Kinetics of dimethoate biodegradation in bacterial system

The present study is an investigation on the kinetics of dimethoate biodegradation and an estimation of residual dimethoate in bacterial culture by spectrophotometry. The methylene chloride extract of the culture medium was used for determination of dimethoate through its reaction with 1 chloro-2, 4 dinitrobenzene to produce methylamine whose absorbance at 505 nm gave an estimation of dimethoate content. The dimethoate standard curve follows Beer’s law at 505 nm with a slope of 0.0129 absorbance units per μg/mL. The regression equation relating concentration of dimethoate (x) with the absorbance is (y): y = 0.037+ 0.0129x. The amount of residual dimethoate after 7 days were 0, 4, 17, 28 and 29 μg/mL; the rate constants were 0.775, 0.305, 0.225, 0.167 and 0.127 each per day, and the efficiency of dimethoate degradation were 100%, 96%, 83%, 72% and 71%, for Bacillus licheniformis, Pseudomonas aeruginosa, Aeromonas hydrophila, Proteus mirabilis and Bacillus pumilus respectively. Dimethoate remediation could be attained through bacterial metabolism of the pesticide and colorimetric analysis might be useful in the estimation of dimethoate within a detection limit of 5-100 μg/mL.


Introduction
Organophosphorus compounds such as dimethoate [O, O-dimethyl S-methyl-carbamoyl-methyl phosphorodithioate], has both plant and animal systemic insecticidal properties 1 and constitutes the largest class of insecticide used worldwide. 2It is nerve toxin, carcinogen, teratogen, has high oral and dermal acute toxicity, is an eye irritant, and is readily absorbed through lungs.In view of its toxicity, it is important to remove dimethoate from the environment.4][5] Bacterial species can utilize an insecticide as a sole source of carbon and energy for growth, which is manifested by consumption of the parent compound, and which can be detected and estimated by various methods. 2,3,5,6Several methods for the determination of dimethoate compound have been reviewed based on column, paper, and thin layer chromatography, paper electrophoresis, colorimetry, gas chromatography, and radiometry, tandem mass spectrometry. 2,3,7,8The toxicity of dimethoate in freshwater airbreathing catfish Heteropneustes fossilis was carried out by bioassay method. 9The investigations, reported herein, were undertaken for the quantitative estimation of dimethoate when it was introduced directly as a sole source of carbon and energy in bacterial system; another aspect of this investigation was to study the dynamics of dimethoate biodegradation using bacterial isolates from different sources.

Culture condition
The bacterial strains were grown in 50 mL Mineral salts (MS) solution 11 containing 100 µg/mL of dimethoate as the sole source of carbon at 28°C for 24 h.From this culture, bacterial inoculum amounting to approximately 5× 10 5 CFU/mL was prepared as described earlier 10 for all the strains.In separate conical flasks containing 100 mL MS solution supplemented with 100 µg/mL of dimethoate, was inoculated 0.1 mL of the bacterial inoculum of 5×10 5 CFU/mL and incubated at 28°C for up to seven days.

Methylene chloride extract of dimethoate
An aliquot of 1 mL of the bacterial culture fluid cells were taken out aseptically from 100 mL stock culture after every 24 h for up to 7 days and treated as described for the preparation of acetonitrile extract. 11The cells were removed from the culture by centrifugation at 4000 × g for 20 min.The supernatant was extracted twice with equal volumes of methylene chloride, shaken vigorously and centrifuged at 4000 × g and the organic fraction was transferred to a separate tube.The methylene chloride extract was used for the estimation of unknown amount of dimethoate.

Preparation of standard curve
The standard curve for dimethoate was prepared, as has been described earlier. 12A range of dimethoate solutions containing 0 to 100 µg/mL of 99% dimethoate was prepared in methylene chloride in a series of glass-stoppered test tubes.One drop of 0.5% lanolin solution was added to each tube to prevent loss during the evaporation.The solvent was evaporated in a 70°C water bath.The dimethoate present in each test tube was hydrolyzed with 1 mL of 0.5 N methanolic sodium hydroxide solution followed by heating for 10 min in a water bath pre-adjusted to 60°C.The resulting reaction mixture was immediately cooled in a cold-water bath.To each of the tubes, 0.1 mL of the 1-chloro 2, 4 dinitrobenzene reagent was N o n -c o m m e r c i a l u s e o n l y added, stoppered and shaken for 10 min.Next, 2 mL of absolute ethyl alcohol was added and swirled.The absorbance of the resulting red colored solution was measured in spectrophotometer at 505 nm.Absolute ethyl alcohol was taken as the blank solution and the standard curve was prepared by plotting the absorbance reading against micrograms of dimethoate.

