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Pesticides and heterotrophic nitrogen fixation in paddy soils
Soil Biol. Biochem. Vol. 12, pp. 1 to 4 © Pergamon Press Ltd 1980. Printed in Great Britain
0038-0717/80/0101-000!$02.00/0
PESTICIDES AND HETEROTROPHIC NITROGEN FIXATION IN PADDY SOILS D. N. NAYAKand V. RAJARAMAMOHANRAO Laboratory of Soil Microbiology, Central Rice Research Institute, Cuttack 753006, India
(Accepted 7 September 1979)
Samnmry--The effect of three pesticides, benomyl, carbofuran and gamma-BHC at 5 parts 106, rates equivalent to recommended field levels, on the heterotrophic N2 fixation in five air-dried, celluloseamended, submerged, tropical soils was investigated employing 15N tracer technique under laboratory conditions. Addition of benomyl, a carbamate fungicide, to alluvial, laterite and two acid sulphate soils resulted in significant increases in N2 fixation, while carbofuran, a methylcarbamate insecticide, exerted a stimulatory effect on N2 fixation in alluvial, laterite and acid saline soils. Gamma-BHC, a chlorinated hydrocarbon insecticide, stimulated N2 fixation in alluvial and acid sulphate pokkali soils, while considerable inhibition of N2 fixation was evident in other soils. Results showed differential responses of specific groups of N2-fixing organisms to the pesticides depending on the soil type.
INTRODUCTION
MATERIALS AND METHODS
Soils
The universal application of pesticides to agricultural crops often exposes the environment to pollution hazards. Although certain pesticides are not directly applied to the soil, it is the eventual sink in one way or another. There has been little progress in understanding the influences involved in soil-pesticidemicroflora relationships (Johnen and Drew, 1977). More so, the benefit of pesticides as pest combatants could be nullified by detrimental effects on microbial processes having a major influence on plant growth, crop productivity and eventually the soil fertility. Raghu and MacRae (1967a) observed marked stimulation of growth of the indigenous algae upon the application of gamma-BHC. Changes in the activities of other soil microorganisms were also observed (Raghu and MacRae, 1967b). The effect of several pesticides on the growth and N 2 fixation was extensively studied in pure culture systems (Venkataraman and Rajyalakshmi, 1972; MacKenzie and MacRae, 1972; Wood and MacRae, 1974; Charyulu and Rao, 1978). Several investigations have been made on pesticide-nitrification and pesticide-nitrogen transformations in the soil (Raghu and MacRae, 1967b; Alexander, 1969; Vlassak and Livens, 1975; Vlassak, 1975; Gowda et al., 1977; Ramakrishna et al., 1978). Investigations concerning the effects of pesticides o n N 2 fixation in submerged soils are less numerous. The influence of pesticides on soil microorganisms and microbial transformations in soil are dependent on physical, chemical and biochemical conditions in the soil in addition to the nature and concentration of the pesticide in the ecosystem. We determined the influence of three commonly-used pesticides on heterotrophic N2 fixation in five cellulose-amended submerged paddy soils differing widely in their properties. S.B.H. 12/I --a
Alluvial (pH 6.2, organic matter 1.6~/~ total N 0.09~o, electrical conductivity 0.6 mmhos.cm- 1), laterite (pH 5.0, organic matter 3.25~, electrical conductivity 0.2 mmhos, crn- L, total N 0.09~), acid sulphate saline soils from Kerala, South India, locally known as pokkali (pH 4.2, organic matter 8.21~, total N0.24~, electrical conductivity 8.5mmhos.cm-X), karl (pH 3.2, organic matter 27.82~o, total N0.36~, electrical conductivity 15.0 mmhos-cm-X) and karapadam (pH 5.0, organic matter 4.3~o, total N 0.24~0 electrical conductivity 6.3 mmhos.cm-~) were used. Pesticides
The pesticides used consisted of two insecticides carbofuran, Furadan ® (2,3-dihydro-2,2-dimethyl-7benzofuranyl methylcarbamate) and gamma-BHC lindane, (gamma- 1,2,3,4,5,6-hexachlorocyclohexan¢) and a fungicide, benomyl, Benlate ® (methyl-l-(butylcarbamoyl)-2-benzimidazole carbamate). Soil treatment
The soils were air-dried, screened (<2mm), and placed in glass vials (1.2 × 5 em) in 5-g amounts. All the soils were amended with 0.5~o finely powdered cellulose and thoroughly mixed with the soil before flooding. Technical grade pesticide formulations were applied to the soils at 5 parts/106 in acetone, the residual acetone was allowed to evaporate for 12h before flooding. Control treatments received only acetone. N2-fixing populations
The populations of different groups of N2-fixing microorganisms in these soils were counted by conventional serial dilution techniques on N-free media. 1
2
D.N. NAYAKand V. RAJARAMAMOHANRAO
Azospirillum was counted following the method suggested by Okon et al. (1977). Populations of anaerobic N2-fixers and Azotobaeter as per Rao et al. (1973). Results presented are the means of five replicates for Azospirillum populations and three replicates for Azotobacter and anaerobic N2 fixers.
