Malathion degradation by soil isolated bacteria and detection of ...

INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 3, No 5, 2013 © Copyright by the authors - Licensee IPA- Under Creative Commons license 3.0 Research article

ISSN 0976 – 4402

Malathion degradation by soil isolated bacteria and detection of degradation products by GC-MS Tamer M.A. Thabit1,2, Medhat A.H. EL-Naggar2 1- Central Agric. Pesticides Lab. (CAPL), Agric. Research Center, Giza, Egypt 2- Research Central Lab., Grain Silos and Flour Mills Org. (GSFMO), Riyadh, KSA [email protected] doi:10.6088/ijes.2013030500017 ABSTRACT Malathion in-vitro biodegradation study was conducted in liquid medium with five bacterial strains labeled S1, S2, S3, S4 and S5 isolated from newly reclaimed agricultural soil. Malathion residues were measured at successive intervals until 30 days after incubation, paralleled with control samples. Malathion Recovery rate was performed at 0.1 and one mg kg-1, achieved values were 91.30 and 98.70%, respectively, limit of detection (LOD) was 0.03 mg kg-1 while limit of quantification (LOQ) was 0.1 mg kg-1. Malathion half-life values (RL50) were 16.68, 20.27, 21.33, 12.72 and 12.49 days for S1, S2, S3, S4 and S5, respectively and control value was 27.50 days. No significant effect on malathion occurred with S2 (Bacillus amyloliquefaciens) and S3 (Staphylococcus sciuri) treatments. S1 (Pseudomonas aeruginosa), S4 (Bacillus pseudomycoides) and S5 (Bacillus licheniformis) treatments showed significant effect that increased malathion degradation rate compared to control treatment. Two main degradation products resulted from bacterial degradation, namely malathion monocarboxylic (MMA) and malathion dicarboxylic acid (MDA), the first one may convert to the latest one over time. Some other degradation products may occur such as ethyl hydrogen fumarate (EHF) but in negligible amount. Keywords: Malathion, biodegradation, bacteria, agricultural soil, degradation products. 1. Introduction Hundreds of pesticides in different chemical moieties are widely used for agricultural purpose, terrestrial ecosystems, water and soil receive large amounts of it even from handling, direct application or else which lead to occasional contamination besides accumulation lead to many health hazards associated with it (Singh et al. 2004). Hence, the degradation process of pesticides in ecosystems universally takes a large space of interest. Recently, use of microbes for effective detoxifying, degrading and removal of toxic compounds from contaminated soil and water has emerged as an efficient technique to clean up polluted environments (Strong and Burgess 2008). From the great microbial population existing in soil, some types show the capability to degrade some types of pesticides thru specific paths, such using it as a nutrients or as carbon and energy sources due to the chemical nature (Aislabie and Lloyd-Jones 1995). One such common pesticides group is organophosphates, some bacteria strains have the ability to convert OP pesticides into sulfons or oxons or some other degradation products (Hill 2003). One of the most used in this group is malathion [S-1,2-bis(ethoxycarbonyl)ethyl O,O-dimethyl phosphorodithioate], it is cholinesterase inhibitor, acts as non-systemic insecticide and acaricide with contact, stomach, and respiratory action, used to control many insect pests in a wide range of crops (Tomlin 2006). An in vitro biodegradation study has conducted to examine the capability of some bacteria strains existing in the newly reclaimed

Received on December 2012 Published on April 2013

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Malathion degradation by soil isolated bacteria and detection of degradation products by GC-MS

