EFFECT OF SOIL PHYSICOCHEMICAL PROPERTIES ON CHLORPYRIFOS TOLERANT BACTERIA FROM CULTIVATED SOILS

Electronic Journal of Environmental Sciences Vol. 4, 17-23 (2011) ISSN: 0973-9505 (Available online at www.tcrjournals.com) Original Article Indexed in: ProQuest database Abstract, USA ( ProQuest Science journals, Techonology Research database, Illustrata Technology, Environment Science collection and Health and Medical complete), EBSCO databases (USA), Indian Science abstract. EFFECT OF SOIL PHYSICOCHEMICAL PROPERTIES ON CHLORPYRIFOS TOLERANT BACTERIA FROM CULTIVATED SOILS SUMIT KUMAR Biotechnology Department, V. V. P. Engineering College, Saurashtra University, Rajkot 360 005. E. mail: btsumit@gmail.com Received: February 1, 2011; Accepted: February 26, 2011 Abstract: The present study investigated the effect of various soil physicochemical properties on the native chlorpyrifos-tolerant bacterial density in the cultivated soils of Rajkot district of Gujarat. Out of the four soil physical properties considered, bulk density showed negative correlation while the rest three viz. porosity, soil moisture and electrical conductivity showed positive correlation. However, except soil moisture (R 2 = 0.625), none of the soil physical properties showed significant effect on chlorpyrifostolerant bacterial density, as indicated by their lower values of R-square. Similarly, out of four soil chemical properties examined, only ph showed negative correlation and the remaining three viz. organic C, organic N and available P showed positive correlation. Except soil ph, the three other chemical properties of soil showed very significant effect on the abundance of chlorpyrifos-tolerant bacteria in the soil. The results of the present study can be utilized for the development of effective bioremediation process for chlorpyrifos-contaminated soil. Key words: Bulk density, Bacterial density, Electrical conductivity, Chlorpyrifos? INTRODUCTION In modern agriculture, pesticides are frequently used in the field to increase crop production. Besides combating insect pests, insecticides also affect the population and activity of beneficial microbial communities in soil. The effect of insecticides on soil microbial communities was variable with insecticide types, their doses and field conditions. Soil microbes had different response to types of insecticides [1]. Pesticides have made a great impact on human health, production and preservation of foods, fibre and other cash crops by controlling disease vectors and by keeping in check many species of unwanted insects and plants. More than 55% of the land used for agricultural production in developing countries uses about 26% of the total pesticides produced in the world. However the rate of increase in the use of pesticides in developing countries is considerably higher than that of the developed countries. Pesticides are necessary to protect crops and losses that may amount to about 45% of total food production world wide [2]. Chlorpyrifos [O,O-diethyl O-(3,5,6-trichloro-2- pyridyl) phosphorothioate] is used worldwide as 17

