Grantee Research Project Results
2005 Progress Report: Phytoremediation of Perchlorate and N-nitrosodimethylamine as Single and Co-contaminants
EPA Grant Number: R831090Title: Phytoremediation of Perchlorate and N-nitrosodimethylamine as Single and Co-contaminants
Investigators: Mbuya, Odemari S. , Nzengung, Valentine A. , Ugochukwu, Ngozi H. , Jain, Amita
Institution: Florida Agricultural and Mechanical University , University of Georgia
EPA Project Officer: Hahn, Intaek
Project Period: October 1, 2003 through September 30, 2005
Project Period Covered by this Report: October 1, 2004 through September 30, 2005
Project Amount: $399,875
RFA: Superfund Minority Institutions Program: Hazardous Substance Research (2002) RFA Text | Recipients Lists
Research Category: Hazardous Waste/Remediation , Land and Waste Management , Safer Chemicals
Objective:
The objectives of this research project are to: (1) determine the long-term fate of perchlorate taken up into plants and the change in concentration of perchlorate in plant leaves prior to and after senescence; (2) develop a process to minimize uptake and phytodegradation of perchlorate while enhancing root zone biodegradation (rhizodegradation); and (3) develop an analytical method for N-nitrosodimethylamine (NDMA), a byproduct of 1,1-dimethylhydrazine used in liquid rocket fuel production. Currently, there is neither U.S. Environmental Protection Agency (EPA) nor peer-reviewed methods for the analysis of NDMA and its metabolites in plant tissues.
Progress Summary:
Uptake and phytodegradation of sodium perchlorate were evaluated on plants growing under natural field conditions, hydroponic bioreactors, and in pots. Potential phytoremediation of NDMA was investigated using willow plants grown in hydroponic bioreactors.
Willow (Salix nigra) and other species of field plants (tamarix, cat tail, sweet gum, pine, and sedge) growing over perchlorate-contaminated soils and groundwater at Longhorn Army Ammunition Plant (LHAAP) in Texas and Las Vegas Wash (LVW) in Nevada were analyzed for perchlorate tissue content. Perchlorate was detected in all field plant species sampled. Perchlorate not only was detected in live plants but also in dead tissues (dry leaves and stems). Sampled willow plants had tissue perchlorate content of up to168 mg kg-1. The highest concentration of perchlorate (866.3 mg kg-1) was measured in grass samples. Dead leaves that had fallen off of tamarix (salt cedar) plants had up to 296 mg kg-1 of perchlorate, whereas trunk tissues had 463 mg kg-1. There was a spatial variation of tissue perchlorate content within plant species, and perchlorate phytoaccumulation also varied between plant species. The spatial variation of tissue perchlorate content was related to soil and/or groundwater perchlorate spatial variation. Perchlorate is present in plant dry tissues because degradation of perchlorate in plant tissue (phytodegradation) is a slow process, eventually resulting in phytoaccumulation. Senescent and dry vegetation of all plant species collected from LVW had higher perchlorate concentration than live plants. Our investigation showed that perchlorate taken up by plants does not readily transform to its metabolites; instead, it slowly accumulates in plant tissues. The phytoaccumulated perchlorate is important ecologically because senesced leaf fall serves as a potential source of perchlorate recycling and recontamination. Harvesting of perchlorate containing vegetation and subsequent treatment of the plant material by anaerobic biodegradation should be included in a phytoremediation design.
Biostimulation and enhancement of rhizodegradation of perchlorate could reduce the amount of perchlorate uptake by plants. In a series of experiments, the root zone environment (rhizosphere) of willow plants grown in soil bioreactors was manipulated using dissolved organic carbon (DOC) from chicken litter and acetate. The soil bioreactors were amended with 300 mg L-1 DOC from chicken litter and acetate. Initial perchlorate concentration of 27 mg L-1 in bioreactors amended with chicken litter DOC was reduced to the method detection limit (MDL) of 2 μg L-1 in 9 days. The reaction is described by the zero-order kinetic equation with a degradation rate constant of 4.8 ± 0.6 mg L-1 day-1. A second dose of 29 mg L-1 perchlorate applied to the same soil bioreactor was removed completely from the bioreactor within 5 days. This reaction is described by the zero-order equation with a degradation rate constant of 6.5 ± 2.9 mg L-1 day-1. Similar results were observed with bioreactors treated with 300 mg L-1 DOC as acetate. Initial concentration of 33 mg L-1 perchlorate was reduced to below the MDL within 9 days. Unlike the DOC amended bioreactors, the rate of perchlorate removal from bioreactors without DOC amendment (control) was much slower, described by first-order or second-order kinetics. Complete removal of perchlorate from control bioreactors took more than 13 days compared to 9 days for chicken litter and acetate-amended bioreactors. Soil bioreactors had a faster perchlorate removal than hydroponic bioreactors.