agricultural soil to degrade malathion. Bacteria strains have isolated from soil after screening, five common strains have selected and identified, inoculated in a pure liquid culture media fortified with a known amount of malathion, then incubated. Samples have taken in intervals of time until 30 days in parallel with control sample. Malathion degradation products have monitored to define the main degradation products resulted from bacterial effect. 2. Material and methods 2.1 Soil samples collection and preparation Sandy loam Soil samples, which used for screening and isolation of bacteria strains, taken from the surface layer soil (0-10 cm), from five different areas at newly reclaimed lands, Nubariya area, Egypt, during the growing season of 2010. Samples were placed into Sterilin autoclaved polyethylene bags, then air dried for 3-5 day at 18 °C and sieved through 2 mm sieve to be representative and homogeneous then kept at 4 °C until use. 2.2 Bacteria isolation and identification Soil dilution technique was used to isolate microorganisms. Prepared soil samples (10 g), in three replicates were shaken in 90 ml of 0.01% agar in sterile water for 10 min, then left standing for further 20 min. Dilution series was made up to 106, then Aliquots (0.5 ml) were spread on Czapek-dox agar medium (CZA, 45.4 g l-1 purified water), Biolog Inc., California, USA. Plates were amended with Triton X-100 (2 ml l-1) as a spreading agent and incubated at 33±2 °C for 2 weeks (Jones and Stewart 1997). Bacteria isolation was carried out using nutrient agar (Charlau chemicals, Spain) as a selective medium. After dilution of soil samples (10-2-10-12), selective agar media was inoculated and incubated at 33±2 °C, for bacterial strain selection, colonies grow within 48-72 hours in usual (Ilyina et al. 2003), bacteria strains were characterized by determining their utilization profiles on microtiter plate designed to test the ability of an organism to oxidize 95 different carbon sources using BIOLOG GIN III system, Biolog Inc., California, USA. Bacteria isolates were grown for 24 hours at 33±2 °C on Biolog Universal Growth Agar medium (BUG, 57 g l-1 purified water) supplemented with 5% sheep blood, according to Civilini (2009). Sub trial was performed to define the proper malathion concentration for bacteria optimum growth, by determining bacteria growth in BUG medium supplied with malathion as sole carbon source in different concentrations (0, 10, 20, 50, 100, 200 mg kg-1). Rate of bacterial growth was estimated based on standard plate count technique by direct determination of viable cell count per ml using plate count agar medium (PCA, 23.5 g l-1 purified water), from Charlau chemicals, Spain 2.3 Preparation of working samples Five common bacteria strains were selected and identified from more than 20 strains. Known concentration of malathion active ingredient (a.i.) 99.00%, from Dr. Ehrenstorfer Reference Materials, Germany, was used to prepare working solution (5 ug malathion a.i for one ml liquid medium) and has been spread in a sterile culture tube, solvent was evaporated under pure nitrogen stream, then BUG pure liquid medium (57 g l-1 purified water), PH 7.3±0.1 was added (9 ml), bacteria strains were inoculated each separately (1 ml), tubes were shaken for 30 min, then incubated at 33±2 °C. Samples were taken at successive intervals after incubation at zero, 3 hours, 1, 2, 4, 7, 10, 15, 21 and 30 days paralleled with control samples at each interval, zero time is the initial concentration directly before incubation (50 ug).

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2.4 Residues extraction and cleaning up procedures Procedure mentioned by López-Blanco et al. (2006) which use SPE cartridge was used after volumes modifications for extraction and cleaning-up of malathion residues from the liquid medium. CUPSA3 SPE cartridge (C18+n-2 aminoethyl, 100 mg ml-1) from United Chemical Technologies (UCT), USA, was conditioned with ethyl acetate (5 mL) followed by methanol (5 mL) and ultrapure water (5 mL) at rate of 3 ml min-1, without allowing the cartridge to dry out. The aqueous sample (10 mL) was loaded on and passed thru the cartridge at rate of 0.8 ml min-1 (sample should be filtered before loading to remove suspended and insoluble materials of bacteria). Cartridge was dried by blowing nitrogen stream (purity 99.999%) over surface for 2 min. Adsorbed pesticide was eluted by ethyl acetate (5 mL). Rapid Trace SPE workstation from Zymark, Caliper Life Sciences was used for SPE handling, solvents used were HPLC grade, from BDH chemicals, UK. Agilent 7890A gas chromatography equipped with Nitrogen-Phosphorus Detector (NPD) and HP-5 capillary column (30 m × 320 um × 0.25 um) from J&W Scientific was used for pesticide residue analysis, injector at 260 °C, splitless mode, detector 320 °C, ignition gases H2 at 3 ml min-1 and air at 45 ml min-1. Oven programmed at 120 °C for one min, ramped at 20 °C min-1 to 270 °C and held for 2 min., carrier gas N2 at flow rate 3 ml min-1, malathion retention time (Rt) was 4.43 min. Perkin Elmer (PE) GC-MS with GC Clarus 500 and MS in electron impact ionization mode (EI) equipped with Elite-5MS capillary column (30 m × 250 um × 0.25 um) was used for detection of malathion degradation products, Injector at 260 ºC, splitless mode, Helium was used as carrier gas at flow rate of 1.0 ml min-1, oven programmed at 80 °C for 2 min, ramped at 10 °C min-1 to 280 °C, held for 8 min, MS source and transfer line temperatures were 230 and 280 ºC, respectively. 2.5 Method validation studies Standard Stock solution of malathion (400 ng µl-1) freshly prepared in ethyl acetate was used for calibration standard preparation which used for calculation, working solutions at 1, 2, 5, 10, 20, 40 mg kg-1 were prepared. Recovery rate was performed using untreated liquid media, which used in treatments and spiked with malathion a.i., solution at two levels 0.1 and one mg kg-1, then procedures of mentioned entire method were performed. Recovery values achieved were 91.30 and 98.70%, respectively. Limit of determination (LOD) and limit of quantitation (LOQ) of the analytical method used were estimated, values were 0.03 and 0.1 mg kg-1, respectively. 2.6 Kinetic studies The degradation rate of malathion was calculated mathematically according to Timme and Frehse (1980), that degradation behavior of pesticide residues can be described mathematically as a pseudo-first order reaction, rate of degradation (K) could be calculated using common logarithms from the following equation:log R = log R0 − 0.434 Kt R0: residue level at the initial time (zero time), R: residue level at interval in days after application. Kt: degradation rate constant at the successive intervals in days, K: mean of Kt


