Soil application of insecticides influences microorganisms and plant nutrients

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1 Applied Soil Ecology 14 (2000) Soil application of insecticides influences microorganisms and plant nutrients A.C. Das, D. Mukherjee Department of Agricultural Chemistry and Soil Science, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur , India Received 9 March 1999; received in revised form 16 September 1999; accepted 20 September 1999 Abstract An experiment was conducted under laboratory conditions to investigate the effect of four insecticides, viz. HCH, phorate, carbofuran and fenvalerate at their field application rates (7.5, 1.5, 1.0 and 0.35 kg a.i. ha 1, respectively), on the growth and development of bacteria, actinomycetes and fungi as well as their role in the transformations and availability of some plant nutrients in laterite soil (Typic Orchragualf). All the insecticides in general, and HCH and phorate in particular, significantly increased the population of microorganisms in soil. The most predominant genera of microorganisms, such as Bacillus, Micrococcus and Aspergillus were not affected by most of the insecticides. However, some of the insecticides stimulated the growth and development of Bacillus, Proteus, Corynebacterium, Streptomyces, Fusarium, Trichoderma and Rhizopus. On the other hand, some insecticide exerted deleterious effect on the proportions of Pseudomonas, Staphylococcus, Nocardia, Micromonospora, Aspergillus and Rhizopus. Incorporation of insecticides also significantly stimulated the mineralization and availability of organic C, N and P in soil. Among the tested substances, the stimulations were more pronounced with HCH followed by phorate and fenvalerate Elsevier Science B.V. All rights reserved. Keywords: Insecticides; Microbial population; Plant nutrients; Laterite soil 1. Introduction Soil is the repository of all types of chemical inputs including insecticides applied to combat insect pests of crops. As organic substance of any kind cannot escape the onslaught of microbial degradation, insecticides are no exception. Insecticidal residues are generally degraded and degradation products assimilated by soil microorganisms (El-Shahaat et al., 1987; Rache and Coats, 1988; Bhuyan et al., 1993) resulting in increased population sizes and activities of microorganisms which in turn influences the trans- Corresponding author. Tel.: ; fax: address: laser@cal2.vsnl.net.in (A.C. Das). formations of plant nutrient elements in soil (Das and Mukherjee, 1994; Jana et al., 1998). On the other hand, there are some insecticides which are not utilizable by soil microorganisms and these types of insecticides are degraded in soil by microorganisms through cometabolism (Bollag and Liu, 1990). Other insecticides exert deleterious effects on microorganisms (Moorman, 1989; Martinez-Toledo et al., 1992). Therefore, no definite conclusion can be made on the effect of insecticides on microorganisms and their associated transformations of nutrients in soil, since different groups of insecticides exhibit manifold variations in toxicity (Matsumura and Boush, 1971; Simon-Sylvestre and Fournier, 1979). It is interesting to note that insecticides frequently applied in modern agriculture mostly belong to the chlorinated hydro /00/$ see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S (99)