Polymerase chain reaction (PCR) experiments confirmed the presence of the chlorite dismutase gene (cld) in pore water collected from planted bioreactors dosed with DOC and the control. The presence of cld in the control soil bioreactors suggests that soils already harbor a high population of the ubiquitous perchlorate-respiring microbes required for biodegradation of perchlorate. The detection of the cld in soil and agricultural waste products used as electron sources confirms the documented ubiquity of perchlorate-degrading microbes. This means that the limiting factor for biodegradation and rhizodegradation of perchlorate contaminated soils is the lack of an adequate supply of DOC which is consumed by perchlorate degraders and used as carbon and electron sources for mineralization of perchlorate.
Development of an analytical method for NDMA in water and plant tissues was achieved. NDMA analysis was performed using a Shimadzu liquid chromatograph (LC) equipped with a 10AD pump and autoinjector connected to a 10A system controller, C18 ODS Hypersil column (5 μm particles, 125 x 4 mm), and a Shimadzu SPD-10AV ultraviolet (UV) detector. The 1.2 mL aqueous samples collected from the bioreactors were filtered through 0.2 μm syringe filters (Fisher Scientific). A 25 μL of the sample was injected into the LC. One hundred percent water was used as mobile phase A and 100 percent methanol was used as mobile phase B. Gradient elusion of NDMA was done by holding at 98 percent A for 2 minutes then changing to 50 percent B in 6 minutes and returning to 98 percent A at 7 minutes and finally holding for 3 minutes longer. The flow rate of the mobile phase was 1 mL min-1. NDMA absorbance was measured at a wavelength of 225 nm. The retention time for NDMA was 3.9 minutes. Black willow (Salix nigra) were rooted in a greenhouse in aerated dilute Peter’s professional® nutrient solution. After 3 weeks, the prerooted cuttings were transferred to 2L Erlenmeyer® flasks (hydroponic bioreactors) containing one-quarter-strength Hoagland® solution. The reactors were wrapped in aluminum foil to prevent algae growth and NDMA photolysis. A predrilled screw cap with Teflon-lined septum was placed around each cutting. The septum and tree cutting interface was sealed with Parafilm® tape. DAP® aquarium sealant (100% silicone) was used to seal the screw cap and septum to the cuttings to prevent loss of water (and NDMA) by volatilization. Triplicate reactors were prepared for planted and unplanted (control) experiments. All the reactors were spiked to contain 900-1,000 μg L-1 NDMA initial concentrations. About 1.2 mL of rhizosphere solution sample was taken for analysis from each bioreactor. Prior to sampling, the volume of water transpired was replenished by adding nutrient solution to the 2L level. The volume of water added to the reactors was recorded for the duration of the experiment. Willow plants growing in NDMA-contaminated water did not show any signs of toxicity at 1,000 μg L-1 NDMA concentration. The willow plants were observed to remove NDMA from the hydroponic solution. In experiment 1, the plants reduced the initial 1,050 μg L-1 NDMA concentration to 120 μg L-1 in 100 days (bioreactors 1 and 2), whereas the initial concentration of 900 μg L-1 NDMA was reduced to 150 μg L-1 NDMA in 30 days during a second experiment (bioreactors 3 and 4). The rate of NDMA removal from the planted bioreactors was at a constant rate shown by a linear decrease in NDMA concentration over time. Zero-order kinetics of NDMA removal from the planted bioreactors indicates that only one mechanism is responsible for the decontamination process. Removal of NDMA from planted bioreactors was purely by plant uptake through the transpiration stream. This was confirmed by a strong linear relationship between mass of NDMA removed and volume of water transpired. There was no evidence of microbial mediated rhizodegradation of NDMA. In the unplanted control bioreactors, the concentration of NDMA remained the same for 67 days of experiment. Between days 67 and 78, the concentration of NDMA had decreased by about 150 μg L-1, possibly from exposure of the bioreactors to UV radiation. Between days 78 and 102 of the experiment, however, NDMA concentration in the control bioreactors remained the same. Uniform concentration of NDMA in the unplanted control bioreactors suggests that neither sorption to the glass nor microbial degradation of NDMA took place in the bioreactors. Because NDMA removal from bioreactors was only by plant uptake through transpiration stream, it is possible to make a prediction of the rate of NDMA removal from the transpiration rate and vice versa.
Two Ph.D. students (Dawit Yifru and William Mwegoha) have been trained and supported by the grant. Both students are expected to graduate in summer 2006.
Future Activities:
We will: (1) evaluate the effect of concentration and rhizodegradation on the uptake of perchlorate in willow plants in soil bioreactors (six treatments will be used: 100, 200, 400, 600, 800, and 1000 mg L-1); and (2) perform more plant tissue analysis. Also, graduate students supported by the grants will continue writing their dissertations.
Journal Articles:
No journal articles submitted with this report: View all 12 publications for this projectSupplemental Keywords:
biostimulation, phytoaccumulation, phytodegradation, rhizodegradation, bioreactor, hydroponic, photolysis, senescence,, RFA, Scientific Discipline, INTERNATIONAL COOPERATION, Waste, Contaminated Sediments, Environmental Microbiology, Hazardous Waste, Bioremediation, Hazardous, microbiology, degradation, Superfund site remediation, plant species, industrial waste, microbial degradation, bioavailability, biodegradation, perchlorate, contaminated sediment, contaminated soil, contaminants in soil, bioremediation of soils, N-Nitrosodimethylamine, biochemistry, phytoremediationProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.