[15N] studies The influence of pesticides on N2 fixation in submerged soils was investigated employing I~N 2 isotope method. Soil samples amended with 0.5~ cellulose and different pesticide formulations at 5parts/106 were transferred to a desiccator wrapped with black paper to prevent algal growth. Excess CO2 produced during the incubation of the soils was absorbed by a 40~o KOH solution placed in the desiccator at the beginning of the experiment. The desiccator was then sealed and the air was evacuated by vacuum and flushed three times with argon. An atmosphere, containing 72.5 atom~o N-15 (Prochem, London), of 02 (0.20atm), Ar (0.50atm), and N2 (0.30atm 14Nz + 1~N2) was maintained during incubation of soil. Fresh portions of 02 were added (2 times a week) to compensate for the consumed 02. After 28 days incubation at 28°C in the dark, the total N in the soil samples (triplicate) was determined by the Kjeldahl method and the distillates were used for the determination of '~N enrichment of the soils. The isotopic ratio analysis was carried out at the Seibersdorf Laboratory of the International Atomic Energy Agency, Vienna, Austria. RESULTS AND DISCUSSION Isotopic analysis of the soil N indicated significant N2 fixation even in the absence of organic amendment in all the five soil types (Table 1). Acid sulphate soils exhibited appreciable N2-fixing activity despite high acidity and salinity. Addition of cellulose stimulated N2 fixation in alluvial, laterite and acid saline karapadam soils, while marked suppression of N2 fixation was evident in acid sulphate pokkali and kari soils (compare Table 1 with no pesticide treatment in Table 2). Significant gains in N2 fixation following the addition of C substrates were observed in several paddy soils (MacRae and Castro, 1967; Rao, 1976, 1978). The influence of two carbamates and a chlorinated hydrocarbon was investigated on N2 fixation in five cellulose-amended submerged paddy soils. The
Table 1. Indigenous N 2 fixation in submerged rice soils Atom~o excess ~SN at the end of the incubation +_ SD
Soil Alluvial Laterite Acid sulphate (pokkali) Acid sulphate (kari) Acid saline (karapadam)
0.123 + 0.021 0.180 ___0.012 0.065 + 0.015 0.010 + 0.002 0.031 + 0,006
influence of pesticides on N2 fixation varied with soils. Benomyl, a carbamate fungicide, exhibited a marked stimulation on N 2 fixation in four out of five soils. Striking stimulation of the population and N 2 fixation by a N2-fixing Azospirillum sp. was observed in a benomyl-amended paddy soil (Charyulu and Rao, 1978). Since benomyl keeps the submerged soil at relatively high potentials for prolonged periods (Pal et al., 1979), provision of favourable redox potential by benomyl application might have favoured the N2 fixation in these cellulose-amended paddy soils. Carbofuran, a methylcarbamate insecticide, significantly enhanced N2 fixation in an alluvial soil, while stimulation of a lesser degree was observed in a laterite soil. Interestingly, carbofuran completely suppressed N2 fixation in acid sulphate pokkali and kari soils, whereas slight increase in the N2 fixation was observed in acid saline karapadam soil (Table 2). Venkateswarlu et al. (1977) investigated the persistence of carbofuran in these soils and established that carbofuran persisted in these soils in the following order: kari > pokkali > laterite > alluvial. They attributed this to the remarkable differences in the pH attained by these soils following flooding. Kari and pokkali soils are acidic at the time of flooding; but, the pH of pokkali soil increases and reaches near neutrality within 15 days after flooding, whereas kari soil is characterized by highly acidic conditions with a pH rise from 3.0 to 4.2 even 40 days after flooding. Presumably, extreme acid conditions in kari soil seem to be unfavourable for the degradation of carbofuran. Also, under continued anaerobiosis of undisturbed flooded soils, carbofuran-phenol, the hydrolysis product, accumulated (Venkateswarlu and Sethunathan, 1978). The greater persistence of carbofuran in acid soils and unfavourable pH and soil conditions might have resulted in the complete suppression of N2 fixation in the present study. Evidently, in soils where rapid degradation of carbofuran occurred