an agricultural insecticide. Its environmental fate has been studied extensively, and the reported half-life in soil varies from 10 to 120 days, with 3,5,6-trichloro-2-pyridinol (TCP) as the major degradation product. This large variation in halflife has been attributed to variation in factors such as ph, temperature, moisture content, organic carbon content, and pesticide formulation [3]. The manufacture and formulation process of chlorpyrifos generates waste that contains the compound, and this has to be treated by physicochemical and biological means [4]. The pesticides reach soil by one way or the other. Although pesticides may not be universally toxic to all species of microorganisms, they have the potential of disturbing microbial events/activities in the environment, polluted by these chemicals [5]. Pesticides contaminate soil environment resulting in alterations in the equilibrium of soil processes for shorter or longer periods. The observed changes in the soil activity depend on the intensity and spectrum of activity as well as persistence of the parent chemicals or its metabolites. Pesticides might affect microorganisms by reducing their numbers, biochemical activity, diversity and changing the microbial community structure [6]. Soil components may also influence the distribution and activity of microorganisms. In the soil, the preferential zones of microbial activity are associated with sites where fresh decomposable organic matter is released in soils, i.e. the root environment and organic fragments from litter fall or crop residues [7]. Biological activities in soils drive many of the key ecosystem processes that govern the global system, especially in the cycling of elements such as carbon, nitrogen and phosphorus. An understanding of the linkages between soil biodiversity and the processes of C and N cycling is essential given the potential impact of both natural events and human activity on soil communities [8]. Since pesticides are very toxic by design, they have the potential to adversely impact on ecosystem health. Organophosphorous (OP) insecticides such as parathion, methamidophos Electronic Journal of Environmental Sciences 18 and chlorpyrifos are a group of highly toxic agricultural chemicals widely used in plant protection. The contamination of soil by these pesticides may lead to occasional contamination of a wide range of water and terrestrial ecosystems. Extensive use of chlorpyrifos contaminates air, ground water, rivers and lakes [9]. The fate of the pesticides in the soil environment in respect of pest control efficacy; non-target organism exposure and offsite mobility has become a matter of environmental concern potentially because of the adverse effects of pesticidal chemicals on soil microorganisms may affect soil fertility [10]. The present study aimed to investigate the effect of soil physicochemical properties on the chlorpyrifos-tolerant bacterial density in the cultivated soil contaminated with chlorpyrifos. MATERIALS AND METHODS Study sites, sample collection and storage: Three different talukas (administrative blocks) viz. Rajkot, Gondal and Jetpur were selected. These study sites were selected on the criteria that they had extensive cultivation of cotton, groundnut and vegetables and the known history of repeated use of chlorpyrifos as pesticide. The soil samples were taken randomly by using a soil auger from 10-12 places from the same field and mixed thoroughly to get one composite sample. The soil samples were collected by using auger up to a depth of 15 cm. Plant material, debris and larger sized gravels were removed from the sample by hand and the soil was sieved using 2 mm mesh. A total of 30 composite samples were collected, 10 samples from each of the three taluka. The collected samples were stored in the sealed plastic bags at room temperature. These stored samples were used for further experimentation. Soil physicochemical analysis: The soil physical properties considered in this study were bulk density, porosity, soil moisture and electrical conductivity. The soil chemical properties determined were soil ph, organic carbon, organic

Sumit Kumar Fig-1(a): Bulk Density vs. y = -0.1953x + 8.9314 R 2 = 0.0756 0.84 0.88 0.92 0.96 1 1.04 1.08 Bulk Density (g/cc) Fig-1(b): Porosity vs. log (CPT bacterial density y = 0.0028x + 8.5647 R 2 = 0.2978 50 60 70 80 90 Porosity (%) nitrogen and available phosphorus. For the measurement of bulk density, an unperturbed block of soil was taken and dried at 105 o C for 24 hours. The weight of the dried sample was measured using sensitive digital balance and the soil volume was determined by measuring the cylinder volume. The weight of oven dried soil (g) divided by volume of soil core (cc) gave the value of bulk density. The particle density of soil was calculated by taking ratio of mass of dry soil to volume of air dried soil. The soil porosity was calculated from the value of bulk density and particle density, by using the following equation: Gravimetric method was used to determine the soil moisture. For measuring soil ph and electrical conductivity, 10 g soil was mixed with boiling distilled water in the ratio of 1:5. A conductivity meter equipped with HACH sensor was used to measure the electrical conductivity, and a sensitive digital ph meter was utilized for determining ph of the clear layer of the soil system. Both electrical conductivity and ph were measured three times and mean of these three readings was taken to minimize the handling error. A standard titrimetric method was used to determine organic carbon of the soil. Soil organic nitrogen was calculated by multiplying % organic carbon with 0.0862. The soil phosphorus is extracted from the soil using Bray No. 1 solution 19

