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Ammonification and nitrification in soils, and nitrogen fixation by Azospirillum sp. as influenced by cypermethrin and fenvalerate
Agriculture, Ecosystems and Environment, 45 ( 1993 ) 311-317
311
Elsevier Science Publishers B.V., Amsterdam
Ammonification and nitrification in soils, and nitrogen fixation by Azospirillum sp. as influenced by cypermethrin and fenvalerate V. Rangaswamy*, K. Venkateswarlu Department of Microbiology, Sri Krishnadevaraya University, Anantapur 515003, India (Accepted 28 December 1992)
Abstract
Mineralization of peptone-nitrogen and oxidation of ammoniacal nitrogen were significantly enhanced in two agricultural soils when treated with up to 5 kg ha- l of cypermethrin or fenvalerate. The rate of ammonification and nitrification after 2 and 4 weeks was fairly rapid in the soils receiving 2.5 kg ha-t of either insecticide. The enhancement of both transformations, mediated by microorganisms, was more pronounced in the fenvalerate-treated soils. Cultures of Azospirillum sp., isolated from insecticide-treated soils, exhibited greater nitrogen-fixing activity that lasted for at least three generations.
Introduction
The study of the effects of insecticides on non-target microbial populations and processes in soil, an ultimate sink, is an important aspect of research into the environmental impact of these xenobiotics. Microbial processes, in particular associated with the nitrogen cycle, are often chosen as test systems owing to the ecological significance of nitrogen transformations in the biosphere (Stratton, 1990). Chemical control by spraying synthetic pyrethroid insecticides such as cypermethrin and fenvalerate has been the common practice in controlling several major pests of groundnut (Arachis hypogeaeL. ) (Das, 1988 ). There is no information available on the interaction between these insecticides and microorganisms that are implicated in the transformation of nitrogen in soil. The present study was, therefore, aimed at determining the influence of cypermethrin and fenvalerate on ammonification and nitrification in soils, and nitrogen fixation by Azospirillum sp., isolated from soil. *Corresponding author.
© 1993 Elsevier Science Publishers B.V. All rights reserved 0167-8809/-93/$06.00
312
v. Rangaswamy, K. Venkateswarlu/Agriculture, Ecosystems & Environment 45 (1993) 311-317
Materials and methods
Soi/s A red laterite soil and a black vertisol, both collected from groundnut fields of the Anantapur district ofAndhra Pradesh, India, were used. The soil sampies, collected randomly from a depth of up to 12 cm from fallow fields, were air-dried and sifted through a 2 m m sieve.
Insecticides Aqueous solutions, prepared from commercial formulations of cypermethrin (Cyperkill 25 EC, Bharat Pulverising Mills, Bombay, India) and fenvalcrate (Fenkem 20 EC, Rallis India, Bangalore, India) were used in soil incubation studies. Stock solutions from technical-grade cypermethrin (a-cyano3-phenoxy phenyl-3-(2,2-dichlorovinyl)-2,2-dimethyl cyclopropane carboxylate; 92.2% pure ) and fenvalerate (cyano- ( 3-phenoxy-ct-phenyl)-methyl 4chloro-( 1-methyl ethyl ) benzene acetate; 93.7% pure ) were prepared in acetone for use in pure culture studies.
Ammonification and nitrification in soils Samples (10 g) of black soil were placed in test tubes (25-200 mm), and were treated with 0.05 ml stock solutions of cypermethrin or fenvalerate to provide final concentrations of 10, 25, 50, 75 and 100/lg g- ~soil (equivalent to 1, 2.5, 5, 7.5, and 10 kg ha -I ). Soil samples receiving 0.05 ml of acetone served as controls. After complete evaporation of the solvent at room temperature, all treatments including the controls were supplemented with either 1000 ppm organic nitrogen as analytical grade peptone, or 200 ppm ammoniacal nitrogen in the form of analytical grade ammonium sulfate. After thorough mixing, all the soil samples were maintained at 60% water-holding capacity (WHC) and incubated at room temperature (28 _+4°C). Seven days after incubation, three samples of each treatment were withdrawn to estimate ammonium by nesslerization (Jackson, 1971 ), nitrite by diazotization (Barnes and Folkard, 1951 ), and nitrate by phenol disulfonic acid method (Bremner, 1965). In another experiment, both black and red soils were treated with 2.5 kg h a - 1 of cypermethrin and fenvalerate. Soil samples with no insecticide treatment served as controls. Peptone ( 1000 ppm organic N ) or ammonium (200 ppm NH~"-N) was added to the untreated and insecticide-treated soil samples. Soil moisture was maintained at 60% WHC and incubated at room temperature. Moisture levels were restored to their initial values during incubation. Three samples of each treatment were withdrawn after l, 2, 3 and 4
V. Rangaswamy, K. Venkateswarlu/Agriculture, Ecosystems & Environment 45 (1993) 311-317
313
weeks to determine the rate of ammonification, and after 2, 4, 6 and 8 weeks to determine the rate of nitrification following the methods adopted by Tu ( 1973 ). All results are expressed on the basis of oven-dried soils.
