Research Journal of Agriculture and Forestry Sciences ______________________________ ____ ISSN 2320 - 6 063 Vol. 1 ( 5 ), 13 - 1 7 , June (201 3 ) Res. J. Agriculture and Forestry Sci. International Science Congress Association 13 Optimization of soil parameters for Benzyl Benzoate degradation by Pseudomonas desmolyticum NCIM 2112 Rokade Kedar B. 1* and Mali Gajanan V. 2 1 JJT University, Vidyanagari, Jhunjhunu, Rajasthan, 333001, INDIA 2 Bharati Vidyapeeth’s, MBSK Kanya Mahavidyalaya, Kadegaon, Dist. Sangli - 415304, MS, INDIA Available online at: www.isca.in Received 5 th April 201 3 , revised 16 th April 201 3 , accepted 21 st May 201 3 Abstract Additives are the chemical compounds generally used in the i nsecticides formulations. Benzyl benzoate is one of such additive generally used in the formulation of neonicotinoid insecticides. Similar to insecticide, additives are also responsi ble for the potential toxicity in soil environment. The present research w ork describes Optimization of soil parameters for benzyl benzoate degradation by Pseudomonas desmolyticum NCIM 2112. Soil degradation study shows the complete degradation of benzyl benzoate in soil with increase in number of soil microflora and CO 2 evoluti on rate. Due to biodegradation of benzyl benzoate by Pseudomonas desmolyticum NCIM 2112 increase in soil fertility was observed . Keywords: Additive, benzyl benzoate, soil, biodegradation, p seudomonas . Introduction Insecticides are widely used in most a reas of the crop production to minimize infestations caused by insects to crops 1 . Use of insecticides constitutes an important aspect of modern agriculture, as they are routinely used by farmers for pest management 2 . Additives are generally used in insecti cide formulation and are responsible for toxicity to the insects 3 . Benzyl benzoate is one of such additive mainly used to eliminate lice and mites 4 . Benzyl benzoate is generally used in many products and formulations therefore it is of major environmental concern 5 . To avoid the loss in agriculture, it is imperative to use insecticides with its additives in the agriculture field to such an extent that will not arise any other ecological factors. But the overexposure of insecticide in the agriculture field results into bioaccumulation and biomagnification of these compounds in the environment 6 . Addition of insecticide affects the microbial components of an ecological niche and therefore effect is observed on biotransformation reaction occurring in soil 7 . In case of soil, smaller particles i.e. clay is responsible for adsorption of insecticide additives 8 . Immobilization of insecticidal residues takes place by the clay particles and if the overexposure of insecticide residues takes place it shows adverse effec t on the growth of microorganisms 9 . Due to adsorption of insecticide additives to clay particles decrease in number of soil microorganisms has been reported and concluded that it results into slower decomposition of organic matter in soil 10 . Humic acid is one of the most active ingredients of soil organic matter. Presence of insecticide residues reduces the mobility of humic acid in agriculture soil 11,12 . Persistence of the particular insecticide additive in the soil depends upon the microbial systems pres ent in that soil. In such cases, where innate microbial population of the soil is unable to degrade insecticides, the external addition of insecticide degrading microflora is recommended 13 . In such case, biodegradation of organophosphorus insecticide chlo rpyriphos by means of Pseudomonas desmolyticum NCIM 2112 has been reported and concluded that by means of external addition of such insecticide degrading bacteria it is possible to reduce the residual toxicity of insecticide in soil 14 . Contaminated land due to repeated use of insecticide with its additives is a potential threat to human health and its continual use leads to adverse effect on environment. This investigation deals with Optimization of soil parameters for benzyl benzoate degradation by Pseud omonas desmolyticum NCIM 2112. Methodology Insecticide