, pp. 103-111. Duncan, D.M., 1955. Multiple range and multiple tests. Biometrics, 42: 1-42. Endo, T., Taiki, K., Nobutsura, T. and Michihiko, S., 1982. Effect of insecticide cartaphydrochloride on soil enzyme activities, respiration and on nitrification. J. Pestic. Sci., 7:101-110. Kiss, S., Stefanic, G. and Dragan-Bularda, M., 1975. Soil enzymology in Romania (Part II). Contributii Botanice Cluj., pp. 197-207. Lethbridge, G. and Burns, R.G., 1976. Inhibition of soil urease by organophosphorus insecticides. Soil Biol. Biochem., 8: 99-102. Narayana Rao, V.V.S., 1988. Effect of pesticides on ammonification. In: R. Lal (Editor), Pesticides and Nitrogen Cycle Vol 2. CRC Press, Boca Raton, FL, pp. 2-41. Naumann, K., 1970. Zur Dynamik der Bodenmikroflora nach Anwendung von Pflanzenschutzmitteln. II. Die Reaction verschiedener physiologisher Gruppen von Bodenbakterien auf den Einsutz yon Parathion-methyl in Flreiland. Zbl. Bakt. Abt. II, 124: 755-765. Naumann, K., 1972. Die Wirkung einiger Umweltafaktoren and die Reaktion der Bodenmikroflora gengenuber Planzenschatzonitteln. Zbl. Bakt. Abt. II, 127: 379-396. Patel, B.R. and Vora, V.J., 1981. Efficacy of different insecticides for the control of groundnut jassid E m p o a s c a kerri Pruthi. Pesticides, 15 ( 10): 33-34. Rangaswamy, V., Charyulu, P.B.B.N. and Venkateswarlu, K., 1989. Effect of monocrotophos and quinalphos on soil population and nitrogen-fixing activity o f A z o s p i r i l l u m sp. Biomed. Environ. Sci., 2:305-311. Rangaswamy, V. and Venkateswarlu, K., 1990. Stimulation of ammonification and nitrification in soils by the insecticides, monocrotophos and quinalphos. Biomed. Environ. Sci., 3: 391396. Rangaswamy, V. and Venkateswarlu, K., 1992. Activities of amylase and invertase as influenced by the application of monocrotophos, quinalphos, cypermethrin and fenvalerate to groundnut soil. Chemosphere, 25: 525-530. Speir, T.W. and Ross, D.J., 1975. Effects of storage on the activities of protease, urease, phosphatase and sulphatase in three soils under pasture. NZ. J. Sci., 18:231-237. Tu, C.M. and Miles, J.R.W., 1976. Interactions between insecticides and soil microbes. Residue Rev., 64:17-66. Tu, C.M., 1980. Influence of five pyrethroid insecticides on microbial populations and activities in soil. Microb. Ecol., 5: 321-327.
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Tu, C.M., 1981. Effect of some pesticides on enzyme activities in an organic soil. Bull. Environ. Contam. Toxicol., 27:109-113. Tu, C.M., 1988. Effect of selected pesticides on activities of invertase, amylase and microbial respiration in sandy soil, Chemosphere, 17:159-163.
319
Elsevier Science Publishers B.V., Amsterdam
Activities of dehydrogenase and protease in soil as influenced by monocrotophos, quinalphos, cypermethrin and fenvalerate V. Rangaswamy*, B.R. Reddy, K. Venkateswarlu Department of Microbiology, Sri Krishnadevaraya University, Anantapur 515003, India (Accepted 21 June 1993)
Abstract
Two organophosphorus insecticides, monocrotophos and quinalphos, and two synthetic pyrethroids, cypermethrin and fenvalerate, were assessed for their effects on the activities of dehydrogenase (in terms of triphenyl formazan formed from triphenyl tetrazolium chloride) and protease (as tyrosine formed from casein) in soil, collected from a fallow groundnut field. The influence of the selected insecticides on enzyme activities was dose-dependent; the activities increased with increasingconcentrations of the insecticides up to 2.5 kg ha- i. Higher rates (5-12.5 kg ha- 1) of the insecticides were either innocuous or toxic to the enzyme activities. The significant stimulation in the activity of dehydrogenase and protease, associated with 2.5 kg ha- 1, lasted up to 14 days and 30 days, respectively. The results of the present study clearly indicate that the insecticides, widely used in cultivation of groundnut, at field application rates enhance the activities ofdehydrogenase and protease in soil.