Electronic Journal of Environmental Sciences Fig-1(d): E. C. vs. y = 0.1459x + 8.6471 R 2 = 0.1794 0.4 0.5 0.6 0.7 0.8 0.9 1 Electrical conductivity (ms/cm) Fig-1(c): Soil moisture vs. y = 0.0027x + 8.6341 R 2 = 0.6253 20 30 40 50 60 Soil moisture (%) and measured calorimetrically based on the reaction with ammonium molybdate and development of the Molybdenum Blue colour. The absorbance was measured at 882 nm using a spectrophotometer, which is directly proportional to the amount of phosphorus extracted from the soil [11]. Chlorpyrifos-tolerant bacterial density in soil: The chlorpyrifos-tolerant bacterial density per gram of soil, in terms of colony forming units (CFUs), was determined using viable plate count technique. One gram of soil was properly dissolved in 9 ml of sterile distilled water and diluted to 10-3 and 10-5 using the sterile distilled water. From these two dilutions, 0.1 ml portion 20 was used to spread the agar plates containing chlorpyrifos 20 mg/l. The plates were incubated at room temperature for 48 hours. Also, N-agar plates not supplemented with chlorpyrifos were used as control to determine the total bacterial count in the untreated soil. The bacterial cells were counted using viable count technique and represented in terms of CFUs. All the plating was performed in triplicates and results were represented as mean. The viable count of bacteria was obtained from this value by reference to the serial dilution used. Statistical analysis: The MS-Excel worksheet was used for classification and statistical analysis of data obtained on soil physicochemical

Sumit Kumar Fig-2(a): Soil ph vs. y = -0.075x + 9.3229 R 2 = 0.3713 7 7.5 8 8.5 Soil ph Fig-2(b): Organic C vs. log (CPT bacterial density y = 0.0238x + 8.5878 R 2 = 0.5593 4 5 6 7 8 9 10 Soil Organic C (g/kg) properties and chlorpyrifos-tolerant bacterial density. The analyzed data were used to prepare result table and graphs. The useful inferences were derived from the interpretation such result table and graphs. RESULTS AND DISCUSSION 21 Soil physicochemical properties: The soil physicochemical properties like bulk density, porosity, soil moisture, soil ph, organic carbon, organic nitrogen, available phosphorus, etc. affect the population and diversity of bacteria in the cultivated soil. Therefore, it is imperative to examine the relation between soil physicochemical properties and abundance of native pesticide-tolerant bacteria. The presence of sufficient moisture content in soil is important for physiological activities of microorganisms. Also, a certain minimum level of different nutrients in soil is essential for the presence of an active microbial population in the soil. In this work, some selected physicochemical properties of the soil were determined, for evaluating their effect on chlorpyrifos-tolerant bacterial density. The soil physicochemical properties are as given in Table 1. Chlorpyrifos-tolerant bacterial density in relation to soil physical properties: Four selected physical properties of soil viz. bulk

Electronic Journal of Environmental Sciences Fig-2(c) Organic N vs. y = 0.2712x + 8.5596 R 2 = 0.6519 0.4 0.5 0.6 0.7 0.8 0.9 1 Soil Organic N (g/kg) Fig-2(d) Available P vs. y = 0.0033x + 8.6613 R 2 = 0.7743 5 15 25 35 45 Soil Available P (mg/kg) density, porosity, soil moisture and electrical conductivity, were investigated for their impact on chlorpyrifos-tolerant bacterial density (CPT) in the cultivated soils. The bulk density of the soil was found to be negatively correlated with CPT bacterial density. However, the remaining three physical properties such as porosity, soil moisture and electrical conductivity were positively correlated with CPT bacterial density. The lower values of R-square for correlation between CPT bacterial density and bulk density (0.075), porosity (0.297) and electrical conductivity (0.179) showed that these physical properties had little impact on native CPT bacterial population (Figs. 1a,b,d). The soil moisture content with R-square value of 0.625 showed strong correlation with CPT bacterial density of soil (Fig. 1c). Therefore, the pesticide degrading native bacterial population could be increased by proper and adequate irrigation facilities to the cultivated soil. Chlorpyrifos-tolerant bacterial density in relation to soil chemical properties: Four selected chemical properties of soil viz. soil ph, organic C, organic N and available P, were investigated for their impact on chlorpyrifostolerant bacterial density (CPT) in the cultivated soils. The soil ph was found to be negatively correlated with CPT bacterial density. However, the remaining three chemical properties such as 22