Nitrogen fixation by Azospirillum sp. Aliquots from stock solutions of the technical grade insecticides, prepared in acetone, were placed in sterilized test tubes (25-200 m m ) to provide a final concentration of 50 gg ml - ~ malate medium. The carrier solvent was evaporated, and 20 ml portions of the steam-sterilized malate medium were introduced into each test tube under aseptic conditions. The residues were equilibrated for 24 h to obtain aqueous solutions of the insecticides (Megharaj et al., 1986). Test tubes containing medium without the insecticide served as controls. Soil suspensions ( 1 : 10 soil to water ratio) from untreated and insecticide-treated ( 5 kg h a - 1 level with commercial formulations ) samples, incubated for 7 days, were prepared in sterilized distilled water. Aliquots (0.1 ml) of these suspensions were used to inoculate 20 ml portions of malate medium with and without the insecticide. After 72 h incubation at 37 °C, three tubes for each treatment were digested with HESO4to estimating total nitrogen (N) by the micro-Kjeldahl method as described earlier (Megharaj et al., 1988 ). The amount of N present in 0.1 ml soil suspensions, used for inoculation, together with that of the medium was deducted from experimental values. Cultures of Azospirillurn sp. were isolated from untreated and insecticidetreated (four times at 10-day intervals) soil samples. To determine whether the increased nitrogen-fixing capacity of Azospirillum sp. isolated from soil samples treated with insecticides would continue further, the isolates were subcultured in the semi-solid malate medium three times at an interval of 7 days, and their rates of nitrogen fixation were compared with those of fresh cultures obtained immediately after isolation from untreated and insecticidetreated soil samples. Results and discussion Application of cypermethrin and fenvalerate at levels of up to 5 kg hasignificantly increased the mineralization of peptone-N and the oxidation of ammoniacal-N (Table 1 ). In particular, there was a pronounced stimulation of ammonification and nitrification at a level of 2.5 kg ha-1, which is close to the field application rate, of the insecticides. Fenvalerate application at 7.5 kg h a - l was innocuous to both microbial transformations. However, cypermethrin at 7.5 kg h a - 1 inhibited ammonification and nitrification. The highest level ( l0 kg h a - 1) of both insecticides significantly inhibited the transformations of nitrogen. However, four organophosphorus insecticides viz., Bay 37289, diazinon, Dursban and zinophos at levels of 100 ppm were reported
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z. Rangaswamy, K. Venkateswarlu/Agriculture, Ecosystems & Environment 45 (1993) 311-317
Table 1 Effect of different concentrations of insecticides on ammonification ~ after 7 days, and nitrification 2 after 2 weeks, in black soil Insecticide concentration (kg ha-1 )
Ammonification Cypermethrin
Fenvalerate
0 1.0 2.5 5.0 7.5 10.0
455.20 ~ 517.00 ¢ 543.35 b 498.65 d 397.30 ¢ 358.20 f
455.20 a 528.10 c 552.70 b 498.45 d 414.05 e 293.50 r
(100) (114) (119) (110) (87) (79)
Nitrification
(100) (116) (121) (110) (91) (65)
Cypermethrin
Fenvalerate
28.65 ¢ 41.00 b 49.63 a 41.63 b 30.75 c 25.38 d
28.65 ¢ 41.50 b 52.00 a 41.75 b 29.50 cd 23.00 d
(100) (143) (173) (145) (107) (89)
(100) (149) (182) (146) (103) (80)
1Values are #g (NH~- + NO~- + NO~- )-N g-~ soil. 2Values are #g ( N O r + NO j- ) -N g - ~soil. Figures in parentheses indicate relative production percentages. Means (n = 3), in each column, followed by the same supercripts are not significantly different ( P < 0 . 0 5 ) from each other according to Duncan's new multiple range test.