Introduction
Soil dehydrogenase, with different enzymes or enzyme systems, has a role in the initial stage of oxidation of soil organic matter (Tu, 1980). Proteases are involved in the initial hydrolysis, in soil, of the protein components of the organic nitrogen to simple amino acids. It has been shown that proteases in soil can hydrolyse not only added proteins (Kiss et al., 1975 ) but also native soil proteins and peptides (Dedeken and Voets, 1965 ). Groundnut (Arachis hypogeaeL. ), the most important oil seed crop of India, is being attacked by several insect pests, resulting in a marked reduction of yield (Ayyanna et al., 1982). Chemical control by spraying insecticides such as monocrotophos and quinalphos (organophosphates) (Patel and Vora, 1981 ), and cypermethrin and fenvalerate (synthetic pyrethroids ) (Das, 1988 ) has been the common practice in combating several major pests of groundnut. Any action altering the life functions of non-target soil microorganisms could *Corresponding author.
© 1994 Elsevier Science Publishers B.V. All rights reserved 0167-8809/94/$07.00
320
v. Rangaswamy et aL / Agriculture, Ecosystems and Environment 47 (1994) 319-326
indirectly affect soil enzyme activities (Tu, 1988). Although many studies are available on non-target effects of pesticides in soil (Adus, 1970; Bollen, 1961; Tu and Miles, 1976 ), knowledge of the action of pesticides on soil enzymes is scarce (Tu, 1980). However, very recently we reported the impact of monocrotophos, quinalphos, cypermethrin and fenvalerate on invertase and amylase activities in soil (Rangaswamy and Venkateswarlu, 1992). The present study was undertaken to further information on the influence of the above insecticides towards activities of dehydrogenase and protease in a vertisol. Materials and methods
Soil
A black vertisol, collected from a fallow groundnut field of Anantapur district (a semi-arid region in Andhra Pradesh, India), to a depth of 12 cm, was air-dried and sifted through a 2 m m sieve before use. The physico-chemical characteristics of the soil included pH 8.05; organic matter, 0.89%; total nitrogen, 0.053%, NaHCO3-extractable phosphorus, 4.75 #g g- ~ soil, and electrical conductivity, 0.348 m mhos. Insecticides
Technical samples of two organophosphorus insecticides, monocrotophos (dimethyl (E)- 1-methyl carbamoyl vinyl phosphate), and quinalpohos (o,odimethyl-o-quinoxaline-2-yl phosphorothioate), and two synthetic pyrethroids, cypermethrin (a-cyano-3-phenoxyphenyl-3-(2,2-dichlorovinyl)-2, 2dimethyl cyclopropane carboxylate), and fenvalerate (cyano- ( 3-phenoxyaphenyl)-methyl 4-chloro- ( l-methyl ethyl) benzene acetate ) were dissolved in acetone. Soil incubation
Aliquots (0.05 ml) from stock solutions of the insecticides, prepared in acetone, were applied with a 0.1 ml pipette to the surface of 10 g soil samples contained in test tubes (25mmX 200mm) as followed by Lethbridge and Burns (1976). The final concentrations (on w/w basis) of each insecticide included 10, 25, 50, 75, 100 and 125/lg g-i soil, which correspond to 1, 2.5, 5.0, 7.5, 10 and 12.5 kg ha -I (Anderson, 1978). The field application dose of the selected insecticides ranged from 2 to 2.5 kg h a - ~ (Anonymous, 1988 ). The soil samples receiving only 0.05 ml acetone served as controls. After complete evaporation of the solvent at room temperature, all the treatments including controls were maintained at 60% water-holding capacity (WHC), and
14.Rangaswamy et al. / Agriculture, Ecosystems and Environment 47 (1994) 319-326
321
incubated in the laboratory at 2 8 + 4 ° C . After 7 or 10 days of incubation, duplicate soil samples were withdrawn for the assay of dehydrogenase (Chendrayan et al., 1980 ) and protease (Speir and Ross, 1975 ). The soil samples, in another experiment, were treated with only 2.5 kg h a of the four insecticides. Soil samples with no insecticide treatment served as controls. Soil moisture was maintained at 60% WHC and incubated in the laboratory at 28 _+4°C. Moisture levels were restored to their initial values during incubation. Duplicate soil samples of each treatment including the controls were withdrawn after 7 days, 14 days, 21 days, 28 days and 35 days, and 10 days, 20 days, 30 days, and 40 days of incubation, for the assay of dehydrogenase and protease, respectively.
Assay of dehydrogenase Dehydrogenase activity in soils was determined following the method of Casida et al. (1964) by the reduction of 2, 3, 5 -triphenyl tetrazolium chloride ( T T C ) . Each soil sample ( 10 g) was treated with 0.1 g CaCO3 and incubated for 24 h at 37°C. The triphenyl formazan formed was extracted from the reaction mixture with methanol and assayed at 485 n m in a Spectronic-20D spectrophotometer.