Bacterial density (CFU/g soil) Total CP-tolerant Bulk Density (g/cc) Soil physical properties Porosity (%) Sumit Kumar Table 1: Chlorpyrifos-tolerant (CPT) bacteria in relation to soil physicochemical properties Soil Moisture (%) E.C. (ms/cm) Soil ph Soil chemical properties Organic C (g/kg) Organic N (g/kg) Available P (mg/kg) 2.23 x 10 10 5.89 x 10 8 0.876 77.398 39.838 0.854 7.344 6.058 0.654 16.877 2.15 x 10 10 5.55 x 10 8 0.934 64.066 52.734 0.557 7.506 8.459 0.851 34.562 2.13 x 10 10 4.82 x 10 8 0.995 57.451 23.97 0.619 7.808 6.669 0.697 36.764 2.33 x 10 10 6.06 x 10 8 1.013 61.023 50.811 0.653 8.16 5.178 0.532 13.721 organic C, organic N and available P were positively correlated with CPT bacterial density. The correlation analysis of CPT bacterial density with soil ph, organic C, organic N and available P gave R-square values of 0.371, 0.559, 0.651 and 0.774 respectively (Figs. 2a to 2d). The results showed that available P in soil had higher impact on native CPT bacterial population, followed by organic N, organic C and soil ph. The effective management of soil chemical properties by adequate soil nutrient management can have positive and favourable impact on native CPT bacterial density, which in turn, can facilitate catabolic recycling of applied chlorpyrifos in the cultivated soil. CONCLUSIONS The results of the current work showed that some of the physical and chemical properties of soil have higher impact on the abundance of chlorpyrifos tolerant/degrading native bacteria in the cultivated soil. Out of eight physicochemical properties investigated in this study, the effect of soil moisture, organic C, organic N and available P, was very significant. Therefore, adequate irrigation facilities along with effective soil nutrient management can have positive impact on CPT bacterial population in the cultivated soil. The findings of present study can be utilized for the development of effective bacterial bioremediation process for chlorpyrifoscontaminated soil. The results can be also utilized for the bioremediation of soil contaminated with other pesticides. REFERENCES [1] Ahmed, A. and Ahmad, M.S.: Pak. Entomol., 28 (2): 63-68 (2006) [2] Murugesan, A.G., Jeyasanthi, T. and Maheswari, S.: African J. Micro. Res., 4(1): 10-13 (2010) [3] Singh B.K., Walker A., Morgan J.A.W. and Wright D.J.: Appl. Env. Micro., 69(9): 5198-5206 (2003). [4] Fulekar M.H. and Geetha M.: J. Appl. Biosci., 12: 657-660 (2008). [5] Ajaz, M., Jabeen, N., Akhtar, S. and Rasool, S.A.: Pak. J. Bot., 37(2): 381-388 (2005). [6] Khaing Thida Zun, Moe Kyaw Thu, Weine Nway Nway Oo and Kyaw Nyein Aye: GMSARN Int. Conf. on sustainable devel-opment: Issues & prospects of GMS, 12-14 Nov. (2008). [7] Kabir, M., Chotte, J.L., Rahman, M., Bally, R. and Monrozier, L.J.: Plant Arid Soil, 163: 243-255 (1994). [8] Fitter, A.H., Gilligan, C.A., Hollingworth, K., Kleczkowski, A., Twyman, R.M. and Pitchford, J.W.: Functional Ecology, 19: 369-377 (2005). [9] Rani, M.S., Lakshmi, K.V., Devi, P.S., Madhuri, R.J., Aruna, S., Jyothi, K., Narasimha, G. and Venkateswarlu, K.: African J. Micro. Res., 2: 26-31 (2008) [10] Chowdhury, A., Pradhan, S., Saha, M. and Sanyal, N.: Indian J. Micro., 48: 114-127 (2008). [11] Bray, R.H. and Kurtz, L.T.: Soil Science, 59: 39-45 (1945). 23.