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Malathion half-life value (RL50) was calculated mathematically according to Moye et al. (1987) from the following equation:RL50 =

Ln 2 K

2.7 Statistical analysis t-test was used for analyzing the obtained data statistically to define the significance levels with the basis outlined by Snedecor and Cochran (1967). 3. Result and conclusion 3.1 Bacteria strains identification Data in Table 1 and Figure 1 show the morphology and characteristics of each strain colonies of five bacteria strains have been tested. S1 (Pseudomonas aeruginosa), Gram-negative, aerobic, rod-shaped bacterium with unipolar motility and has the ability to grow at 42 °C. S2 (Bacillus amyloliquefaciens), Gram-positive, catalase positive, aerobic, rod-shaped and motile, found naturally in soil and has the ability to degrade proteins extracellular, which excretes a particular enzyme called subtilisin degrades proteins that it encounters, that has been found to be useful in some industries such as laundry detergents and contact lens cleansers. S3 (Staphylococcus sciuri), Gram positive, oxidase-positive, coagulase-negative, sciuri was originally used to categorize 35 strains shown to utilize cellobiose, galactose, sucrose, and glycerol. S4 (Bacillus pseudomycoides) is a Gram positive forms chains of cells, non-motile, can hydrolyze starch, Casein and Gelatin. S5 (Bacillus licheniformis) is a Gram positive, commonly found in soil, so found on bird feathers such as sparrows and aquatic species such as ducks. It is a thermophilic bacterium, optimal growth temperature is around 30 °C and can survive at higher temperature. The optimal temperature for enzyme secretion is 37 °C so it can exist in spore form to resist harsh environs or in a vegetative state when conditions are fine. 3.2 Malathion residues Data in Table 2 and Figure 2 show the amount of malathion as µg found after incubation with tested bacteria strains and the degradation rate compared to control treatment. Data revealed that the most influential bacteria strains on the acceleration of malathion degradation rate were S5 (Bacillus licheniformis), S4 (Bacillus pseudomycoides) and S1 (Pseudomonas aeruginosa), respectively. Calculated residue half-life values (RL50) for S5, S4 and S1 treatments were 12.49, 12.72 and 16.68 days, respectively and were 20.27 and 21.33 days for S2 and S3, respectively compared to control treatment that was 27.50 days. Results indicated that P values for S5, S4 and S1 were 0.0079, 0.0077 and 0.0149, respectively less than the null hypothesis H0 (no significant effect) at P ˂ 0.05. Results indicated that S5, S4 and S1 treatments significantly accelerated malathion degradation rate. On the other hand, p values of S2 and S3 treatments were more than the probability limit (P ˂ 0.05), so had no significant effect (0.0619 and 0.0705, respectively). Results indicated that some Bacillus types had significant influence on malathion degradation rate in particular and organophosphorous in general, that was in the same trend with results obtained by Kamal Zeinat et al. (2008), also some Pseudomonas types have the same effect as reported by Ghosh Poorva et al. (2010) and Kanekar Pradnya et al. (2004), they indicated Tamer M.A. Thabit, Medhat A.H. EL-Naggar International Journal of Environmental Sciences Volume 3 No.5, 2013

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that Pseudomonas aeruginosa and Pseudomonas stutzeri have the ability to degrade organophosphorous pesticides by the utilization as carbon and energy sources. Similar findings Found by Horne et al. (2002), that Agrabacterium radiobacter has the ability to hydrolyze a wide range of organophosphorus insecticides. Deshpande et al. (2001) Found that Pseudomonas aeruginosa MCMB-427 demonstrated the ability to degrade dimethoate insecticide. Table 1: Morphology and characteristics of tested bacteria strains Strain label