Colorimetric estimation of residual dimethoate from bacterial cultures
Each of the bacterial culture taken at regular interval of day 1, 2, 3, 4, 5, 6, and 7 was extracted as described above with methylene chloride and dimethoate in the culture was treated with methanolic sodium hydroxide and 1 chloro-2, 4 dinitrobenzene to form the red colored compound, whose absorbance was measured at 505 nm.The unknown amount of residual dimethoate present due to biodegradation was determined from the standard dimethoate curve. 12
C114 was examined at an initial concentration of 100 µg/mL dimethoate, after a period of seven days. 12The efficiency of dimethoate degradation was evaluated with respect to their percent biodegradation, rate constants, and half-lives for dimethoate degradation.

Results
The dimethoate standard curve follows Beer's law at 505 nm, in the range of 5-100 µg/mL of dimethoate, and it has a slope of 0.0129 absorbance units per µg/mL.The blank solution (absolute ethyl alcohol) gives an average absorbance of 0.034.The regression equation relating concentration of dimethoate (x) with the absorbance is (y): y = 0.037 + 0.0129x.Herein it was found that 1 µg/mL dimethoate was equivalent to an absorbance of 0.0142 (Figure 1).
A comparative study of ln (N 0 /N) against time in days for the five bacterial isolates, Ps. aeruginosa W171, B. licheniformis F102, Pr. mirabilis C114 strain, B. pumilus KS23 and A. hydrophila O102 strains, at day 1, 2, 3, 4, 5, 6, and 7 is depicted in Figure 2, where N 0 is the initial concentration of dimethoate and N is the dimethoate left at the end of 24 h incubation period.Residual dimethoate left after bacterial degradation of 100 µg/mL of dimethoate in the liquid culture of B. licheniformis F102 culture was 2 µg/mL at day 3, after which peri-od no residual dimethoate was found.In cultures of the other four dimethoate degraders: Ps. aeruginosa W171, A. hydrophila O102, P. mirabilis C114 and B. pumilus KS23, the residual dimethoate obtained after day 7 were 4, 17, 28 and 29 µg/mL (Figure 2).
The percentage biodegradation of dimethoate in liquid culture for the above five strains after 7 days is presented in Figure 3.The B. licheniformis F102 strain demonstrated 100% dimethoate degradation at day four.After incubation for 7 day, the rates of degradation of dimethoate were 96%, 83%, 72%, and 71% respectively for Ps.aeruginosa W171, A. hydrophila O102, Pr. mirabilis C114, and B. pumilus KS23.