Table 2. Effect of pesticides on heterotrophic N 2 fixation in submerged paddy soils Atom~,o excess a5N at the end of incubation + standard deviation* Acid sulphate saline Treatment No pesticide + Carbofuran + Benomyl + Gamma-BHC
Alluvial 0.277 __+0.168(4.7) 0.786 + 0.067(13.4) 0.542 +___0.113(9.2) 0.754 + 0.071 (12.8)
Laterite
Pokkali
Kari
0.236 + 0.077(5.7) 0.005 + 0(0.2) 0.012 + 0.002(1.5) 0.296 __+0.063(7.2) 0(0) 0 (0) 0.427 __+0.044(10.3) 0.017 __+0.001 (0.6) 0.046 _+ 0.006 (5.6) 0.161 + 0.022(4.0) 0.033 __+0.005(1.2) 0.013 + 0.003 (1.6)
* LSD between treatments at P 1 and 50/0is 0.063 and 0.050 respectively. Figures in the parentheses indicate N2 fixed rag. kg-~ soil. Pesticides were applied at 5 pg. g- ~ soil.
Acid saline Karapadam 0.055 + 0.010(1.6) 0.067 + 0.006 (2.Q) 0.028 + 0.004 (0.8) 0.046 _+0.005(1.3)
Pesticides and N 2 fixation Table 3. N2-fixing populations in pesticide-amended submerged soils Azospirillum sp.
Soil Alluvial Laterite Karapadam (acid saline)
Azotobacter
Anaerobic N 2 fixers
No No No pesticide Carbofuran Benomyl pesticide Carbofuran Benomyl pesticide Carbofuran Benomyl (106 g- 1 dry soil) (103 g- 1 dry soil) (106 g- 1 dry soil) 5.4 48 3.2
26 0.48 1.6
34 32.0 9.0
1.3 0.4 1.0
1.5 1.7 0.8
1.3 0.2 0.6
4.8 2.2 18.4
18.4 22 5.6
34 0.26 7.0
Soils were amended with 0.5~o cellulose and pesticides were applied at 5 ,ug.g-1
greater N 2 fixation was noticed. Vlassak and Livens (1975) observed depression of nitrification by carbamate compounds in soil systems and indicated that the influence depends upon the soil properties. Our results further indicate that carbamate insecticides exhibit both stimulatory and inhibitory influences on N 2 fixation depending upon soil type. Gamma-BHC, a chlorinated-hydrocarbon insecticide, stimulated N2 fixation in an alluvial and acid sulphate pokkali soil (Table 2). In other soils this insecticide inhibited N2 fixation considerably. Application of gamma-BHC resulted in significant increase in N 2 fixation in two Philippine soils (Raghu and MacRae, 1976b). They ascribed this stimulation to N 2 fixation by algae and heterotrophic NE-fixing bacteria. G a m m a - B H C application to the pure culture of Azotobacter vinelandii had no significant effect upon growth or acetylene reduction (MacKenzie and MacRae, 1972). However, in the present study there was no apparent growth of algae and the incubation was carried out under protected light conditions t o eliminate the algae. Microbiological analysis showed that the effects of pesticides on the population of N2-fixing organisms were related to the pesticide used, soil type and specific groups of N 2 fixers (Table 3). Thus, Azospirillum was stimulated by benomyl in alluvial, !aterite, and karapadam soils, and by carbofuran only in alluvial soil. With respect to anaerobic N 2 fixers benomyl was stimulatory only in alluvial soil, and carbofuran in alluvial and laterite soils. Azotobacter was not markedly influenced by either pesticide irrespective of the soil used. Clearly, these results demonstrate differential effects of pesticides on specific groups of N2 fixers accounting for differences in N2 fixation in flooded soils.