Colony Name

Shape

Color

S1

Pseudomonas aeruginosa

flat and irregular

yellowgreen and fluorescent

negative

Unipolar motility

S2

Bacillus amyloliquefaciens

spreading and irregularly

Creamy

positive

motile

S3

Staphylococcus sciuri

S4

Bacillus pseudomycoides

S5

Bacillus licheniformis

Yellowish gray white to cream

Circular Singly and short chains round, matt and granular

Motility

Nonmotile Nonmotile

positive positive

greenish

positive

S3

S2

S1

Gram Character

motile

S5

S4

Figure 1: Morphological shape of tested bacteria strains Table 2: Malathion µg found after incubation with tested bacteria strains

0*

50

3**

49.76 ±1. 45 49.16 ±1. 15 48.38 ±1.22 47.17 ±1.32 43.55 ±2.37 39.64 ±2.36

1 2 4 7 10

0.0 0.48 1.68 3.24 5.66 12.90 20.72

50 49.74 ±1.22 48.83 ±1.33 47.38 ±1.29 45.08 ±2.42 40.57 ±2.31 36.93 ±1.31

00.0 0.51 2.33 5.24 9.84 18.86 33.30

50 49.73 ±0.81 48.60 ±1.11 47.92 ±2.43 44.08 ±4. 35 41.88 ±3.39 37.35 ±1.32

0.0 0.54 2.80 4.16 11.84 16.24 25.30

50 49.77 ±0.92 48.55 ±2.10 46.17 ±2.34 44.05 ±1.28 42.73 ±2.22 38.75 ±1.37

0.0 0.46 2.90 7.66 11.90 14.54 22.50

50 49.77 ±0.64 48.95 ±1.40 45.86 ±2.34 42.18 ±1.23 36.82 ±1.32 26.71 ±2.25

0.0 0.46 2.10 8.28 15.60 26.30 46.50


50 49.73 ±0.67 48.88 ±1.25 45.57 ±1.38 43.13 ±2.15 36.80 ±1.36 25.77 ±1.9

%Loss

Residue

S5 %Loss

Residue

S4 %Loss

Residue

S3 %Loss

Residue

S2 %Loss

Residue

S1 %Loss

Residue

Time

C

0.0 0.54 2.24 8.86 13.70 26.40 48.40

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15 21 30

34.16 ±2.28 24.65 ±1.25 15.91 ±0.89

31.68 50.70 68.18

33.35 ±1.21 14.20 ±0.82 3.92 ±0.91

46.10 71.60 92.10

24.02 ±1.21 20.14 ±1.18 16.83 ±0.73

51.96 59.70 66.3

28.92 ±1.23 24.71 ±0.93 16.80 ±0.54

42.16 50.58 66.40

16.62 ±1.13 10.36 ±0.45 3.23 ±0.21

66.70 79.20 93.50

16.88 ±0.85 10.82 ±0.32 2.97 ±0.13

66.20 78.30 94.00

K

0.0252

0.0415

0.0341

0.0324

0.0544

0.0554

RL50 (days)

27.50

16.68

20.27

21.33

12.72

12.49

P˂ 0.05

---

0.0149

0.0619

0.0705

0.0077

0.0079

*: Zero time (the initial concentration before incubation), **: Samples were taken three hour after incubation, RL50: Half-life value, K: Degradation rate constant, p: probability level at P ˂ 0.05, Mean± Standard Deviation (SD).

Figure 2: Malathion biodegradation rate by tested bacteria strains 3.3 Malathion degradation products identification Malathion insecticide is dithiophosphate (phosphorothiolothionate) moiety (Figure 3), which considered one of the main chemical groups belongs to organophosphorous group as mentioned by Hassal (1990).