2 56 A.C. Das, D. Mukherjee / Applied Soil Ecology 14 (2000) carbon, organophosphate, carbamate and synthetic pyrethroid groups, but their comparative residual effects on microorganisms under a particular soil conditions have rarely been reported (El-Shahaat et al., 1987; Singh and Prasad, 1991). Based on the above reports, an experiment was conducted to investigate the effects of HCH (hexachlorocyclohexane), phorate (O,O-diethyl-S-ethylthiomethyl dithiophosphate), carbofuran (2,2-dimethyl-2,3-dihydrobenzofuran-7-yl-Nmethyl carbamate) and fenvalerate [(RS)-cyano (3-phenoxyphenyl) (RS) methyl-4-chloro- (1- methylethyl) benzene acetate], representing the chlorinated hydrocarbon, organophosphate, carbamate and synthetic pyrethroid groups of insecticides, respectively, at their recommended field application rates, on the growth and distribution of representative soil microorganisms and the mineralization of C, N and P. 2. Materials and methods 2.1. Experimental methods A laterite soil (Typic Orchragualf) having the general characteristics as presented in Table 1 was collected from Midnapore district in West Bengal, India by taking several thin slices from the surface soil layer (0 10 cm) by means of a spade as outlined by Jackson (1973). The composite soils were air-dried (at C) in shade and passed through a 2 mm Table 1 General characteristics of the soil used in the experiment Soil taxonomy (USDA, 1975) Typic Orchragualf Textural class sandy clay Sand (%) 44.4 Silt (%) 15.0 Clay (%) 40.6 Density (g cm 3 ) 1.41 Water holding capacity (%) 42.2 ph H2 O (1 : 2.5) 5.4 Cation exchange capacity [cmol (p + )kg 1 ] 4.45 Electrical conductivity (dsm 1 ) 0.33 Organic C (g kg 1 ) 2.25 Total N (g kg 1 ) 0.24 Available N (mg kg 1 ) 30.5 Available P (mg kg 1 ) 1.72 Total bacteria (c.f.u g 1 ) a 29.3 Actinomycetes (c.f.u g 1 ) 15.6 Fungi (c.f.u g 1 ) 8.5 a c.f.u.: colony-forming unit. (4 8 mesh cm 1 ) sieve. The processed soils were then stored in screw-cap jars and were used for the experiment. Four insecticides, HCH (50% active ingredient (a.i.) in wettable powder formulation; Rhone Poulenc), phorate (10% a.i. in granular formulation; Pesticide India), carbofuran (3% a.i. in granular formulation; Bayer India) and fenvalerate (20% a.i. in emulsifiable concentrate formulation; Rallis India) at rates of 7.5, 1.5, 1.0 and 0.35 kg a.i. ha 1, respectively were mixed thoroughly and separately with 1 kg air-dried and sieved soil ( 2 mm) and were placed in earthenware pots. The water content of the soil was adjusted to 60% of water holding capacity and maintained throughout the experiment by the periodic addition of distilled water. To avoid photogradation of insecticides and evaporation loss of water from soil, the pots were kept covered with black polyethylene sheet and were incubated in the dark at 30 ± 1 C for 60 days. Treatments were replicated three times. During incubation, soil samples (approximately 50 g) were collected at periodic intervals from the replicated pots of each treatment for analysis of growth and distribution of microorganisms and the mineralization of nutrient elements. Soil moisture content was measured from subsamples Soil sampling and analysis Soil samples were collected from each pot by taking soil cores by pushing an open-ended glass tube (12.5 cm length, 1.8 cm inner diameter) into the soil to a depth of 10 cm. The subsamples were immediately analyzed to determine microbial populations and chemical transformations Analysis of microbial population Soil samples were analyzed immediately after collection to enumerate the colony-forming units (c.f.u.) of bacteria, actinomycetes and fungi following the serial dilution technique and pour plate method (Salle, 1973) in asparagine-mannitol agar (Thornton, 1922), dextrose-casein agar (Jensen, 1930) and rose-bengal agar (Martin, 1950) media, respectively. The agar plates were incubated at 30 C ± 1 C for 7 days. After 5, 15, 30, 45 and 60 days of incubation, the c.f.u. of bacteria, actinomycetes and fungi were counted by the method of Salle (1973).

