Accumulation, Distribution And Dissipation Of Pentachlorophenol And Its Impact On The Microbial Community Of Rhizospheric Soil

Persistent organic pollutants (POPs) are organic compounds which enter in the environment through industry as by products as well as through human and agricultural actives, these compounds show some resistance to degrade in the environment. Pentachlorophenol (PCP) is one of the persistent organic pollutants and is moderately persistent in the soil, with a reported field half-life of 23 to 178 days. Although the use of PCP has decreased in recent years, but still causes environmental problems in soil and aquatic sediments at many locations. In China the main sources of PCP contamination occur where Na-PCP was used to kill snails and for the cleaning of ponds; about 60% of the national production of Na-PCP is applied for this purpose. Considering the potential health risks of PCP, China restricted the production and use of Na-PCP in 1997. However, with the re-emergence of schistosomiasis in the traditional epidemic areas (Li and Cai,2004), the production and use of PCP for snail elimination and schistosomiasis control has increased once again. The annual national output reached approximately 3000 t in 2003, although no data have been reported yet regarding the figure for more recent years. The increased PCP use likely has resulted in more environmental contamination and potential health risks.In this series of experiment PCP was used as major pollutant and we select rice as test crop. Micro-Clark type oxygen, pH microsensor and redox electrodes were used to measure oxygen, pH and redox potential in the root soil interface as well as dynamic changes in their concentration with the change in light intensity. Further we conducted pot experiment with two PCP degraded intermediate 3,5-DCP (3,5-Dichlorophenol) and 2,4,6-TCP (2,4,6-Trochlorophenol). To study their impact on the rice leaf and root so we determined antioxidants enzymes activity, ultrastructural changes in cell as well as we study the dissipation process of chlorinated compounds. Further we study the dissipation process and change in electron acceptors and donors activity in root soil interface as well as dissipation rate at different distance from the root by using especially design multi compartment rhizobox. To further understand in detail about the dissipation process of PCP in the rice rhizosphere. We further use rhizobox and measured different fractions of PCP in the rhizospheric environment and structural changes in microbial community. In detail main results obtained are as follows. (1) To investigate the dynamic changes taken place in the root soil interface of rice plant, an experiment was conducted with the objectives to understand the role of root in changing oxygen, pH and redox potential in root soil interface with the changing day light. Micro-Clark type oxygen and pH microsensor and redox electrodes were used to measure oxygen, pH and redox potential as had been described by Revsbech and Jorgensen. Date was determent after 15 days of rice transplantation, results showed that dynamic changes in the oxygen, pH and redox potential activates in root soil interface. We measured 48 hours consecutive data and found that release of oxygen in the root soil interface by root through aerenchyma tissues played significant changes in the pH and redox potential. In the Results we found significant higher concentration of oxygen (88%) in the rhizosphere between 12:30 to 13:00 hours when the light intensity was (48500 Lux). Same time we found significant increase in redox potential (230.43mv) that showed significant relation with the concentration of released oxygen in the rhizosphere. This change in oxygen and redox also revealed effect on the change in pH. We found that at 21:00 hours light intensity on plant canopy was zero and oxygen concentration in the rhizosphere was significantly lower (0.408%). We also found significant lower redox potential values at 23:00 houes (138.76mv) when all the oxygen might be diffused out from the rhizosphere and caused the change in redox potential. Under the control light conditions we also found significant change as compare to natural light conditions, under the 16000 Lux fixed light intensity we found maximum oxygen release in the soil is nearly 35% that was significant less concentration as compare to natural light condition so due to this change in redox and pH also get effected. So experimental results revealed that changed in light intensity had significant effects on the release of oxygen in the root soil interface and ultimately affected the pH and redox potential. So this experimental result helps us understand the dynamic changes taken place in the root soil interface due to changes in light intensity, as well as helpful for further experiment especially to decide sampling time and study the activity of chlorinated compounds in root soil interfaces.(2) In order to further understand the behaviors of chlorinated compounds in the rhizosphere of rice, an experiment was conducted to study the effect of 3,5-DCP,2,4,6- TCP and PCP on the antioxidants enzymes activity, lipid peroxidase and ultrastructural changes in the leaves and root cells as well as dissipation of these chlorinated compounds. To check the real effect of rhizosphere in the pot, we grew plants in nylon mesh bags containing 500g soil inside the pot. Soil was spiked with 45±0.5 mg kg-1 (dry weight). To prepare the xenobiotic spiked soil. PCP,2,4,6-TCP and 3,5-DCP solutions using acetone as the solvent were mixed uniformly with a small portion of soil. The spiked soil was then vented for 24 h to let the acetone vaporize and then mixed thoroughly with a large portion of uncontaminated soil (1:14, w/w). The spiked soils were equilibrated in the greenhouse for one week at field capacity, and flooded with water for 24 hours before transplanting rice(Oryza sative L.) seedlings. Two week old rice seedlings were transplanted in flooded water pots and data was collected after 20 days and 40 