to stimulate ammonification after 1 week of incubation in a sandy loam soil (Tu, 1970). The level of 2.5 kg ha-~ was selected to determine the rate of ammonification and nitrification in both soils, because this particular concentration effected a pronounced stimulatory effect (Table 1 ). In general, mineralization of the added organic nitrogen in soils with no insecticide was significantly more rapid after 2 weeks of incubation (Table 2 ). Thus, about 50% of added organic nitrogen was mineralized during this period. An increased incubation period resulted in the formation of little available nitrogen (NH~-+ NO~- + NOj- ), presumably owing to biological immobilization. Such an observation was also made by Tu (1973) with a loamy sand. However, application of cypermethrin or fenvalerate markedly stimulated ammonification. Moreover, this mineralization process was fairly rapid in soils treated with fenvalerate. Likewise, four nematicides, such as carbofuran, Dasanit, D-D, and Vortex, either had no effect or slightly stimulated ammonification in a loamy sand incubated at 28°C (Tu, 1973 ). The rate of nitrification followed a trend similar to that of ammonification. Ammonium was oxidized fairly rapidly to nitrate in soils (Table 2). No attempt was made to distinguish the implication of either autotrophic or heterotrophic nitrifiers. There was a greater accumulation of NO~- and NO~after 4 weeks, but this was followed by a decrease with increasing period of incubation. The decrease in oxidized forms of nitrogen after 8 weeks accounted for nearly 80% of that accumulated after 4 weeks. The application of cypermethrin and fenvalerate to both soils significantly enhanced the oxidation of NH~-N throughout the incubation. Fenvalerate exhibited a striking stimulation of nitrification. Several other studies have also demonstrated an
v. Rangaswamy, K. Venkateswarlu/Agriculture, Ecosystems & Environment 45 (1993) 311-317
315
Tab le 2 I nf luence o f c y p e r m e t h r i n a n d f e n v a l e r a t e ( b o t h at 2.5 kg h a - ~) on a m m o n i f i c a t i o n a n d n i t r i f i c a t i o n in soils Treatment
Ammonification
Nitrification
Il
2
3
4
2
4
6
8
478.85 a 543.30 b 650.95 b
557.63" 628.63 b 646.63 c
500.75 ~ 514.20 b 524.70 b
389.48" 399.90" 400.10 ~
34.13 ~ 49.63 b 52.25 b
192.00 a 225.00 b 228.38 c
67.00 a 75.50 ab 81.25 b
34.88 a 35.75" 35.20 ~
400.10 a 481.70 b 531.90 c
475.93 a 546.88 b 551.80 b
466.80 a 491.15 b 481.55 b
381.05 a 389.18 ~b 399.65 b
32.50" 44.50 b 50.88 ¢
172.88" 204.75 b 215.25 b
39.85 a 43.68 b 42.48 b
30.25 a 32.25 a 32.50 ~
Black soil Control Cypermethrin Fenvalerate
R e d soil Control Cypermethrin Fenvalerate
t l n c u b a t i o n , in weeks. M e a n s (n = 3 ), in each c o l u m n for a soil type, followed by the s a m e letter are not s i gni fi c a nt l y different ( P < 0.05 ) from each o t h e r a c c o r d i n g to D u n c a n ' s new m u l t i p l e range test. Refer to Table 1 for o t h e r footnotes.
inhibition of nitrification by pesticides such as HCH (Gaur and Misra, 1977; Ray et al., 1980), carbaryl (Bartha et al., 1967; Ramakrishna and Sethunathan, 1983), and benomyl (Gowda et al., 1977; Ramakrishna et al., 1979). However, ammoniacal nitrogen incorporated in a loamy sand was oxidized significantly after 8 weeks of incubation at 28°C with carbofuran, D-D, Dasanit, and Vortex (Tu, 1973 ). Similarly, carbofuran application, at 10 and 100 ppm, to rice rhizosphere soil suspension markedly stimulated the autotrophic oxidation of ammonium (Ramakrishna and Sethunathan, 1982 ). Cultures of Azospirillum, obtained after 7 days of soil incubation, from unamended soils exhibited appreciable nitrogen-fixing activity (Table 3 ). A significant stimulation of nitrogen fixation was evident in cultures from soils treated with the two insecticides at a level of 5 kg ha- 1 when compared with cultures from untreated soils. The extent of nitrogen fixation by the cultures observed in the present study are comparable with those of Azospirillum cultures isolated from the same soils amended with monocrotophos and quinalphos for 7 days (Rangaswamy et al., 1989), and those cultures isolated from a rice soil amended with benomyl and incubated for 30 days (Charyulu and Rao, 1978). The cultures from untreated soil, when inoculated into the medium supplemented with either cypermethrin or fenvalerate, at 50/zg ml -~, exhibited greater nitrogen-fixing activity. However, the stimulation in nitrogen fixation was more pronounced in cultures ofAzospirillum sp. isolated from insecticide-treated (5 kg ha- 1) soil and inoculated to the medium containing 50/zg ml- ~of the insecticide. A comparison of the results on ammonification,
316
v. Rangaswamy, K. Venkateswarlu/Agriculture, Ecosystems&Environment 45 (1993) 311-317
Table 3 Influence of cypermethrin and fenvalerate on nitrogen fixation (mg N g-~ malate) by Azospirillum lipoferum Soil type
Culture from untreated soil
Culture from insecticide-treated soiP
Untreated
250/tg m l - ~
Untreated
250/tg m l -
Cypermethrin Black soil 6.30" Red soil 5.89"
10.97 c 9.86 c
9.51 b 8.46 b
13.13 d 11.61 d
Fen valerale Black soil Red soil
11.96 c 10.62 ~
9.98 b 8.75 b
13.83 d 12.83 a
7.18" 6.53"
~The soil sample was treated with commercial formulation of the insecticide (5 kg h a - ~) and culture was isolated after 7 days. 2Semi-solid malate medium was supplemented with technical sample of the insecticide ( 50 #g m l medium ) before incubation with the culture. Means (n = 3), in each row, followed by different superscripts are significant ( P < 0.05) from each other according to Duncan's multiple range test.