3 Table 2 Effects of insecticides on the populations of microorganisms in soil A.C. Das, D. Mukherjee / Applied Soil Ecology 14 (2000) Treatments Sampling days Mean Bacteria (c.f.u g 1 ) Control 43.3 ± 5.2 a 72.8 ± ± ± ± HCH 63.6 ± ± ± ± ± Phorate 45.9 ± ± ± ± ± Carbofuran 52.2 ± ± ± ± ± Fenvalerate 50.4 ± ± ± ± ± Mean LSD 0.05 treatment 9.4; sampling date 9.3; interaction n.s. Actinomycetes (c.f.u g 1 ) Control 15.1 ± ± ± ± ± HCH 24.6 ± ± ± ± ± Phorate 25.2 ± ± ± ± ± Carbofuran 20.6 ± ± ± ± ± Fenvalerate 26.4 ± ± ± ± ± Mean LSD 0.05 treatment 5.6; sampling date 5.5; interaction 12.5 Fungi (c.f.u g 1 ) Control 9.8 ± ± ± ± ± HCH 14.9 ± ± ± ± ± Phorate 10.3 ± ± ± ± ± Carbofuran 10.9 ± ± ± ± ± Fenvalerate 12.4 ± ± ± ± ± Mean LSD 0.05 treatment 3.2; sampling date 3.2; interaction n.s. a Mean ± s.d.; n.s.: not significant Isolation and identification of microorganisms Individual colonies of bacteria, actinomycetes and fungi were isolated from and cultured on their respective agar media. Fourteen isolates of each type of microorganism were selected at random from the agar plates of each treatment and the isolates were transferred to agar slants containing the same medium. After purification, the isolates were identified to generic level following the guide of Skerman (1967) as modified by Bowie et al. (1969) for bacteria, the key of Skerman (1975) for actinomycetes and procedure of Gilman (1957) for fungi Chemical analysis Soil samples were also analyzed for changes in organic C, total N and available N (NH NO 3 ) following the methods outlined by Jackson (1973). The water soluble P in soil was extracted in sodium bicarbonate and quantified using a colorimetric procedure (Olsen and Dean, 1982) Data analysis The results were evaluated by analysis of variance (ANOVA) and the statistical significance (P = 0.05) of difference between means within factors (insecticides and incubation time) was evaluated using Fisher s protected LSD method (Petersen, 1994). 3. Results and discussion 3.1. Effect on microbial population Application of insecticides in general significantly increased the population of bacteria, actinomycetes and fungi in soil (Table 2) with HCH having the most pronounced effect followed by phorate and fenvalerate. These findings support earlier workers (Rache and Coats, 1988; Das et al., 1995) who reported that numbers of soil microoganisms were stimulated through the utilization of chlorinated hydrocarbon, organophosphate and synthetic pyrethroid insecti-

4 58 A.C. Das, D. Mukherjee / Applied Soil Ecology 14 (2000) Table 3 Effects of insecticides on the occurrence of predominant genera of microorganisms in soil No. of isolates a Microorganisms Treatments Control HCH Phorate Carbofuran Fenvalerate Bacteria Bacillus sp Micrococcus sp Pseudomonas sp Staphylococcus sp Corynebacterium sp Proteus sp Azotobacter sp Diplococcus sp Lactobacillus sp Erysipelothrix sp Actinomycetes Streptomyces sp Nocardia sp Micromonospora sp Fungi Aspergillus sp Penicillium sp Fusarium sp Trichoderma sp Rhizopus sp Helminthosporium sp a For each group of microorganisms, 14 isolates were identified at each sampling. Therefore, during five sampling periods the total isolates under any treatment was 14 5 = 70. cides. Population sizes of bacteria were greatest 30 days following application of all four insecticides. Stimulation of actinomycete and fungal populations was less pronounced, with maximum increases occurring between 15 and 60 days for different insecticides. This indicated greater utilization of insecticidal residues as well as their degraded products (Bhuyan et al., 1993) by the soil microorganisms to derive energy and other nutrient elements for their cellular metabolism (El-Shahaat et al., 1987). Another source of nutrients to the microorganisms was that application of insecticides had an effect on a certain part of the microbial, and perhaps also the soil animal, biomass of the soil resulting in death of that sensitive part and consequently released nutrients. Microorganisms not so sensitive to the insecticides utilized the released nutrients and increased in their numbers. It was also revealed that among the microorganisms, actinomycetes and fungi were highly responsive to the application of HCH (60.4 and 60.3%) and phorate (44.5 and 46.6%) and this trend was also followed for actinomycetes