CHARYULU P. B. B. N. and RAO V. R. (1978) Nitrogen fixation by ,4zospirillum sp. isolated from benomylamended rice soil. Current Science 47, 822-823. GOWDA T. K. S., RAO V. R. and SETHUNATHAN N. (1977)
Heterotrophic nitrification in simulated oxidized zone of a flooded soil amended with benomyl. Soil Science 123, 171-175. JOHNEN B. G. and DREW E. A. (1977) Ecological effects of pesticides on soil microorganisms. Soil Science 123, 319-324. MACKENZIE K. A. and MACRAE I. C. (1972) Tolerance of
the nitrogen-fixing system of Azotobacter vinelandii for four commonly used pesticides. Antonie can Leeuwenhoek 38, 529-535. MACRAE I. C. and CASTROT. F. (1967) Nitrogen fixation in some tropical rice soils. Soil Science 103, 277-280. OKON Y., ALBRECHT S. L. and BURRIS R. H. (1977) Methods for growing Spirillum lipoferum and for counting it in pure culture in association with plants. Applied and Encironmental Microbiology 33, 85-88. PAL S. S., SUDHAKAR-BARIKand SETHUNATHANN. (1979)
Effect of benomyl on iron reduction and redox potential in flooded soil. Journal of Soil Science 30, 155-158. RAGHU K. and MACRAE I. C. (1967a) The effect of the gamma isomer of benzene hexachloride upon the microflora of submerged rice soils--I. Effect upon algae. Canadian Journal of Microbiology 13, 173-180. RAGHU K. and MACRAE I. C. (1967b) The effect of the gamma isomer of benzene hexachloride upon the microflora of submerged rice soils--II. Effect upon nitrogen mineralization and fixation and selected bacteria. Canadian Journal of Microbiology 13, 622-627. RAMAgRISHNAC., RAO V. R. and SETHUNATHANN. (1978) Nitrification in simulated oxidized surface layer of a flooded soil amended with carbofuran. Soil Biology & Biochemistry 10, 555-556. RAg V. R., KALININSKAYAZ. A. and MILLER U. M. (1973) The activity of non-symbiotic nitrogen fixation in soils of rice fields studied with ~N. Microbiologiya 42, 729-734. RAO V. R. (1976) Nitrogen fixation as influenced by moisture content, ammonium sulphate and organic sources in a paddy soil. Soil Biology & Biochemistry 8, 445-448. RAO V. R. (1978) Effect of carbon sources on asymbiotic nitrogen fixation in a paddy soil. Soil Biology & BiDchemistry 10, 319 321,
Acknowledgements--We thank Dr H. K. Pande, Director, for his keen interest and encouragement. This study was supported by the International Atomic Energy Agency, Vienna, Austria, and the Department of Atomic Energy, Government of India. The senior author acknowledges financial support from University Grants Commission.