days. Results showed significant increase in the antioxidant enzymes activity and lipid peroxidase (MDA) activity. In 3,5-DCP treated sample we found maximum MDA activity in the leaves and root (33.60 and 41.80 nmol(gfw)-1 respectively) that showed cell membrane damage in the leaves and roots of rice plants as well as concentration of superoxide dismutase (SOD) also increased (97.36 and 123.79U(gfw)-1respectively) in leaves and roots significantly that showed the production of H2O2, to hunt this H2O2 we found significant increase in peroxidase (POD) and ascorbate peroxidase (APX) activity (309.87 and 683.79 OD470/gfwmin & 146.62 and 168.86 nmol(gfw)-1 respectively). Over all result showed that 3,5-DCP caused more toxicity in the rhizosphere as compare to 2,4,6-TCP and PCP. In transmission electron microscopy we found that in the PCP treated soil, there were no high ultrastructural damages in the root and leave cell samples as compare to 2,4,6-TCP and 3,5-DCP. We also found that PCP treated rice plant showed good growth and played significant roles in the dissipation of PCP as compare to control (79.59% PCP loss). So from this experiment we found that rice rhizsphere played significant roles in the dissipation of PCP and PCP did not cause much damage to rice plants.(3) On the basis of previous experimental data we designed next experiment to investigate the dissipation of pentachlorophenol in the aerobic-anaerobic interfaces established by the rhizosphere of rice (Oryza sative L.) roots. To investigate the dissipation behavior of PCP in the aerobic-anaerobic interfaces established by the rhizosphere of rice roots, a glasshouse experiment was conducted using a specially designed rhizobox. The possible biogeochemical mechanisms were also studied through illustration of the dynamic behavior of important electron acceptors and donors which potentially involved in the reductive dechlorination and aerobic catabolism processes of PCP. The soil was spiked with 20±0.5 and 45±0.5 mg PCP kg-1. Soil in the rhizobox was divided into five different compartments at various distances from the root surface. Maximum dissipation of PCP in planted soil was observed at 3 mm distance from the root zone as well as rapid changes in concentrations of sulfate, chloride, nitrate and ammonium at the same distance from the root. In contrast, in the unplanted soil, no difference was observed in the PCP concentration with increasing distance. After 45 days, a significantly more concentration of PCP was dissipated in planted soil compared to unplanted soil; in the unplanted microcosms, about 45% of the initial PCP was lost at both low and high added rates, respectively. This was, proportionately, a significantly smaller percentage compared to the planted rhizosphere (an average of 66% and 64.5% respectively). Moreover, the correlations of PCP dissipation with SO42-, NO3- and Fe3+ were significantly negative, while the correlations of PCP dissipation with NH4+, Fe2+ and Cl were significantly positive. This suggested the oxidization of soil constituents could inhibit aerobic catabolism of PCP by consuming O2, and the reduction of soil constituents could inhibit anaerobic reductive dechlorination of PCP. Therefore, the significance of the rhizosphere in phytoremediation of chlorinated compounds such as PCP differs significantly between wetland and rainfed systems.(4) From previous experimental results we got some good ideas about the role of rice in the dissipation of PCP, so to understand the rice rhizosphere effects on the dynamic changes of various fractions of PCP and the microbial community, a glasshouse experiment was conducted by using a rhizobox in which rice seedlings were grown for 45 days. The soil was spiked with 20 or 45 mg kg-1 PCP. Soil in the rhizobox was divided into five compartments at various distances from the root. Sequential PCP extractions were conducted by using three extractants:CaCl2, butanol, and DCM. The butanol extractable form of PCP showed significantly higher concentration compared to the other two extractable fractions of PCP at different distance from the root. Among three different fractions 45 days after rice transplantation, the polar and soluble neutral form of PCP was significantly higher at 4 mm and 5 mm distance from the root at 20 and 45 mg kg-1 PCP treatments, respectively. Butanol extraction showed a significant difference at 3mm distance from the root at both PCP levels (59.24 and 29.10% respectively). The DCM extractable form of PCP showed significant differences at 5 mm distance from the root at the two different PCP treatment levels (15.63 and 20.42% respectively). After 15 days. significantly higher concentrations of the polar and soluble neutral form of PCP were found compared to the control as well as to the other two fractions of PCP, but its concentration decreased with the increase of time. We found significantly higher concentration of the butanol extractable form of PCP compared to other extractable forms of PCP after 45 days.In DCM extraction after 45 days the PCP concentration significantly decreased at both 20 mg kg-1 and 45 mg kg-1 PCP treatments compared to the control. Thirty four phospholipids fatty acids (PLFAs) were identified in the two different PCP concentrations of the rice rhizosphere. The total soil PLFA concentration in the planted soils ranged from 29 to 52 nmol g-1. The highest concentration was at 3 mm distance from the roots at both PCP concentrations. After 45 days, there were distinct changes in the PLFAs of different signature groups in planted, compared to unplanted, PLFA concentrations of gram-negative bacteria groups (i15:0, a15:0,il6:016:1ω7c, i17:0,cy17;0,17:0,18:1 co7c and cy19:0ω8c), gram-positive bacteria (i14:0, i15;0, i16;0,i17:0,a15:0 and a17:0), actinomycete (19:0(10Me)) and fungal groups (18:2ω6,9c and 18:1ω9c). The results suggest that the aerobic-anaerobic interface established by the root in the rhizosphere of rice showed pronounced effects on the xenobiotic activity as well as on the microbial activity in the rhizosphere.