Assay of protease Untreated and insecticide-treated soil samples (2 g) were incubated for 24 h at 30°C with 10 ml of 0.1 M tris (2-amino-2 (hydroxymethylmethyl)-propane-1 : 3-diol, pH, 7.5 ) containing sodium caseinate (2% w / v ) . Four milliliters of aqueous solution ( 17.5% w / v ) of trichloro acetic acid was then added and the mixture was centrifuged. A suitable aliquot of the supematant was treated with 3 ml of 1.4 M NaCO3 followed by the addition of 1.0 ml FolinCicalteu reagent (33.3% v / v ) . The blue colour was read after 30 min at 700 n m in a spectrophotometer. Tyrosine was used as a standard.
Statistical analysis The concentrations of the enzymes were calculated on a soil weight (ovendried ) basis. The insecticide treatments were contrasted with untreated controis and the significant differences ( P < 0.05 ) between values of each sampiing and each insecticide were performed using Duncan's new multiple range ( D M R ) test (Duncan, 1955).
322
• Rangaswamy et al. / Agriculture, Ecosystems and Environment 47 (1994) 319-326
R e s u l t s and d i s c u s s i o n
Soil dehydrogenase, one of the indices of microbial activity, measured in terms of triphenyl formazan accumulated from TTC, has been employed in the present study, as this enzyme is likely to be affected by addition of pesticides (Cervelli et al., 1978 ). Concentrations of monocrotophos, quinalphos, cypermethrin, and fenvalerate that effected a significant stimulation in enzyme activity during a 7 day incubation in soil samples ranged from 1 to 7.5 kg h a - 1 (Table 1 ). The accumulation of formazan was more striking at the 2.5 kg h a - 1 level. Even 10 and 12.5 kg h a - 1 levels of the selected insecticides (except 12.5 kg ha-1 of monocrotophos) were either stimulatory or had no influence on dehydrogenase activity. Likewise, methylparathion at 15 kg h a - 1 was reported to stimulate soil dehydrogenase activity (Naumann, 1970 ). But, Table 1 Effect of different concentrations of the insecticides after 7 days of incubation on activities of dehydrogenase ~in black soil Insecticide Monocrotophos concentration (kg ha-1 )
Quinalphos
Cypermethrin
Fenvalerate
0 1.0 2.5 5.0 7.5 10.0 12.5
38" 66 b 158 ~ 120 d 64 b 43 a 30"
38" 76 b 210 c 158 d 114 e 71 b 25"
(100) (200) (552) (146)
38 "b 61 ¢ 201 d 149 e
(300)
97 f
(255)
(187) (66)
51 be 23 a
(134) (61)
38 bc 62 ~ 178 e 92 f 49 Cd 28 ab 18b
(100) (163) (468) (242) (128) (74) (47)
(100) (173) (414) (315) (168) (113) (78)
(100) (161) (529) (392)
l#g formazan per gram of soil formed after 24 h incubation with TTC. Figures in parentheses indicate relative production percentages. Means, in each column, followed by the same letter are not significantly different ( P < 0.05 ) from each other according to DMR test. Table 2 Effect of the insecticides at 2.5 kg h a - 1 on activities of dehydrogenasel in soil Insecticide
Soilincubation (days) 7
Control Monocrotophos Quinalphos Cypermeyhrin Fenvalerate
38 a 178 b 158 b 210 ¢ 201 c
14 (100) (468) (415) (552) (258)
1Refer to Table 1 for footnotes.