Table 4 Impact ofsubculturing ofA. lipoferum isolated from cypermethrin- and fenvalerate-treated soil samples 1 on nitrogen fixation (mg N g - 1 malate) Soil type
Black soil Red soil
Fresh isolate from untreated soil
9.80" 7.88"
Isolate from cypermethrintreated soil
Isolate from fenvaleratetreated soil
Fresh
After third subculturing
Fresh
After third subculturing
17.62 b 15.95 b
16.99 b 15.28 b
17.33 b 16.63 c
16.86 b 16.51 c
lSoil samples were treated four times with insecticide at 5 kg h a - i level. Refer to Table 3 for other footnotes.
nitrification (Table 2) and nitrogen-fixing capacity ofAzospirillum sp. (Table 3) under the impact of selected insecticides clearly indicates that fenvalerate, at a level of 5 kg ha- l, not only enhances the rate of ammonification and nitrification but also stimulates nitrogen fixation significantly. An attempt was made to determine whether the observed nitrogenase activity would continue upon subsequent subcultures of the diazotroph. Although fresh cultures from the insecticide-treated soil exhibited greater nitrogen fixation when compared with those from untreated soils, subculturing of the isolates three times had no effect on nitrogen fixation in the cultures of Azospirillum sp., exposed to the selected pyrethroids. The present study clearly shows that soil application of cypermethrin and fenvalerate enhances the rate
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317
ofammonification and nitrification and that the increased nitrogen-fixing capacity ofAzospirillum sp., isolated from soils treated with cypermethrin and fenvalerate, lasts for longer periods. Acknowledgment V. Rangaswamy is grateful to the University Grants Commission, New Delhi, India, for financial assistance. References Barnes, H. and Folkard, A.R., 1951. The determination of nitrite. Analyst, 76: 599-603. Bartha, R., Lanzilotis, R.P. and Pramer, D., 1967. Stability and effects of some pesticides in soil. Appl. Microbiol., 15: 67-75. Bremner, J.M., 1965. Inorganic form of nitrogen. In: C.A. Black (Editor), Methods of Soil Analysis Part 2. American Society of Agronomy, Madison, WI, pp. 1179-1237. Charyulu, P.B.B.N. and Rao, V.R., 1978. Nitrogen fixation by Azospirillum sp. in benomyl amended rice soil. Curr. Sci., 47: 822-823. Das, B.B., 1988. Efficiency of some synthetic pyrethroids and conventional insecticidal spray against groundnut pest complex. Pesticides, 22: 10-12. Gaur, A.C. and Misra, K.C., 1977. Effect of simazine, lindane and ceresan on soil respiration and nitrification rates. Plant Soil, 46: 5-15. Gowda, T.K.S., Rao, V.R. and Sethunathan, N., 1977. Heterotrophic nitrification in the simulated oxidized surface of a flooded soil amended with benomyl. Soil Sci., 123:171-175. Jackson, M.L., 1971. Soil Chemical Analysis. Prentice-Hall, India, New Delhi. Megharaj, M., Venkateswarlu, K. and Rao, A.S., 1986. Growth response of four species of soil algae to monocrotophos and quinalphos. Environ. Pollut., A42:15-22. Megharaj, M., Venkateswarlu, K. and Rao, A.S., 1988. Effect of insecticides and phenolics on nitrogen fixation by Nostoc linckia. Bull. Environ. Contam. Toxicol., 41: 277-281. Ramakrishna, C. and Sethunathan, N., 1982. Stimulation of autotrophic ammonium oxidation in rice rhizosphere soil by the insecticide carbofuran. Appl. Environ. Microbiol., 44: 1-4. Ramakrishna, C. and Sethunathan, N., 1983. Inhibition ofheterotrophic and autotrophic nitrification in bacterial cultures by carbaryl and 1-naphthol. J. Appl. Bacteriol., 54:191-195. Ramakrishna, C., Gowda, T.K.S. and Sethunathan, N., 1979. Effect of benomyl and its hydrolysis products MBC and AB on nitrification in a flooded soil. Bull. Environ. Contam. Toxicol., 21: 328-334. Rangaswamy, V., Charyulu, P.B.B.N. and Venkateswarlu, K., 1989. Effects of monocrotophos and quinalphos on soil population and nitrogen-fixing activity ofAzospirillum sp. Biomed. Environ. Sci., 2:305-311. Ray, R.C., Ramakrishna, C. and Sethunathan, N., 1980. Nitrification inhibition in a flooded soil by hexachlorocyclohexane and carbofuron. Plant Soil, 56:165-168. Stratton, G.W., 1990. Effects of the insecticide endosulfan on nitrification in low pH agricultural soils. Toxicity Assessment, 5:319-336. Tu, C.M., 1970. Effect of four organophosphorus insecticides on mircobial activities in soil. Appl. Microbiol., 19: 479-484. Tu, C.M., 1973. The temperature-dependent effect of residual nematicides on the activities of soil microorganisms. Can. J. Microbiol., 19: 855-859.