VENKATARAMANG. S. and RAJYALAKSHMIB. 0972) Relative tolerance of nitrogen-fixing blue-green algae to pes-
REFERENCES
VENKATESWARLU,K. and SETHUNATHAN N. (1978) Degradation of carbofuran in rice soils as influenced by
ALEXANDERM. (1969) Microbial degradation and biological effects of pesticides in soil. In Soil Biology, Reviews of Research, Natural Resources Research, UNESCO 9, 209 240.
ticides. Indian Journal of Agricultural Sciences 42, 119-121. repeated applications and exposure to aerobic conditions following anaerobiosis. Journal of Agricultural and Food Chemistry 26, 1148 1151. VENKATESWARLU K., GOWDA T. K. S. and SETHUNATHAN
N. (1977) Persistence and biodegradation of carbofuran
4
D.N. NAYAK and V. RAJARAMAMOHANRAO
in flooded soils. Journal of Agricultural and Food Chemistry. 25, 533-536. VLASSAK K. (1975) Effects of some pesticides on biological nitrogen fixation and on mineralization of nitrogen in soil. Roczniki Gteboznawcze 25, 191-199. VLASSAK K. and LIVENS J. (1975) Effect of some pesticides
on nitrogen transformations in soil. The Science of the Total Environment 3, 363-372. WOOD P. A. and MAcRAE I. C. (1974) The effect of several organophosphorus insecticides upon the acetylene reduction activity of Azotobacter vinelandii. Bulletin of Em~ironmental Contamination and Toxicology 12, 26--31.
0038-0717/80/0101-000!$02.00/0
PESTICIDES AND HETEROTROPHIC NITROGEN FIXATION IN PADDY SOILS D. N. NAYAKand V. RAJARAMAMOHANRAO Laboratory of Soil Microbiology, Central Rice Research Institute, Cuttack 753006, India
(Accepted 7 September 1979)
Samnmry--The effect of three pesticides, benomyl, carbofuran and gamma-BHC at 5 parts 106, rates equivalent to recommended field levels, on the heterotrophic N2 fixation in five air-dried, celluloseamended, submerged, tropical soils was investigated employing 15N tracer technique under laboratory conditions. Addition of benomyl, a carbamate fungicide, to alluvial, laterite and two acid sulphate soils resulted in significant increases in N2 fixation, while carbofuran, a methylcarbamate insecticide, exerted a stimulatory effect on N2 fixation in alluvial, laterite and acid saline soils. Gamma-BHC, a chlorinated hydrocarbon insecticide, stimulated N2 fixation in alluvial and acid sulphate pokkali soils, while considerable inhibition of N2 fixation was evident in other soils. Results showed differential responses of specific groups of N2-fixing organisms to the pesticides depending on the soil type.
INTRODUCTION
MATERIALS AND METHODS
Soils
The universal application of pesticides to agricultural crops often exposes the environment to pollution hazards. Although certain pesticides are not directly applied to the soil, it is the eventual sink in one way or another. There has been little progress in understanding the influences involved in soil-pesticidemicroflora relationships (Johnen and Drew, 1977). More so, the benefit of pesticides as pest combatants could be nullified by detrimental effects on microbial processes having a major influence on plant growth, crop productivity and eventually the soil fertility. Raghu and MacRae (1967a) observed marked stimulation of growth of the indigenous algae upon the application of gamma-BHC. Changes in the activities of other soil microorganisms were also observed (Raghu and MacRae, 1967b). The effect of several pesticides on the growth and N 2 fixation was extensively studied in pure culture systems (Venkataraman and Rajyalakshmi, 1972; MacKenzie and MacRae, 1972; Wood and MacRae, 1974; Charyulu and Rao, 1978). Several investigations have been made on pesticide-nitrification and pesticide-nitrogen transformations in the soil (Raghu and MacRae, 1967b; Alexander, 1969; Vlassak and Livens, 1975; Vlassak, 1975; Gowda et al., 1977; Ramakrishna et al., 1978). Investigations concerning the effects of pesticides o n N 2 fixation in submerged soils are less numerous. The influence of pesticides on soil microorganisms and microbial transformations in soil are dependent on physical, chemical and biochemical conditions in the soil in addition to the nature and concentration of the pesticide in the ecosystem. We determined the influence of three commonly-used pesticides on heterotrophic N2 fixation in five cellulose-amended submerged paddy soils differing widely in their properties. S.B.H. 12/I --a