Discussion
Biological remediation strategies are environmentally desirable, and considered a costeffective option.In the present study, we have demonstrated the removal of dimethoate (at 100 μg/mL) in enrichment cultures using five different bacterial strains.Several authors have reported on the quantitative determination of dimethoate compound.Pandey et al. 9 used bioassay method to determine the toxicity of dimethoate in catfish Heteropneustes fossilis and found out the LC 50 values for dimethoate to be 2.98-3.38 mg/L for upto 4 days.Pagliuca et al. 1 reported that 37 among 135 raw milk samples were positive for traces, and 10 samples showed organophosphorus pesticide contamination in a range 5-18 µg/kg.Xiang et al. 3 monitored dimethoate utilization by Ps. aeruginosa using SPQC (series piezoelectric quartz crystal) and HPLC (high performance liquid chromatography) with determination limit of 1.08 ng in HPLC.Hadjidemetriou et al. 13 studied the dissipation of dimethoate in citrus foliage by gas-liquid chromatography using 5 % OV-101 coated on a Carbowax 20M surface-modified support (Ultra-Bond 20M).Gamón et al. 8 quantified pesticides in fruit and vegetables using gas chromatography/tandem mass spectrometry by spectral confirmation of the matrixes after    extracting the residues from samples with acetone followed by a mixture of dichloromethane-petroleum ether.Szymczyk and Malczewska 13 presented a GC method for the determination of six organophosphorous pesticide residues including dimethoate in cabbage, in which the chopped cabbage was blended with acetone, extracted with n-hexane:methylene chloride (1:1) mixture.The extract was then purified on a GC column and eluted with the mixture of methylene chloride:cyclohexane (1:1) at the flow rate of 0.5 mL/min.Dimethoate was extracted from hexane solution with HBr followed by hydrolysis and determination of the evolved hydrogen sulphide from dimethyl sulphate, as methylene blue.Herein, the parent compound dimethoate was extracted with methylene chloride, followed by alkaline hydrolysis, and colorimetric determination of the resultant methylamine by reaction with 1-chloro-2, 4-dinitrobenzene.Both dimethoate and omethoate react to form the color by the dinitrochlorobenzene colorimetric procedure, but omethoate cannot be determined in the same sample, because gradually it tends to get lost in the clean up procedure.
The B. licheniformis F102 strain isolated from fish intestine degraded 100% of dimethoate in liquid culture at day four.The rate constant of dimethoate degradation was 0.775 per day for the F102 strain, and half-life of dimethoate was calculated as 0.894 day when the strain was incubated in liquid media.Another bacillus, B. pumilus KS23 strain, a soil bacterium, exerted 71% degradation of dimethoate at day 7 with a rate constant 0.127 per day, and for this strain half-life of dimethoate was 5.456 days.The water bacteria Ps. aeruginosa W171 strain demonstrated an efficiency of degrading 96% dimethoate at day 7 with rate constant 0.305 per day, and a 2.268 days half-life of dimethoate as was calculated for this strain.For other two strains, P. mirabilis C114 and A. hydrophila O102, that showed the efficiency of degrading respectively 72% and 83% of dimethoate, the respective rate constants was calculated as 0.167 and 0.225 per day, with half-life of dimethoate as 4.135 days and 3.072 days, respectively.
Deshpande et al. 14 found that Ps. aeruginosa and B. megaterium demonstrated an efficiency of more than 95% for dimethoate degradation after 8 days of incubation, and for the other strains used in their study, the efficiency of dimethoate degradation ranged between 64% and 90%.Liang et al. 4 showed that a Raoultella sp. was able to remove up to 75% of dimethoate via co-metabolism through optimization of carbon and energy source using response surface methodology.Okeke et al., 15 using Pandoraea sp., reported that after 8 weeks of incubation, 89.9% and 93.3% degradation were achieved with γ and α hexachlorohexane (150 μg/mL for each) isomers, respectively.The rate constants for γ and α hexachlorohexane, were 0.28 and 0.32, respectively, and the half-lives were 2.51 weeks and 2 weeks, respectively.Kanrar et al. 16 determined the dissipation pattern, residue level and half live values of imidacloprid in soil, water, plant, grain husk and straw samples in rice ecosystem.Schmalko et al. 17 studied degradation kinetics of dimethoate in yerba mate plants by capillary gas chromatography technique.Half-life times in plants ranked between 9.8 and 11.8 days and the dimethoate concentration decayed to a 22.7% of its initial value (in dry basis); while during seasoning step (at 45°C), half-life time was 17.3 days.Bo and Xin-Huai 18 found out that the half life periods of dimethoate and other organophosphates were in a range of 4.0 to 10.3 h, at 63°C-100°C; and that lactic bacteria in Rhodia and Danisco starter cultures increased the degradation of dimethoate in bovine milk to 11.7% and 20.4% respectively.Hui-mei 1 et al. 19 determined the half-lives of dimethoate in cabbage and soil to be 2 and 1.5 days respectively, through GC detection, with 90.25-110.39%recovery and a variation coefficient of 1.51-5.83%.Hadjidemetriou et al. 13 showed that dimethoate residues were best characterised by two first-order kinetic processes; the half-life values of dimethoate were 2.2 days for the 1-10 days portion, and 7.0 days for the 10-49 days portion.
In this communication, it has been established for the first time that the colorimetric estimation is useful in detecting dimethoate (within a limit of 5-100 µg/mL) in bacterial system, in order to study the dynamics of the pesticide (dimethoate) biodegradation.

Figure 2 .
Figure 2. Comparative study of ln (N0/N) with time in days for the five bacterial isolates, where N 0 is the initial concentration of dimethoate and N is the dimethoate left at the end of 24 h incubation period.

Figure 3 .
Figure 3. Percent biodegradation of dimethoate by five bacterial isolates.

Figure 4 .
Figure 4. Kinetics, expressed as rate constant (per 24 h) and half life (day), of dimethoate degradation reaction using the bacterial isolates.

Figure 1 .
Figure 1.Standard curve for the estimation of dimethoate at 505 nm.The regression equation is y=0.037+0.0129x.