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Elsevier Science Publishers B.V., Amsterdam
Ammonification and nitrification in soils, and nitrogen fixation by Azospirillum sp. as influenced by cypermethrin and fenvalerate V. Rangaswamy*, K. Venkateswarlu Department of Microbiology, Sri Krishnadevaraya University, Anantapur 515003, India (Accepted 28 December 1992)
Abstract
Mineralization of peptone-nitrogen and oxidation of ammoniacal nitrogen were significantly enhanced in two agricultural soils when treated with up to 5 kg ha- l of cypermethrin or fenvalerate. The rate of ammonification and nitrification after 2 and 4 weeks was fairly rapid in the soils receiving 2.5 kg ha-t of either insecticide. The enhancement of both transformations, mediated by microorganisms, was more pronounced in the fenvalerate-treated soils. Cultures of Azospirillum sp., isolated from insecticide-treated soils, exhibited greater nitrogen-fixing activity that lasted for at least three generations.
Introduction
The study of the effects of insecticides on non-target microbial populations and processes in soil, an ultimate sink, is an important aspect of research into the environmental impact of these xenobiotics. Microbial processes, in particular associated with the nitrogen cycle, are often chosen as test systems owing to the ecological significance of nitrogen transformations in the biosphere (Stratton, 1990). Chemical control by spraying synthetic pyrethroid insecticides such as cypermethrin and fenvalerate has been the common practice in controlling several major pests of groundnut (Arachis hypogeaeL. ) (Das, 1988 ). There is no information available on the interaction between these insecticides and microorganisms that are implicated in the transformation of nitrogen in soil. The present study was, therefore, aimed at determining the influence of cypermethrin and fenvalerate on ammonification and nitrification in soils, and nitrogen fixation by Azospirillum sp., isolated from soil. *Corresponding author.
© 1993 Elsevier Science Publishers B.V. All rights reserved 0167-8809/-93/$06.00
312
v. Rangaswamy, K. Venkateswarlu/Agriculture, Ecosystems & Environment 45 (1993) 311-317
Materials and methods
Soi/s A red laterite soil and a black vertisol, both collected from groundnut fields of the Anantapur district ofAndhra Pradesh, India, were used. The soil sampies, collected randomly from a depth of up to 12 cm from fallow fields, were air-dried and sifted through a 2 m m sieve.
Insecticides Aqueous solutions, prepared from commercial formulations of cypermethrin (Cyperkill 25 EC, Bharat Pulverising Mills, Bombay, India) and fenvalcrate (Fenkem 20 EC, Rallis India, Bangalore, India) were used in soil incubation studies. Stock solutions from technical-grade cypermethrin (a-cyano3-phenoxy phenyl-3-(2,2-dichlorovinyl)-2,2-dimethyl cyclopropane carboxylate; 92.2% pure ) and fenvalerate (cyano- ( 3-phenoxy-ct-phenyl)-methyl 4chloro-( 1-methyl ethyl ) benzene acetate; 93.7% pure ) were prepared in acetone for use in pure culture studies.
Ammonification and nitrification in soils Samples (10 g) of black soil were placed in test tubes (25-200 mm), and were treated with 0.05 ml stock solutions of cypermethrin or fenvalerate to provide final concentrations of 10, 25, 50, 75 and 100/lg g- ~soil (equivalent to 1, 2.5, 5, 7.5, and 10 kg ha -I ). Soil samples receiving 0.05 ml of acetone served as controls. After complete evaporation of the solvent at room temperature, all treatments including the controls were supplemented with either 1000 ppm organic nitrogen as analytical grade peptone, or 200 ppm ammoniacal nitrogen in the form of analytical grade ammonium sulfate. After thorough mixing, all the soil samples were maintained at 60% water-holding capacity (WHC) and incubated at room temperature (28 _+4°C). Seven days after incubation, three samples of each treatment were withdrawn to estimate ammonium by nesslerization (Jackson, 1971 ), nitrite by diazotization (Barnes and Folkard, 1951 ), and nitrate by phenol disulfonic acid method (Bremner, 1965). In another experiment, both black and red soils were treated with 2.5 kg h a - 1 of cypermethrin and fenvalerate. Soil samples with no insecticide treatment served as controls. Peptone ( 1000 ppm organic N ) or ammonium (200 ppm NH~"-N) was added to the untreated and insecticide-treated soil samples. Soil moisture was maintained at 60% WHC and incubated at room temperature. Moisture levels were restored to their initial values during incubation. Three samples of each treatment were withdrawn after l, 2, 3 and 4
V. Rangaswamy, K. Venkateswarlu/Agriculture, Ecosystems & Environment 45 (1993) 311-317
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weeks to determine the rate of ammonification, and after 2, 4, 6 and 8 weeks to determine the rate of nitrification following the methods adopted by Tu ( 1973 ). All results are expressed on the basis of oven-dried soils.