with carbofuran (41.2%) and fenvalerate (48.4%) as compared to that of untreated control soil Effect on the distribution of microorganisms Most of the insecticides did not have marked effects on the distribution of Bacillus and Micrococcus, the most dominant bacteria present in soil (Table 3). This trend was also recorded with HCH and carbofuran in regard to the proportions of Pseudomonas and Staphylococcus, respectively, while the distribution of the latter genera was retarded with phorate and fenvalerate and that of Pseudomonas with carbofuran and Staphylococcus with HCH. On the other hand, Bacillus with fenvalerate, Corynebacterium with carbofuran and Proteus with phorate were stimulated. This corroborates the findings of earlier workers (Turco and

5 A.C. Das, D. Mukherjee / Applied Soil Ecology 14 (2000) Konopka, 1990; Sakata et al., 1992) who pointed out that different bacteria utilized the degraded products of the insecticides to derive energy, carbon and other nutrients for their growth and development resulting in an increase in their populations in soil. Sustaining the earlier report (Das et al., 1995), Streptomyces followed by Nocardia and Micromonospora were the predominant genera of actinomycetes isolated from soil (Table 3). The results of the present experiment clearly indicated that the proportions of Streptomyces were highly increased due to the incorporation of insecticides while those of Nocardia and Micromonospora were reduced. This indicated that Streptomyces was the most active actinomycete to utilize the insecticidal residues in soil. Aspergillus followed by Penicillium and Fusarium were the most dominant fungi in soil treated with different insecticides (Table 3). This was also in accordance with the report of Srimathi et al. (1986). Application of HCH, carbofuran and fenvalerate did not have marked effects on the distribution of Aspergillus while the proportion of Fusarium was highly increased by these insecticides and more so with HCH and fenvalerate, indicating that Fusarium utilized chlorinated hydrocarbon (HCH) and synthetic pyrethroid (fenvalerate) insecticides more effectively for their growth and metabolism (Levavon, 1993). Similar response was also shown by Trichoderma to phorate and carbofuran, and by Rhizopus to phorate. On the other hand, no insecticide was beneficial to Penicillium in soil. This was also true for HCH and fenvalerate in the case of Rhizopus and for phorate in the case of Aspergillus and Fusarium. From the results (Table 3), it was also revealed that most of the predominant genera of microorganisms remained unchanged due to the application of insecticides while some secondary genera were more sensitive to the insecticides and were repressed in soil. The nutrients released due to the death of the secondary genera as well as the degraded products of the insecticides were utilized by the predominant and occasional genera of microorganisms resulting in an increase in their proportions in soil Effect on organic C and total N The mineralization rate of organic C in soils treated with different insecticides was higher compared with that of the control (Table 4). This indicated that insecticides stimulated the growth and activities of heterotrophic microorganisms (Murthy et al., 1991) which in turn, mineralized the soil organic matter to derive energy and carbon for their cellular constituents (Debnath et al., 1994) resulting in lower retention of organic C in soil. Among the insecticides, the increase in mineralization of organic C was highest under HCH (18.8%) followed by phorate (15.2%) and fenvalerate (13.5%), reflecting trends in the populations of microorganisms discussed earlier (Table 2). Similar trends were recorded for total N content (Table 4), reflecting denitrification following enhanced microbial mineralization of organic N in soil. The stimulating influence of the insecticides on microbial mineralization of organic N (Singh and Prasad, 1991; Rangaswamy and Venkateswarlu, 1993) brought about a significant reduction of total N content which was more pronounced with HCH (22.7%) followed by phorate (19%) and fenvalerate (18.1%). Similar results were also demonstrated by Gaikawad et al. (1973) with chlorinated hydrocarbon and organophosphate insecticides Effect on available N and P The greater mineralization of N following