Alluvial (pH 6.2, organic matter 1.6~/~ total N 0.09~o, electrical conductivity 0.6 mmhos.cm- 1), laterite (pH 5.0, organic matter 3.25~, electrical conductivity 0.2 mmhos, crn- L, total N 0.09~), acid sulphate saline soils from Kerala, South India, locally known as pokkali (pH 4.2, organic matter 8.21~, total N0.24~, electrical conductivity 8.5mmhos.cm-X), karl (pH 3.2, organic matter 27.82~o, total N0.36~, electrical conductivity 15.0 mmhos-cm-X) and karapadam (pH 5.0, organic matter 4.3~o, total N 0.24~0 electrical conductivity 6.3 mmhos.cm-~) were used. Pesticides
The pesticides used consisted of two insecticides carbofuran, Furadan ® (2,3-dihydro-2,2-dimethyl-7benzofuranyl methylcarbamate) and gamma-BHC lindane, (gamma- 1,2,3,4,5,6-hexachlorocyclohexan¢) and a fungicide, benomyl, Benlate ® (methyl-l-(butylcarbamoyl)-2-benzimidazole carbamate). Soil treatment
The soils were air-dried, screened (<2mm), and placed in glass vials (1.2 × 5 em) in 5-g amounts. All the soils were amended with 0.5~o finely powdered cellulose and thoroughly mixed with the soil before flooding. Technical grade pesticide formulations were applied to the soils at 5 parts/106 in acetone, the residual acetone was allowed to evaporate for 12h before flooding. Control treatments received only acetone. N2-fixing populations
The populations of different groups of N2-fixing microorganisms in these soils were counted by conventional serial dilution techniques on N-free media. 1
2
D.N. NAYAKand V. RAJARAMAMOHANRAO
Azospirillum was counted following the method suggested by Okon et al. (1977). Populations of anaerobic N2-fixers and Azotobaeter as per Rao et al. (1973). Results presented are the means of five replicates for Azospirillum populations and three replicates for Azotobacter and anaerobic N2 fixers.
[15N] studies The influence of pesticides on N2 fixation in submerged soils was investigated employing I~N 2 isotope method. Soil samples amended with 0.5~ cellulose and different pesticide formulations at 5parts/106 were transferred to a desiccator wrapped with black paper to prevent algal growth. Excess CO2 produced during the incubation of the soils was absorbed by a 40~o KOH solution placed in the desiccator at the beginning of the experiment. The desiccator was then sealed and the air was evacuated by vacuum and flushed three times with argon. An atmosphere, containing 72.5 atom~o N-15 (Prochem, London), of 02 (0.20atm), Ar (0.50atm), and N2 (0.30atm 14Nz + 1~N2) was maintained during incubation of soil. Fresh portions of 02 were added (2 times a week) to compensate for the consumed 02. After 28 days incubation at 28°C in the dark, the total N in the soil samples (triplicate) was determined by the Kjeldahl method and the distillates were used for the determination of '~N enrichment of the soils. The isotopic ratio analysis was carried out at the Seibersdorf Laboratory of the International Atomic Energy Agency, Vienna, Austria. RESULTS AND DISCUSSION Isotopic analysis of the soil N indicated significant N2 fixation even in the absence of organic amendment in all the five soil types (Table 1). Acid sulphate soils exhibited appreciable N2-fixing activity despite high acidity and salinity. Addition of cellulose stimulated N2 fixation in alluvial, laterite and acid saline karapadam soils, while marked suppression of N2 fixation was evident in acid sulphate pokkali and kari soils (compare Table 1 with no pesticide treatment in Table 2). Significant gains in N2 fixation following the addition of C substrates were observed in several paddy soils (MacRae and Castro, 1967; Rao, 1976, 1978). The influence of two carbamates and a chlorinated hydrocarbon was investigated on N2 fixation in five cellulose-amended submerged paddy soils. The