Nitrogen fixation by Azospirillum sp. Aliquots from stock solutions of the technical grade insecticides, prepared in acetone, were placed in sterilized test tubes (25-200 m m ) to provide a final concentration of 50 gg ml - ~ malate medium. The carrier solvent was evaporated, and 20 ml portions of the steam-sterilized malate medium were introduced into each test tube under aseptic conditions. The residues were equilibrated for 24 h to obtain aqueous solutions of the insecticides (Megharaj et al., 1986). Test tubes containing medium without the insecticide served as controls. Soil suspensions ( 1 : 10 soil to water ratio) from untreated and insecticide-treated ( 5 kg h a - 1 level with commercial formulations ) samples, incubated for 7 days, were prepared in sterilized distilled water. Aliquots (0.1 ml) of these suspensions were used to inoculate 20 ml portions of malate medium with and without the insecticide. After 72 h incubation at 37 °C, three tubes for each treatment were digested with HESO4to estimating total nitrogen (N) by the micro-Kjeldahl method as described earlier (Megharaj et al., 1988 ). The amount of N present in 0.1 ml soil suspensions, used for inoculation, together with that of the medium was deducted from experimental values. Cultures of Azospirillurn sp. were isolated from untreated and insecticidetreated (four times at 10-day intervals) soil samples. To determine whether the increased nitrogen-fixing capacity of Azospirillum sp. isolated from soil samples treated with insecticides would continue further, the isolates were subcultured in the semi-solid malate medium three times at an interval of 7 days, and their rates of nitrogen fixation were compared with those of fresh cultures obtained immediately after isolation from untreated and insecticidetreated soil samples. Results and discussion Application of cypermethrin and fenvalerate at levels of up to 5 kg hasignificantly increased the mineralization of peptone-N and the oxidation of ammoniacal-N (Table 1 ). In particular, there was a pronounced stimulation of ammonification and nitrification at a level of 2.5 kg ha-1, which is close to the field application rate, of the insecticides. Fenvalerate application at 7.5 kg h a - l was innocuous to both microbial transformations. However, cypermethrin at 7.5 kg h a - 1 inhibited ammonification and nitrification. The highest level ( l0 kg h a - 1) of both insecticides significantly inhibited the transformations of nitrogen. However, four organophosphorus insecticides viz., Bay 37289, diazinon, Dursban and zinophos at levels of 100 ppm were reported
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Table 1 Effect of different concentrations of insecticides on ammonification ~ after 7 days, and nitrification 2 after 2 weeks, in black soil Insecticide concentration (kg ha-1 )
Ammonification Cypermethrin
Fenvalerate
0 1.0 2.5 5.0 7.5 10.0
455.20 ~ 517.00 ¢ 543.35 b 498.65 d 397.30 ¢ 358.20 f
455.20 a 528.10 c 552.70 b 498.45 d 414.05 e 293.50 r
(100) (114) (119) (110) (87) (79)
Nitrification
(100) (116) (121) (110) (91) (65)
Cypermethrin
Fenvalerate
28.65 ¢ 41.00 b 49.63 a 41.63 b 30.75 c 25.38 d
28.65 ¢ 41.50 b 52.00 a 41.75 b 29.50 cd 23.00 d
(100) (143) (173) (145) (107) (89)
(100) (149) (182) (146) (103) (80)
1Values are #g (NH~- + NO~- + NO~- )-N g-~ soil. 2Values are #g ( N O r + NO j- ) -N g - ~soil. Figures in parentheses indicate relative production percentages. Means (n = 3), in each column, followed by the same supercripts are not significantly different ( P < 0 . 0 5 ) from each other according to Duncan's new multiple range test.