additive : Benzyl benzoate of 99.99% purity was purchased from Loba Chemicals, Mumbai, India. Growth of Bacteria: Growth of P.desmolyticum NCIM 2112 was monitored in mineral based medium 14 , having com position as, 0.3% NaNO 3 , 0.1% K 2 HPO 4 , 0.05% MgSO 4 , 0.05% KCl, 0.0001% FeSO 4 , 0.05% yeast extract, glucose 1.0 % and pH 7.00 at 28 0 C under aerobic conditions on rotary shaker at 120 rpm. Toxicity of benzyl benzoate on different soil parameters: Soil sampl ing and preparation: 100g (dry weight equivalent) of soil sample having no known insecticide exposure was sieved through a mesh with 2mm diameter pores. The clay content was determined 15 . As soil energy transfer processes takes place in presence of moistur e its maintenance during the experiment was done by adding five milliliters of distilled water in the soil. Research Journal of Agriculture and Forestry Sciences ___ ______________________________ __________ ____ ISSN 2320 - 6063 Vol. 1 ( 5 ), 13 - 1 7 , June (201 3 ) Res. J. Agriculture and Forestry Sci. International Science Congress Association 14 Benzyl benzoate at 10μg.g - 1 of concentration was added in soil and mixed well. Microbial inoculum of P.desmolyticum NCIM 2112 at 3.5 x 10 5 cfu /ml cell density was used throughout the experiment to study the biodegradation of benzyl benzoate in soil. A control set of fertile soil containing neither benzyl benzoate nor P.desmolyticum NCIM 2112 was run parallel to the experiment. Toxicity on organi c carbon and organic matter content of soil : The toxicity of benzyl benzoate on organic carbon and organic matter content of soil was assessed by preparing two sets as - one containing the benzyl benzoate but without P.desmolyticum NCIM 2112 while another set contains benzyl benzoate and P.desmolyticum NCIM 2112 . The soils samples were analyzed after every 5 days interval up to 15 days 16 . Toxicity on microbial flora of soil : The toxicity of benzyl benzoate on soil microorganisms was studied by treating t he soil with benzyl benzoate and compared with the soil having P.desmolyticum NCIM 2112 inoculum. Effect of degradation on soil microbial flora was studied at every 5 days interval up to 15 days, for both soil samples. Toxicity on humic acid and trace element content of soil : Initial humic acid content and trace elements of soil was determined as per the methods described previously 17 . Toxicity of benzyl benzoate on different trace elements mainly Cu ++ , Fe ++ , Mn ++ , Zn ++ at 10μg.g - 1 of concentration alo ne and in presence of P.desmolyticum NCIM 2112 was studied. Detection of trace elements was done after 5, 10 and 15 days of incubation with P.desmolyticum NCIM 2112 by using atomic absorption spectrometer (Perkin Elmer A analyst 300). Soil respiration stu dy: Amount of CO 2 evolved from soil in presence of benzyl benzoate as well as in presence of both benzyl benzoate and P.desmolyticum NCIM 2112 inoculum was determined as per the method described previously 18 . In this experiment, 5 ml of benzyl benzoate so lution having 10 ppm concentration was mixed with 100 gms of sieved soil. This mixture was taken in 250ml flask. To correlate the degradation of benzyl benzoate with soil respiration, 10 ml cell suspension of Pseudomonas desmolyticum NCIM 2112 with 3.5 x 1 0 5 cfu /ml cell density was added at the bottom of flask. A control set containing only 100 gms of sieved soil was also run parallel to it. To trap the CO 2 evolved due to microbial activities during incubation, 30ml of 1M NaOH solution was taken in small g lass beaker and it was placed inside the flask. The flask were then sealed and incubated at room temperature (27±1 0 C). During incubation, the NaOH was renewed at every 2, 5 and 10 days of interval. The trapped CO2 was titrated with standard 0.1N HCl and pH was measured by pH meter (PICO, Lab India). The amount of CO 2 was expressed as μg of CO 2 - C evolved g - 1 of soil. Statistical analysis : All the experiments were carried out in triplicate. Analysis of the variants was carried out on all data at P0.05 using Graph Pad software. (Graph Pad Instat version 3.00, Grap