Table 1. Indigenous N 2 fixation in submerged rice soils Atom~o excess ~SN at the end of the incubation +_ SD
Soil Alluvial Laterite Acid sulphate (pokkali) Acid sulphate (kari) Acid saline (karapadam)
0.123 + 0.021 0.180 ___0.012 0.065 + 0.015 0.010 + 0.002 0.031 + 0,006
influence of pesticides on N2 fixation varied with soils. Benomyl, a carbamate fungicide, exhibited a marked stimulation on N 2 fixation in four out of five soils. Striking stimulation of the population and N 2 fixation by a N2-fixing Azospirillum sp. was observed in a benomyl-amended paddy soil (Charyulu and Rao, 1978). Since benomyl keeps the submerged soil at relatively high potentials for prolonged periods (Pal et al., 1979), provision of favourable redox potential by benomyl application might have favoured the N2 fixation in these cellulose-amended paddy soils. Carbofuran, a methylcarbamate insecticide, significantly enhanced N2 fixation in an alluvial soil, while stimulation of a lesser degree was observed in a laterite soil. Interestingly, carbofuran completely suppressed N2 fixation in acid sulphate pokkali and kari soils, whereas slight increase in the N2 fixation was observed in acid saline karapadam soil (Table 2). Venkateswarlu et al. (1977) investigated the persistence of carbofuran in these soils and established that carbofuran persisted in these soils in the following order: kari > pokkali > laterite > alluvial. They attributed this to the remarkable differences in the pH attained by these soils following flooding. Kari and pokkali soils are acidic at the time of flooding; but, the pH of pokkali soil increases and reaches near neutrality within 15 days after flooding, whereas kari soil is characterized by highly acidic conditions with a pH rise from 3.0 to 4.2 even 40 days after flooding. Presumably, extreme acid conditions in kari soil seem to be unfavourable for the degradation of carbofuran. Also, under continued anaerobiosis of undisturbed flooded soils, carbofuran-phenol, the hydrolysis product, accumulated (Venkateswarlu and Sethunathan, 1978). The greater persistence of carbofuran in acid soils and unfavourable pH and soil conditions might have resulted in the complete suppression of N2 fixation in the present study. Evidently, in soils where rapid degradation of carbofuran occurred
Table 2. Effect of pesticides on heterotrophic N 2 fixation in submerged paddy soils Atom~,o excess a5N at the end of incubation + standard deviation* Acid sulphate saline Treatment No pesticide + Carbofuran + Benomyl + Gamma-BHC
Alluvial 0.277 __+0.168(4.7) 0.786 + 0.067(13.4) 0.542 +___0.113(9.2) 0.754 + 0.071 (12.8)
Laterite
Pokkali
Kari
0.236 + 0.077(5.7) 0.005 + 0(0.2) 0.012 + 0.002(1.5) 0.296 __+0.063(7.2) 0(0) 0 (0) 0.427 __+0.044(10.3) 0.017 __+0.001 (0.6) 0.046 _+ 0.006 (5.6) 0.161 + 0.022(4.0) 0.033 __+0.005(1.2) 0.013 + 0.003 (1.6)
* LSD between treatments at P 1 and 50/0is 0.063 and 0.050 respectively. Figures in the parentheses indicate N2 fixed rag. kg-~ soil. Pesticides were applied at 5 pg. g- ~ soil.
Acid saline Karapadam 0.055 + 0.010(1.6) 0.067 + 0.006 (2.Q) 0.028 + 0.004 (0.8) 0.046 _+0.005(1.3)
Pesticides and N 2 fixation Table 3. N2-fixing populations in pesticide-amended submerged soils Azospirillum sp.