Figure 3: Dithiophosphate (phosphorothiolothionate) chemical core and malathion structure Malathion degradation products demonstrated in figure 4, 5 and 6 were monitored by GC/MS at 10, 21 and 30 days after incubation. Malathion was detected as a parent compound at 15.60 min, molecular formula C10H19O6PS2, and M.W 330.360. The most common degradation Tamer M.A. Thabit, Medhat A.H. EL-Naggar International Journal of Environmental Sciences Volume 3 No.5, 2013

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product detected at 10 days for all treatments also was the existing one at 21 and 30 days for S2, S3 and C treatments was malathion mono carboxylic acid (MMA), Rt at 14.58 min, molecular formula C8H15O6PS2, and M.W 302.307. In S1, S4 and S5 treatments at 21 and 30 days, malathion dicarboxylic acid (MDA), Rt at 12.80 min, molecular formula C6H11O6PS2, and M.W 274.280, was also detected. In the significant treatments, MMA and MDA degradation products were in the scale together that may MMA partially converted to MDA at the end of treatments, while MMA was the highest concentration and common spread one for all treatments and control. In S1 treatment at 21 and 30 days, ethyl hydrogen fumarate (EHF) was found in a small and negligible amount, Rt at 6.46 min, formula C6H8O4 and M.W 144.125. The obtained data indicated that the main degradation product of malathion which resulted from bacterial degradation is malathion monocarboxylic acid which may convert to malathion dicarboxylic acid over time. Results were in accordance with the one obtained by Bourquin (1977), which demonstrated that malathion could be degraded by salt-marsh microorganisms to malathion monocarboxylic acid which converted partially to malathion dicarboxylic acid and then to various phosphothionates by increasing time. Results were in the same trend with those obtained by Kamal Zienat et al. (2008), which declared that strain of Bacillus thuringiensis MOS-5 (Bt) was able to utilize malathion as a sole carbon and energy source and degrade it to main compounds, malathion monocarboxylic acid (MMA) and malathion dicarboxylic acid (MDA).

Figure 4: Malathion main degradation products resulting from bacterial degradation

Figure 5: Malathion and degradation products separation by GC/MS Tamer M.A. Thabit, Medhat A.H. EL-Naggar International Journal of Environmental Sciences Volume 3 No.5, 2013

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Results declared by Roberts (1998) revealed that malathion major metabolites detected in human and rat urine were dicarboxylic and monocarboxylic acids, minor metabolites detected in humans were the same as in rats except monomethyl and dimethyl phosphate were found in humans, but thiomalic acid and monoethyl fumarate were not. In laboratory and field, the most common degradation products were identified, malathion mono and dicarboxylic acids, malaoxon, ethyl hydrogen fumarate, diethyl thiosuccinate, and CO2 as reported by Newhart Kaylynn (2006).

Figure 6: Mass spectrum of malathion (A), MMA (B), MDA (C) and EHF (D) by GC/MS Malaoxon is the primary metabolite of malathion under certain abiotic environmental conditions, which may form from environmental degradation so, malaoxon formation may be greater on dry soils however it is less stable and can be quickly degraded to non-toxic metabolites, so considered minor metabolite in soil (Odenkirchen and Wente 2007), Although malathion may be degraded by chemical processes in soil such as chemical hydrolysis, but the amount of microbial degradation is far greater than chemical degradation in natural systems (Mulla et al. 1981). 4. Conclusion Some bacteria strains isolated from the agricultural soil, namely Pseudomonas aeruginosa, Bacillus pseudomycoides and Bacillus licheniformis have the ability to degrade malathion insecticide in-vitro. Malathion degradation products identified after bacterial biodegradation were malathion monocarboxylic acid (MMA), which considered the main and common spread degradation product and partially convert to malathion dicarboxylic acid (MDA) over time. Other degradation product namely ethyl hydrogen fumarate (EHF) was identified only with Pseudomonas aeruginosa treatment but in negligible amount. Tamer M.A. Thabit, Medhat A.H. EL-Naggar International Journal of Environmental Sciences Volume 3 No.5, 2013