to stimulate ammonification after 1 week of incubation in a sandy loam soil (Tu, 1970). The level of 2.5 kg ha-~ was selected to determine the rate of ammonification and nitrification in both soils, because this particular concentration effected a pronounced stimulatory effect (Table 1 ). In general, mineralization of the added organic nitrogen in soils with no insecticide was significantly more rapid after 2 weeks of incubation (Table 2 ). Thus, about 50% of added organic nitrogen was mineralized during this period. An increased incubation period resulted in the formation of little available nitrogen (NH~-+ NO~- + NOj- ), presumably owing to biological immobilization. Such an observation was also made by Tu (1973) with a loamy sand. However, application of cypermethrin or fenvalerate markedly stimulated ammonification. Moreover, this mineralization process was fairly rapid in soils treated with fenvalerate. Likewise, four nematicides, such as carbofuran, Dasanit, D-D, and Vortex, either had no effect or slightly stimulated ammonification in a loamy sand incubated at 28°C (Tu, 1973 ). The rate of nitrification followed a trend similar to that of ammonification. Ammonium was oxidized fairly rapidly to nitrate in soils (Table 2). No attempt was made to distinguish the implication of either autotrophic or heterotrophic nitrifiers. There was a greater accumulation of NO~- and NO~after 4 weeks, but this was followed by a decrease with increasing period of incubation. The decrease in oxidized forms of nitrogen after 8 weeks accounted for nearly 80% of that accumulated after 4 weeks. The application of cypermethrin and fenvalerate to both soils significantly enhanced the oxidation of NH~-N throughout the incubation. Fenvalerate exhibited a striking stimulation of nitrification. Several other studies have also demonstrated an
v. Rangaswamy, K. Venkateswarlu/Agriculture, Ecosystems & Environment 45 (1993) 311-317
315
Tab le 2 I nf luence o f c y p e r m e t h r i n a n d f e n v a l e r a t e ( b o t h at 2.5 kg h a - ~) on a m m o n i f i c a t i o n a n d n i t r i f i c a t i o n in soils Treatment
Ammonification
Nitrification
Il
2
3
4
2
4
6
8
478.85 a 543.30 b 650.95 b
557.63" 628.63 b 646.63 c
500.75 ~ 514.20 b 524.70 b
389.48" 399.90" 400.10 ~
34.13 ~ 49.63 b 52.25 b
192.00 a 225.00 b 228.38 c
67.00 a 75.50 ab 81.25 b
34.88 a 35.75" 35.20 ~
400.10 a 481.70 b 531.90 c
475.93 a 546.88 b 551.80 b
466.80 a 491.15 b 481.55 b
381.05 a 389.18 ~b 399.65 b
32.50" 44.50 b 50.88 ¢
172.88" 204.75 b 215.25 b
39.85 a 43.68 b 42.48 b
30.25 a 32.25 a 32.50 ~
Black soil Control Cypermethrin Fenvalerate
R e d soil Control Cypermethrin Fenvalerate
t l n c u b a t i o n , in weeks. M e a n s (n = 3 ), in each c o l u m n for a soil type, followed by the s a m e letter are not s i gni fi c a nt l y different ( P < 0.05 ) from each o t h e r a c c o r d i n g to D u n c a n ' s new m u l t i p l e range test. Refer to Table 1 for o t h e r footnotes.
inhibition of nitrification by pesticides such as HCH (Gaur and Misra, 1977; Ray et al., 1980), carbaryl (Bartha et al., 1967; Ramakrishna and Sethunathan, 1983), and benomyl (Gowda et al., 1977; Ramakrishna et al., 1979). However, ammoniacal nitrogen incorporated in a loamy sand was oxidized significantly after 8 weeks of incubation at 28°C with carbofuran, D-D, Dasanit, and Vortex (Tu, 1973 ). Similarly, carbofuran application, at 10 and 100 ppm, to rice rhizosphere soil suspension markedly stimulated the autotrophic oxidation of ammonium (Ramakrishna and Sethunathan, 1982 ). Cultures of Azospirillum, obtained after 7 days of soil incubation, from unamended soils exhibited appreciable nitrogen-fixing activity (Table 3 ). A significant stimulation of nitrogen fixation was evident in cultures from soils treated with the two insecticides at a level of 5 kg ha- 1 when compared with cultures from untreated soils. The extent of nitrogen fixation by the cultures observed in the present study are comparable with those of Azospirillum cultures isolated from the same soils amended with monocrotophos and quinalphos for 7 days (Rangaswamy et al., 1989), and those cultures isolated from a rice soil amended with benomyl and incubated for 30 days (Charyulu and Rao, 1978). The cultures from untreated soil, when inoculated into the medium supplemented with either cypermethrin or fenvalerate, at 50/zg ml -~, exhibited greater nitrogen-fixing activity. However, the stimulation in nitrogen fixation was more pronounced in cultures ofAzospirillum sp. isolated from insecticide-treated (5 kg ha- 1) soil and inoculated to the medium containing 50/zg ml- ~of the insecticide. A comparison of the results on ammonification,
316
v. Rangaswamy, K. Venkateswarlu/Agriculture, Ecosystems&Environment 45 (1993) 311-317
Table 3 Influence of cypermethrin and fenvalerate on nitrogen fixation (mg N g-~ malate) by Azospirillum lipoferum Soil type
Culture from untreated soil
Culture from insecticide-treated soiP
Untreated
250/tg m l - ~
Untreated
250/tg m l -
Cypermethrin Black soil 6.30" Red soil 5.89"
10.97 c 9.86 c
9.51 b 8.46 b
13.13 d 11.61 d
Fen valerale Black soil Red soil
11.96 c 10.62 ~
9.98 b 8.75 b
13.83 d 12.83 a
7.18" 6.53"
~The soil sample was treated with commercial formulation of the insecticide (5 kg h a - ~) and culture was isolated after 7 days. 2Semi-solid malate medium was supplemented with technical sample of the insecticide ( 50 #g m l medium ) before incubation with the culture. Means (n = 3), in each row, followed by different superscripts are significant ( P < 0.05) from each other according to Duncan's multiple range test.