184 ~ 249 b 237 b 284 c 276 c
21 (100) (135) (128) (154) (150)
357" 426 b 393 ~b 438 c 393 ~b
28 (100) (119) (110) (122) (110)
215 ~ 215" 241 b 222 b 229 b
35 (100) (100) (112) (103) (107)
152" 149 a 155" 158" 152"
(100) (98) (102) (104) (100)
323
V. Rangaswamy et aL / Agriculture, Ecosystems and Environment 47 (1994) 319-326
Table 3 Effect of different concentrations of the insecticides after 10 days of incubation on activities of protease t in soil Insecticide concentration (kg ha-1 )
Monocrotophos
Quinalphos
Cypermethrin
Fenvalerate
0 1 2.5 5 7.5 10 12.5
403 ab 630 d 672 e 555 f 475 ¢ 439 be 383 ~
403 ~b 588 Cd 639 a 565 ¢ 450 b 389 "b 362"
403 ~ 520 b 608 ¢ 560 d 494 e 434 f 345 s
403 a 515 b 691 ¢ 612 d 547 e 540 f 371"
(100) (156) (166) (138) (118) (109) (95)
(100) (146) (159) (140) (112) (97) (90)
(100) (129) (151) (139) (123) (108) (88)
(100) (128) (172) (152) (136) (117) (92)
tag tyrosine per gram of soil formed after 2 h incubation at 30°C with 1% casein. Refer to Table 1, for other footnotes. Table 4 Effect of the insecticides at 2.5 kg h a - l on activity of protease t in soil Insecticide
Soil incubation (days) l0
Control Monocrotophos Quinalphos Cypermethrin Fenvalerate
403" 672 d 639 ¢ 608 b 691 d
20 (100) (166) (158) (150) (171)
520" 688 b 672 b 677 b 723 ¢
30 (100) (132) (129) (130) (139)
350" 420 b 434 c 399 b 446 c
40 (100) (120) (124) (114) (127)
277" 289" 293" 277" 280 a
(100) (104) (106) (100) (101)
'/lg tyrosine per gram of soil formed after 2 h incubation at 30 °C with casein. Refer to Table 1 for other footnotes.
complete inhibition of the enzyme activity was noticed with 150-300 kg h a - 1 methylparathion at temperatures of 12-15 °C (Naumann, 1972 ). The extent of dehydrogenase activity of soil samples under the impact of the selected insecticides at 2.5 kg h a - 1 was also determined after incubating the insecticide-treated samples for 7, 14, 21, 28 and 35 days (Table 2). In general, the dehydrogenase activity was relatively less in the soils maintained under non-flooded conditions as reported by Chendrayan et al. (1980). This can be expected because dehydrogenase activity is significantly more pronounced in flooded soils as most dehydrogenases are of anaerobic origin (Chendrayan et al., 1980). There was a progressive increase in the accumulation of formazan with increasing period of incubation up to 21 days which gradually decreased further. In fact, application of insecticides to soils led to an initial striking increase in dehydrogenase activity.By the end of 5 weeks of soil incubation, insecticide application had virtually no influence on the enzyme activity. On the contrary, application ofturbufos, triazophos and trich-
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lornat at 5 and 10 #g g- 1 to an organic soil initially inhibited dehydrogenase activity followed by the stimulatory effect at the end of 2 weeks of incubation at 28°C (Tu,1981). Similarly, carbaryl at 10, 50 and 100 p.p.m, was known to inhibit dehydrogenase activity in three soils supplemented with glucose (Narayana Rao, 1988 ). However, significant increase in dehydrogenase activity was noticed with lower concentrations (0.5 and 5/tg g- ~) of five pyrethroid insecticides, permethrin (FMC 33297 ), FML 45498, Shell WL 41706, Shell WL 43467 and Shell WL 43775, after 3 weeks (Tu, 1980). The soil samples, treated with concentrations ranging from 1 to 12.5 kg h a - 1 of the insecticides, and incubated for 10 days, were supplemented with 1% casein in order to determine the non-target effects of these insecticides on protease activity, measured in terms of tyrosine formed after 2 h at 30°C. Application of quinalphos up to 5 kg h a - 1, or monocrotophos up to 7.5 kg h a - 1, and cypermethrin or fenvalerate up to 10 kg h a - ~greatly enhanced the activity of protease in soil (Table 3 ). The activity of protease was significantly more pronounced in soil samples that received 2.5 kg ha-~ of the four insecticides. Except the 12.5 kg h a - 1 level of cypermethrin, which was inhibitory, the higher concentrations (up to 12.5 kg h a - ~) of the insecticides were non-toxic to the soil enzyme. Similarly, population ofAzospirillum sp. in soil, the same soil used in the present study, and its nitrogen-fixing activity (Rangaswamy et al., 1989), and also ammonification and nitrification (Rangaswamy and Venkateswarlu, 1990) were either unaffected or enhanced by the application, even up to a level of 5 kg h a - 1, of monocrotophos or quinalphos. When the selected insecticides were applied at 2.5 kg ha-1 (the concentration which was seemingly stimulatory to the enzyme) to the soil samples, protease activity was enhanced significantly until 30 days of soil incubation (Table 4). However, incubation of insecticide-treated samples up to 40 days resulted in no stimulation of the enzyme activity. However, Endo et al. ( 1982 ) reported depression in protease activity of soils treated with 100 and 1000 p.p.m, of cartaphydrochloride under upland conditions. The results of the present study clearly indicate that the insecticides, widely used in cultivation of groundnut, at field application rates enhance the activities of dehydrogenase and protease in soil.
Acknowledgements One of us (VRS) is grateful to the University Grants Commission, New Delhi, India, for financial assistance.
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