h Pad software, San Diego, CA, USA). Results and Discussion Effect of benzyl benzoate on various soil parameters: Effect on Organic carbon and Organic matter content of soil: Effects of benzyl benzoate on soil parameters before and after degradation are summarized in table - 1. It indicates that soil containing benzyl benzoate shows constant percentage of organic carbon as well as organic matter. On the contrary in presence of P.desmolyticum NCIM 2112 the organic carbon and organic matter content decreas es along with the biodegradation of benzyl benzoate. Effect on microbial flora of soil: The effect of benzyl benzoate on soil microorganisms indicates that 10μg.g - 1 of benzyl benzoate concentration is toxic for the growth of soil microorganism whereas i n the presence of P.desmolyticum NCIM 2112, the number of soil microflora was found to be increased, indicating degradation of benzyl benzoate in soil. Table - 1 Effect on Organic carbon and Organic matter content of soil Day Sample Clay (%) pH Temperatu re ( 0 C) Standard plate count (SPC) cfu/ml Organic carbon (%) Organic matter (%) - Agricultural Fertile soil (Control) 37.4±0.115 7±0.333 30±0.333 180±0.577 2.27±0.008 1.32 ±0.011 5 WB 37.4±0.115 7±0.333 30±0.333 180±0.577 2.27±0.008 1.32±0.011 - WBI 37.4±0.115 7±0.333 30±0.333 190±1 2.15±0.008 1.25±0.011 10 WB 37.4±0.115 7±0.333 30±0.333 167±0.666 2.27±0.008 1.32±0.011 - WBI 37.4±0.115 7±0.333 30±0.333 240±0.333 1.70±0.008 0.99±0.005 15 WB 37.4±0.115 7±0.333 28±0.333 67±0.333 2.15±0.008 1.32±0. 020 - WBI 37.4±0.115 7±0.333 30±0.333 300±0.333 1.24±0.008 0.72±0.020 - Values are mean of ±SEM of three experiments, WB - With benzyl benzoate, WBI - With benzyl benzoate and P.desmolyticum NCIM 2112 inocula. Research Journal of Agriculture and Forestry Sciences ___ ______________________________ __________ ____ ISSN 2320 - 6063 Vol. 1 ( 5 ), 13 - 1 7 , June (201 3 ) Res. J. Agriculture and Forestry Sci. International Science Congress Association 15 As shown in table - 2, presence of P.desmolyti cum NCIM 2112 in soil results into biodegradation of benzyl benzoate which reduces the soil toxicity level and thus simultaneous effect was observed on increase in trace element content of soil. Thus, correlation is observed in between organic matter degra dation and increase in trace element content of soil. The ecotoxicity of insecticide additive with reference to organic matter, organic carbon and phytotoxicity has been reported recently and found that these additives are responsible for loss in soil fert ility 19 . Soil respiration study: The effect of benzyl benzoate on organic matter content of soil in absence as well as in presence of the P.desmolyticum NCIM 2112 was determined in terms of CO 2 - C evolved in µg.g - 1 of soil after 2, 5 and 10 days of incu bation. The amount of CO 2 evolved was increased in presence of P.desmolyticum NCIM 2112 indicating the degradation of organic matter along with benzyl benzoate. Presence of benzyl benzoate in soil shows inhibitory effect on soil environment on the contrar y in presence of P.desmolyticum NCIM 2112, stimulatory effects on soil microbial respiration was observed as shown in (figure - 1). Degradation of the insecticide along with its additive by the microorganisms not only depends upon the enzyme systems but a lso on the different environmental conditions such as, temperature, pH, water potentials and available nutrients 20 . Some of the insecticides are readily degraded by the microbes however; some are recalcitrant in nature 21 . It was previously reported that pl ants can fulfilled the phosphorus requirement because of the soil microorganisms having capability of insecticide degradation 22 - 24 . Presence of insecticides along with its additives affects this mineralization process which ultimately results in to inhibi tion of nitrification or N 2 fixation in soil 25 . It has been previously reported that it is possible to degrade such hazardous insecticide additives by microbes which have genetic diversity and metabolic versatility 26 . Biodegradation of organophosphorus in secticide additive tallowamine