the application of insecticides resulted in an increase in the amounts of mineralized N (NH 4 + and NO 3 ) in soil (Fig. 1). The increase was most pronounced with HCH and phorate. This indicated that insecticides in general, and HCH and phorate in particular, stimulated the growth and activities of ammonifying and nitrifying bacteria which were mainly responsible for the mineralization of organic N to NH 4 + and oxidation of NH 4 + to NO 3, respectively (Rangaswamy and Venkateswarlu, 1993). As ammonification was followed by nitrification, the greater accumulation of available N in soils treated with HCH and phorate during the early days of incubation indicated that the process of ammonification was much faster than that of nitrification in these treated soils. Moreover, the insecticides indirectly stimulated ammonification by killing a part of the biomass in the soil and thus gave an increase in available nitrogen 5 days after the start of the experiment. The available nitrogen was then immolilized in the new microbial biomass created by those microorganisms not sensitive to the insecticides and

6 60 A.C. Das, D. Mukherjee / Applied Soil Ecology 14 (2000) Table 4 Effects of insecticides on organic C and total N in soil Treatments Sampling days Mean Amount of organic C (g kg 1 ) Control 2.25 ± 0.02 a 2.23 ± ± ± ± HCH 2.23 ± ± ± ± ± Phorate 2.23 ± ± ± ± ± Carbofuran 2.21 ± ± ± ± ± Fenvalerate 2.22 ± ± ± ± ± Mean LSD 0.05 treatment 0.06; sampling date 0.05; interaction n.s. Amount of total N (g kg 1 ) Control 0.24 ± ± ± ± ± HCH 0.22 ± ± ± ± ± Phorate 0.21 ± ± ± ± ± Carbofuran 0.23 ± ± ± ± ± Fenvalerate 0.22 ± ± ± ± ± Mean LSD 0.05 treatment 0.02; sampling date n.s.; interaction n.s. a Mean ± s.d.; n.s.: not significant. Fig. 1. Effect of insecticides on the availability of N in soil. The treatments include control ( ), HCH ( ), phorate ( ), carbofuran, ( ) and fenvalerate ( ). Bars represent standard deviations.

7 A.C. Das, D. Mukherjee / Applied Soil Ecology 14 (2000) Fig. 2. Effect of insecticides on the availability of P in soil. For explanations, see Fig. 1. therefore able to utilize the released nutrients for their growth and development. Similar observations were also recorded by earlier workers (Das and Mukherjee, 1994; Jana et al., 1998) with different types of insecticides in soil. Incorporation of insecticides also brought about a significant rise in the availability of soluble P in soil (Fig. 2) suggesting that insecticides significantly increased the activities of phosphate mineralizing/solubilizing microorganisms (Arora and Gaur, 1979; Das and Mukherjee, 1994). The availability of soluble P was greatly increased on the 5th day with carbofuran and fenvalerate and up to the 60th day with HCH and phorate, manifesting greater utilization of chlorinated hydrocarbon and organophosphate insecticides as well as their degraded products by the phosphate solubilizing/mineralizing microorganisms for their growth and metabolism (Ogunseitan and Odeymi, 1985). 4. Conclusions The results of the present investigation clearly indicate that application of different insecticides, HCH and phorate in particular, at their recommended field rates, killed part of the soil microbial, perhaps also soil animal, biomass resulting in an increase of available N and P in soil. Some microorganisms, such as bacteria, actinomycetes and fungi not sensitive to the insecticides tested were able to utilize the nutrients with a resultant increase in their populations in soil. This also indicated that the tested insecticides at their recommended field rates did not have any deleterious effect on the growth and activities of the major soil microorganisms responsible for the transformations of C, N and P and thus the insecticides can be safely used to combat the insect pests for better crop production. References Arora, D., Gaur, A.C., Microbial solubilization of different inorganic phosphates. Ind. J. Exptl. Biol. 17, Bhuyan, S., Sreedharan, B., Adhya, T.K., Sethunathan, N., Enhanced biodegradation of -hexachlorocyclohexane ( -HCH) in HCH (commerial) acclimatized flooded soil: factors affecting its development and persistence. Pestic. Sci. 38, Bollag, J.M., Liu, S.Y., Biological transformation processes of pesticides. In: Cheng, H.H. (Ed.), Pesticide in the

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