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Acknowledgments We are grateful to Dr. Dalia I. H. El-Geddawy (Sugar Crop Research Institute, Agriculture research center, Egypt) for review and revision of this manuscript. 5. References 1. Aislabie J. and Lloyd-Jones G., (1995), A review of bacterial degradation of pesticides, Australian journal of soil research, 33, pp 925-942. 2. Bourquin A. W., (1977), Degradation of malathion by salt-marsh microorganisms, Applied and environmental microbiology, 33, pp 356-362. 3. Civilini M., (2009), Identification and characterization of bacteria isolated under selective pressure of volatile organic compounds, Journal of environmental biology, 30, pp 99-105. 4. Deshpande N. M., Dhakephalkar P. K., and Kanekar P. P., (2001), Plasmid-mediated dimethoate degradation in Pseudomonas aeruginosa MCMB-427, Letters in applied microbiology, 33, pp 275-279. 5. Ghosh Poorva G., Sawant Neha A., Patil S. N., and Aglave B. A., (2010), Microbial biodegradation of organophosphate pesticides, International journal of biotechnology and biochemistry, 6, pp 871-876. 6. Hassal, A. K., (1990), Organophosphorous insecticides: The biochemistry and uses of pesticides, ELBS publication, London, pp 81-124 7. Hill, E. F., (2003), Wildlife toxicology of organophosphorus and carbamate pesticides: Handbook of Ecotoxicology, in D. J. Hoffman, B. A. Rattner, G. A. Burton Jr., J. Cairns Jr. (Eds.), Lewis publishers, Boca Raton, Florida, pp 281-312. 8. Horne I., Sutherland T. D., Harcourt R. L., Russell R. J., and Oakeshott J. G., (2002), Identification of an opd (organophosphate degradation) gene in an Agrobacterium isolate, Applied environmental microbiology, 68(7), pp 3371-3376. 9. Ilyin, A., Castillo S. M. I., Villarreal S. J. A., Ramirez E. G., and Candelas R. J., (2003), Isolation of soil bacteria for bioremediation of hydrocarbon contamination, BECTH. MOCK. YH-TA. CEP. 2. XHMH3, 44, pp 88-91. 10. Jones E. E. and Stewart A., (1997), Biological control of Sclerotinia minor in lettuce using Trichoderma species, Proceedings of 50th New Zealand plant protection conference, pp 154-158. 11. Kamal Zienat M., Fetyan Nashwa A. H., Ibrahim M. A., and El-Nagdy S., (2008), Biodegradation and detoxification of Malathion by of Bacillus Thuringiensis MOS-5, Australian journal basic and applied sciences, 2, pp 724-732. 12. Kanekar Pradnya p., Bhadbhade Bharati J., Deshpande Neelima M., and Sarnaik Seema, S., (2004), Biodegradation of organophosphorous pesticides, Proceedings of Indian National Science Academy, B70, pp 57-70. Tamer M.A. Thabit, Medhat A.H. EL-Naggar International Journal of Environmental Sciences Volume 3 No.5, 2013

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13. Lopez-Blanco C., Gomez-Alvarez S., Rey-Garrote M., Cancho-Grande B., and SimalGandara J., (2006), Determination of pesticides by solid phase extraction followed by gas chromatography with nitrogen-phosphorous detection in natural water and comparison with solvent drop microextraction, Analytical and bioanalytical Chemistry, 384, pp 1002-1006. 14. Moye H. A., Malagodi M. H., Yoh J., Leibee G. L., Ku C. C., and Wislocki P. G., (1987), Residues of avermectin B1a rotational crop and soils following soil treatment with (14C) avermectin B1a, Journal of agriculture and food Chemistry, 35, pp 859864. 15. Mulla M. S., Mian L. S., and Kawecki J. A., (1981), Distribution, transport, and fate of the insecticides malathion and parathion in the environment: Residue reviews, in F. A. Gunther and J. D. Gunther (Eds.), Springer, New York. 16. Newhart Kaylynn, (2006), Environmental fate of Malathion, California: Environmental protection agency, Department of pesticide regulation, Environmental monitoring branch. 17. Odenkirchen E., and Wente S.P., (2007), Risks of malathion use to federally listed California red-legged frog (Rana aurora draytonii), Pesticide effects determination. Washington, DC, Environmental protection agency, Office of pesticide programs, Environmental fate and effects division, U.S. government printing office. 18. Roberts T. R., (1998), Metabolic Pathways of Agrochemicals - Part 2: Insecticides and Fungicides, The royal society of Chemistry, Cambridge, pp 360-367 19. Singh K., Brajesh K., Walker A., Alum J., Morgan W., and Wright D. J., (2004), Biodegradation of chlorpyrifos by Enterobacter strain B-14 and its use in biodegradation of contaminated soils, Applied environmental microbiology, 70, pp 4855-4863. 20. Snedecor G. V. and Cochran W. G., (1967), Statistical methods 6th Ed. Iowa: Iowa state Univ. Press Ames. 21. Strong P. J. and Burgess J. E., (2008), Treatment methods for wine-related and distillery wastewaters: a review, Bioremediation journal, 12, pp 70-87. 22. Timme G., and Frehse H., (1980), Statistical interpretation and graphic representation of the degradation behaviour of pesticide residues, Pflanzenschutz Nachrichten Bayer, 33, pp 47-60. 23. Tomlin C. D. S., (2006), The pesticide manual. 14th Ed. World compendium. British Crop Protection Council (BCPC), Alton, Hampshire.


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