Table 4 Impact ofsubculturing ofA. lipoferum isolated from cypermethrin- and fenvalerate-treated soil samples 1 on nitrogen fixation (mg N g - 1 malate) Soil type
Black soil Red soil
Fresh isolate from untreated soil
9.80" 7.88"
Isolate from cypermethrintreated soil
Isolate from fenvaleratetreated soil
Fresh
After third subculturing
Fresh
After third subculturing
17.62 b 15.95 b
16.99 b 15.28 b
17.33 b 16.63 c
16.86 b 16.51 c
lSoil samples were treated four times with insecticide at 5 kg h a - i level. Refer to Table 3 for other footnotes.
nitrification (Table 2) and nitrogen-fixing capacity ofAzospirillum sp. (Table 3) under the impact of selected insecticides clearly indicates that fenvalerate, at a level of 5 kg ha- l, not only enhances the rate of ammonification and nitrification but also stimulates nitrogen fixation significantly. An attempt was made to determine whether the observed nitrogenase activity would continue upon subsequent subcultures of the diazotroph. Although fresh cultures from the insecticide-treated soil exhibited greater nitrogen fixation when compared with those from untreated soils, subculturing of the isolates three times had no effect on nitrogen fixation in the cultures of Azospirillum sp., exposed to the selected pyrethroids. The present study clearly shows that soil application of cypermethrin and fenvalerate enhances the rate
14.Rangaswamy, K. Venkateswarlu/Agriculture, Ecosystems & Environment 45 (1993) 311-317
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ofammonification and nitrification and that the increased nitrogen-fixing capacity ofAzospirillum sp., isolated from soils treated with cypermethrin and fenvalerate, lasts for longer periods. Acknowledgment V. Rangaswamy is grateful to the University Grants Commission, New Delhi, India, for financial assistance. References Barnes, H. and Folkard, A.R., 1951. The determination of nitrite. Analyst, 76: 599-603. Bartha, R., Lanzilotis, R.P. and Pramer, D., 1967. Stability and effects of some pesticides in soil. Appl. Microbiol., 15: 67-75. Bremner, J.M., 1965. Inorganic form of nitrogen. In: C.A. Black (Editor), Methods of Soil Analysis Part 2. American Society of Agronomy, Madison, WI, pp. 1179-1237. Charyulu, P.B.B.N. and Rao, V.R., 1978. Nitrogen fixation by Azospirillum sp. in benomyl amended rice soil. Curr. Sci., 47: 822-823. Das, B.B., 1988. Efficiency of some synthetic pyrethroids and conventional insecticidal spray against groundnut pest complex. Pesticides, 22: 10-12. Gaur, A.C. and Misra, K.C., 1977. Effect of simazine, lindane and ceresan on soil respiration and nitrification rates. Plant Soil, 46: 5-15. Gowda, T.K.S., Rao, V.R. and Sethunathan, N., 1977. Heterotrophic nitrification in the simulated oxidized surface of a flooded soil amended with benomyl. Soil Sci., 123:171-175. Jackson, M.L., 1971. Soil Chemical Analysis. Prentice-Hall, India, New Delhi. Megharaj, M., Venkateswarlu, K. and Rao, A.S., 1986. Growth response of four species of soil algae to monocrotophos and quinalphos. Environ. Pollut., A42:15-22. Megharaj, M., Venkateswarlu, K. and Rao, A.S., 1988. Effect of insecticides and phenolics on nitrogen fixation by Nostoc linckia. Bull. Environ. Contam. Toxicol., 41: 277-281. Ramakrishna, C. and Sethunathan, N., 1982. Stimulation of autotrophic ammonium oxidation in rice rhizosphere soil by the insecticide carbofuran. Appl. Environ. Microbiol., 44: 1-4. Ramakrishna, C. and Sethunathan, N., 1983. Inhibition ofheterotrophic and autotrophic nitrification in bacterial cultures by carbaryl and 1-naphthol. J. Appl. Bacteriol., 54:191-195. Ramakrishna, C., Gowda, T.K.S. and Sethunathan, N., 1979. Effect of benomyl and its hydrolysis products MBC and AB on nitrification in a flooded soil. Bull. Environ. Contam. Toxicol., 21: 328-334. Rangaswamy, V., Charyulu, P.B.B.N. and Venkateswarlu, K., 1989. Effects of monocrotophos and quinalphos on soil population and nitrogen-fixing activity ofAzospirillum sp. Biomed. Environ. Sci., 2:305-311. Ray, R.C., Ramakrishna, C. and Sethunathan, N., 1980. Nitrification inhibition in a flooded soil by hexachlorocyclohexane and carbofuron. Plant Soil, 56:165-168. Stratton, G.W., 1990. Effects of the insecticide endosulfan on nitrification in low pH agricultural soils. Toxicity Assessment, 5:319-336. Tu, C.M., 1970. Effect of four organophosphorus insecticides on mircobial activities in soil. Appl. Microbiol., 19: 479-484. Tu, C.M., 1973. The temperature-dependent effect of residual nematicides on the activities of soil microorganisms. Can. J. Microbiol., 19: 855-859.