ethoxylate has been reported recently and it was concluded that such additives shows soil toxicity as well as phytotoxicity 27 . Presence of xenobiotic compound such as insecticides, dyes in the environment are always responsib le for ecotoxicity, degradation of such compound was previously reported and concluded that by means of bacterial degradation, toxicity of such compounds can be lowered 28 . Use of fungi in the biodegradation of recalcitrant compounds from the environment ha s also been reported and concluded that fungi plays important role in the biodegradation of xenobiotic compounds 29, 30 . Table - 2 Effect on Humic acid and trace element content of soil Day Sample Clay (%) Humic acid (465nm) Leachability (ppm) Cu ++ Fe ++ Mn ++ Zn ++ - Agricultural soil (Control) 38.4±0.115 0.15±0.005 0.56±0.003 3.1±0.066 1.64±0.003 1.21±0.003 5 WB 38.4±0.115 0.15±0.005 0.45±0.003 2.1±0.066 1.54±0.003 1.15±0.003 - WBI 39.4±0.115 0.15±0.005 0.52±0.003 4.1±0.066 1.84±0.003 1.31±0.003 10 WB 38.4±0.115 0.15±0.005 0.35±0.003 1.97±0.066 1.24±0.003 1.05±0.003 - WBI 38.4±0.115 0.19±0.005 0.54±0.003 3.27±0.003 1.95±0.003 1.63±0.003 15 WB 38.4±0.115 0.14±0.005 0.10±0.003 0.97±0.066 1.00±0.003 1.00±0.003 - WBI 38.4±0.115 0.20±0.003 0.56±0.003 3 .96±0.003 2.16±0.003 1.92±0.003 - Values are mean of ±SEM of three experiments, WB - With benzyl benzoate, WBI - With benzyl benzoate and microbial inocula, Cu - Copper, Fe - Ferrous, Mn - Manganese, Zn - Zinc . Figure - 1 Soil respiration study in t erms of CO 2 - C evolution in µg / gm of soil - Control, with benzyl benzoate, with benzyl benzoate and microbial inocula. Results Obtained are mean values ± SD, n = 5. Research Journal of Agriculture and Forestry Sciences ___ ______________________________ __________ ____ ISSN 2320 - 6063 Vol. 1 ( 5 ), 13 - 1 7 , June (201 3 ) Res. J. Agriculture and Forestry Sci. International Science Congress Association 16 Conclusion Soil parameters influence the degradation of inse cticide additives in soil. By means of external addition of microorganisms like P.desmolyticum NCIM 2112 biodegradation of benzyl benzoate in soil is possible. Significant correlation is observed in between presence of benzyl benzoate with organic matter c ontent of soil. By means of biodegradation, toxicity of benzyl benzoate in soil can be lowered. Similarly, the soil respiration study shows that the organic matter decomposition rate is enhanced with more CO 2 evolution due to degradation of benzyl benzoate by P. desmolyticum NCIM 2112. To avoid the problem concerned with bioaccumulation and biomagnfication of insecticide residues through soil by plant uptake we recommend the use of P. desmolyticum NCIM 2112 for the remediation of insecticide contaminated la nd. Acknowledgement The authors are very grateful to the Principal, Bharati Vidyapeeth’s MBSK Kanya Mahavidyalaya, Kadegaon, Dist. Sangli (M.S.) for providing the laboratory facilities to complete the investigation. References 1. Damalas C.A., Understandi ng benefits and risks of pesticide use, Sci Res and Essay. , 4(10), 945 - 949 (2009) 2. Gouma S., Biodegradation of mixtures of pesticides by bacteria and white rot fungi , Ph.d. Thesis, School of Health Cranfield University, 416 (2009) 3. Ravier I., Haouisee E., C lement M., Seux R. and Briand O., Field experiments for the evaluation of pesticide spray - drift on arable crops, Pest Manag Sci., 61(8) , 728 - 36 (2005) 4. Olsen O.T., Jacobsen L. and Svendsen U.G., Effectiveness of benzyl benzoate in elimination of house dust, Aerobiol., doi : 10.1007/BF02248117 (1996) 5. Tovey E. and Marks G., Methods and effectiveness of environmental control, J Aller Clini Immunol., 103 , 179 - 191 (1999) 6. Singh J., and Singh D.K., Dehydrogenase and phosphomonoesterase activities in groundnut ( Arac his hypogaea L.) field after diazinon, imidacloprid and lindane treatments, Chemosphere, 60 , 32 – 42 (2005) 7. Bhardwaj V. and Garg N., Importance of Exploration of Microbial Biodiversity, I. Res. J. Biological Sci., 1(3), 78 - 83 (2012) 8. Frederick T., Soils and Soil Fertility, 6 th Edition, John Wiley & Sons, ISBN 978 - 81 - 265 - 1787 - 9 (2008) 9. Bashan Y., Inoculants of plant growth promoting bacteria for use in agriculture, Biotech adv., 16(4) , 729 - 770 (1998) 10. Omar S.A., Availability of phosphorus and sulfur of insectici de origin by fungi, Biodegrad., 9 , 327 - 336 (1998) 11. Senesi N., Binding mechanisms of pesticides to soil humic substances, Sci Total Environ., 123/124, 63 – 76 (1992) 12. Wang Q. and Lemley A.T., Kinetic effect of humic acid on alachlor degradation by anodic Fento n treatment, J. Environ Qual., 33, 2343 – 2352 (2004) 13. Singh D.K., Biodegradation and bioremediation of pesticide in soil: concept, method and recent developments, Indian J of Microbiol. , 48 , 35 - 40 (2008) 14. Rokade K.B., and Mali G.V., Biodegradation of chlorpyr ifos by Pseudomonas desmolyticum NCIM 2112 , Int J Pharm Bio Sci., 4(2) , 609 – 616 (2013) 15. Day P.R., Particle fractionation and particle - size analysis, Methods of soil analysis, Part 1: American Society of Agronomy, Inc., Madison, Wisconsin, in Black, C.A, (ed s), 545 - 567 (1965) 16. Walkely A., and Black I,A., An examination method for determining soil organic matter and a proposed modification of the chromic acid titration method, Soil Sci., 37 , 29 - 38 (1934) 17. Faithfull N.T., Methods in agricultural chemical analysis : a practical handbook, 1 st edn. CABI Publishing, UK, 68 - 70 (2002) 18. Latif M.A., Razzaque M.A., Rehman M.M., Impact of some selected insecticide application on soil microbial respiration, Pakistan J Biol Sci., 11(16) , 2018 - 2022 (2008) 19. Ghosh J.S., and Rokade K.B., Biodegradation of 2 - mercaptobenzothiazolyl - (Z) - (2 - aminothiazol - 4 - yl) - 2 - (tertbutoxycarbonyl) isopropoxyiminoacetate by Pseudomonasdesmolyticum NCIM 2112, Appl Microbiol Biotechnol., 93 , 753 – 761 (2012) 20. Karpouzas D.G. and Walker A., Factors influencin g the ability of Pseudomonas putida epI to degrade ethoprophos in soil, Soil Bio and Biochem., 32 , 1753 - 1762 (2002) 21. Richins D., Kaneva I., Mulchandani A. and Chen W., Biodegradation of organophosphorus pesticides by surface expressed organophophorous hydro lase, Nature Biotechnol , 15, 984 - 987 (1997) 22. Subramanian G., Sekhar S. and Sampoornam S., Biodegradation and utilization of organophosphorus pesticides by cyanobacteria, Int Biodeter and Biodegrad. , 33 , 129 - 143 (1994) 23. Omar S.A., Effect of some pesticides on soil - borne fungi and some soil microbial biochemical processes with special reference to some processes involved in the nitrogen cycle. Ph. D. Botany Dept, University of Assiut, Egypt (1991) 24. Abd - Alla M.H., Use of organic phosphorus by Rhizobium leguminosa rum biovar viceae phosphatase, Biol Fertil Soils., 18, 216 – 218 (1994) Research Journal of Agriculture and Forestry Sciences ___ ______________________________ __________ ____ ISSN 2320 - 6063 Vol. 1 ( 5 ), 13 - 1 7 , June (201 3 ) Res. J. Agriculture and Forestry Sci. International Science Congress Association 17 25. Sardar D., and Kole R.K., Metabolism of chlorpyrifos in relation to its effect on the availability of some plant nutrients in soil, Chemosphere , 46 , 506 - 510 (2005) 26. Siddavattam D., Khajam ohiddin S., Manavathi B., Pakala S.B., and Merrick M., Transposon – like organization of the plasmid - borne organophosphate degradation (opd) gene cluster found in Flavobacterium sp., Appl and Environ Microbiol. , 69 , 2533 - 2539 (2003) 27. Rokade K.B., and Mali G .V., Biodegradation of tallowamine ethoxylate by Pseudomonas desmolyticum NCIM 2112, I. Res. J. Biological Sci., 1(4), 1 - 8 (2012) 28. Praveen Kumar G.N. and Bhat Sumangala K., Decolorization of Azo dye Red 3BN by Bacteria, I. Res. J. Biological Sci., 1(5) , 46 - 52 (2012) 29. Praveen Kumar G.N. and Bhat Sumangala K., Fungal Degradation of Azo dye - Red 3BN and Optimization of Physico - Chemical Parameters, I. Res. J. Biological Sci., 1(2) , 17 - 24, (2012) 30. Namdhari B.S., Rohilla S.K., Salar R.K., Gahlawat S.K., Bansal P., a nd Saran A.K., Decolorization of Reactive Blue MR, using Aspergillus species Isolated from Textile Waste Water, I. Res. J. Biological Sci., 1(1) , 24 - 29, (2012)