Soil Alluvial Laterite Karapadam (acid saline)
Azotobacter
Anaerobic N 2 fixers
No No No pesticide Carbofuran Benomyl pesticide Carbofuran Benomyl pesticide Carbofuran Benomyl (106 g- 1 dry soil) (103 g- 1 dry soil) (106 g- 1 dry soil) 5.4 48 3.2
26 0.48 1.6
34 32.0 9.0
1.3 0.4 1.0
1.5 1.7 0.8
1.3 0.2 0.6
4.8 2.2 18.4
18.4 22 5.6
34 0.26 7.0
Soils were amended with 0.5~o cellulose and pesticides were applied at 5 ,ug.g-1
greater N 2 fixation was noticed. Vlassak and Livens (1975) observed depression of nitrification by carbamate compounds in soil systems and indicated that the influence depends upon the soil properties. Our results further indicate that carbamate insecticides exhibit both stimulatory and inhibitory influences on N 2 fixation depending upon soil type. Gamma-BHC, a chlorinated-hydrocarbon insecticide, stimulated N2 fixation in an alluvial and acid sulphate pokkali soil (Table 2). In other soils this insecticide inhibited N2 fixation considerably. Application of gamma-BHC resulted in significant increase in N 2 fixation in two Philippine soils (Raghu and MacRae, 1976b). They ascribed this stimulation to N 2 fixation by algae and heterotrophic NE-fixing bacteria. G a m m a - B H C application to the pure culture of Azotobacter vinelandii had no significant effect upon growth or acetylene reduction (MacKenzie and MacRae, 1972). However, in the present study there was no apparent growth of algae and the incubation was carried out under protected light conditions t o eliminate the algae. Microbiological analysis showed that the effects of pesticides on the population of N2-fixing organisms were related to the pesticide used, soil type and specific groups of N 2 fixers (Table 3). Thus, Azospirillum was stimulated by benomyl in alluvial, !aterite, and karapadam soils, and by carbofuran only in alluvial soil. With respect to anaerobic N 2 fixers benomyl was stimulatory only in alluvial soil, and carbofuran in alluvial and laterite soils. Azotobacter was not markedly influenced by either pesticide irrespective of the soil used. Clearly, these results demonstrate differential effects of pesticides on specific groups of N2 fixers accounting for differences in N2 fixation in flooded soils.
CHARYULU P. B. B. N. and RAO V. R. (1978) Nitrogen fixation by ,4zospirillum sp. isolated from benomylamended rice soil. Current Science 47, 822-823. GOWDA T. K. S., RAO V. R. and SETHUNATHAN N. (1977)
Heterotrophic nitrification in simulated oxidized zone of a flooded soil amended with benomyl. Soil Science 123, 171-175. JOHNEN B. G. and DREW E. A. (1977) Ecological effects of pesticides on soil microorganisms. Soil Science 123, 319-324. MACKENZIE K. A. and MACRAE I. C. (1972) Tolerance of
the nitrogen-fixing system of Azotobacter vinelandii for four commonly used pesticides. Antonie can Leeuwenhoek 38, 529-535. MACRAE I. C. and CASTROT. F. (1967) Nitrogen fixation in some tropical rice soils. Soil Science 103, 277-280. OKON Y., ALBRECHT S. L. and BURRIS R. H. (1977) Methods for growing Spirillum lipoferum and for counting it in pure culture in association with plants. Applied and Encironmental Microbiology 33, 85-88. PAL S. S., SUDHAKAR-BARIKand SETHUNATHANN. (1979)
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Acknowledgements--We thank Dr H. K. Pande, Director, for his keen interest and encouragement. This study was supported by the International Atomic Energy Agency, Vienna, Austria, and the Department of Atomic Energy, Government of India. The senior author acknowledges financial support from University Grants Commission.
VENKATARAMANG. S. and RAJYALAKSHMIB. 0972) Relative tolerance of nitrogen-fixing blue-green algae to pes-
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