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Final 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. , Jain, Amita , Nzengung, Valentine A. , Ugochukwu, Ngozi H.
Institution: Florida Agricultural and Mechanical University , University of Georgia
EPA Project Officer: Savage, Nora
Project Period: October 1, 2003 through September 30, 2005
Project Amount: $399,875
RFA: Superfund Minority Institutions Program: Hazardous Substance Research (2002)
Research Category: Hazardous Waste/Remediation
Description:
Objective:Environmental contamination by two emerging contaminants, perchlorate (ClO4ˉ) and N-nitrosodimethylamine (NDMA), pose significant health risks to water resources in several regions of the United States. A growing number of ongoing research efforts are focused on investigating the source, transport, health effects, and treatment options for these contaminants. There is an immediate need, however, for low-cost and sustainable treatment technologies that minimize the ecotoxicological risks of exposure to perchlorate and NDMA. This research investigated the efficacy of phytoremediation of NDMA, the fate of perchlorate taken up into plants growing in subhumid and arid climates, the enhancement of rhizodegradation of perchlorate to reduce perchlorate plant uptake, and the effect of perchlorate concentration on phytoremediation.
Specific objectives of the project were 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); (3) develop an analytical method for NDMA, a byproduct of 1,1-dimethylhydrazine used in liquid rocket fuel production (currently, there is neither an EPA nor peer reviewed method for the analysis of NDMA and its metabolites in plant tissues); (4) establish whether woody plants selectively take up perchlorate or NDMA when they occur as co-contaminants; and (5) determine the effect of concentration of perchlorate phytodegradation.
Previous greenhouse hydroponic and soil bioreactor studies provided evidence on the potential effectiveness of phytoremediation of perchlorate. Two main phytoremediation mechanisms identified by these studies included slow uptake and phytodegradation of perchlorate in leaf tissue and rapid degradation in rhizosphere (rhizodegradation). These mechanisms were shown to apply to both aquatic and terrestrial plant systems. Perchlorate taken up into plants is phytodegraded slowly by perchlorate reductase enzyme to chloride via intermediate products, chlorate (ClO3ˉ) and chlorite (ClO2ˉ). The slow phytodegradation results in accumulation of perchlorate in plant tissues, which potentially is recycled in some cases into the environment via leaching from decaying senesced leaves. Phytoaccumulation of a fraction of the perchlorate taken up by plants has been confirmed by the detection of perchlorate in food crops, dairy milk, and human breast milk. This research provides a feasible solution to the nationally recognized problem of perchlorate accumulation in plants. A method for biostimulation of perchlorate-degrading microorganisms in the rhizosphere using natural and artificial electron donors is described for hydroponics system and soil bioreactors.
Most previous experiments on uptake of perchlorate by plants were performed in controlled laboratory conditions for relatively short durations. Therefore, little or no data were available for long-term experiments that simulate the length of the growing season experienced in the field where varying environmental conditions may affect perchlorate plant uptake. Environmental factors that may influence perchlorate plant uptake include distance from source of contamination, exposure duration, availability of organic carbon in the subsurface, and the recycling potential of perchlorate from deciduous leaves. Former rocket fuel manufacturing facilities at the Las Vegas Wash (LVW) in Las Vegas, Nevada, and Longhorn Army Ammunition Plant (LHAAP) in Karnack, Texas, are among more than 150 facilities contaminated by perchlorate nationwide. This study collected and analyzed multiple plant samples surrounding the LHAAP and the LVW to determine the long-term fate of perchlorate accumulated in plant tissue (mainly leaves), spatial and seasonal variability of perchlorate concentration in plants, and the difference in perchlorate concentrations among plant species. The findings of this study, together with recommendations of approaches that should be included in the design of phytoremediation of perchlorate contaminated sites, will be submitted to the journal Ecotoxicology and Environmental Safety.
NDMA is a very potent emerging carcinogen that has been detected in drinking water wells near rocket engine testing facilities and in areas where chlorinated wastewater was used for aquifer recharge. The commonly used treatment technology for NDMA is ultraviolet (UV) radiation, which is expensive and cost-prohibitive in some applications. The U.S. Department of Defense has funded a number of initiatives to develop bioremediation methods for NDMA. Until this study, the efficacy of phytoremediation of NDMA was not known. A set of experiments was conducted to test the hypothesis that because of its water miscibility, NDMA should be readily taken up by phreatophytes. A greenhouse experiment was conducted using black willow (Salix nigra) and hybrid poplar (Populus deltoides ´ nigra) trees to remove NDMA from hydroponic bioreactors. Radiolabeled 14C-NDMA was used as a tracer to investigate the fate of NDMA taken up by these trees and a mass balance analysis was performed. The findings of this experiment were peer reviewed and accepted for publication in the journal Environmental Science & Technology in a special issue that focuses on emerging contaminants.
NDMA and perchlorate frequently occur as co-contaminants at a number of sites because both are used in the production of rocket fuel. The fate of perchlorate and NDMA, however, in the environment when present as co-contaminants is lacking in the literature. This research project investigated whether phreatophytes selectively take up perchlorate or NDMA and the effect of NDMA on the microbial degradation of perchlorate in the rhizosphere. Results of this experiment together with the findings from phytoremediation of NDMA were peer reviewed and accepted for publication in the journal Environmental Science & Technology.
The ability of plants to degrade perchlorate was evaluated at concentrations ranging from 50 mg L-1 to 10,000 mg L-1 in hydroponic and soil bioreactors. This study established the perchlorate concentration threshold toxic and lethal to young willow plants. The concentration threshold could be used as a guide in designing a viable phytoremediation project in which young willow plants will be used.
Summary/Accomplishments (Outputs/Outcomes):Many field and greenhouse experiments were conducted to accomplish the specific objectives outlined in this research project. Findings from each experiment are summarized below.
Long-term Fate of Perchlorate in Vegetation at LHAAP and LVW
All vegetation species sampled from LHAAP and LVW contained quantifiable levels of perchlorate, implying evidence of uptake of perchlorate by the plant species tested. Plant perchlorate concentrations varied between plant species, initial perchlorate concentration in the rhizosphere, and stage of plant maturity. At LHAAP, the highest perchlorate concentration was measured in S. nigra, crabgrass (Digitaria spp.), and bermudagrass (Cynodon dactylon). Salt cedar (Tamarix ramosissima) contained the highest perchlorate concentration at the LVW. Vegetation of the same plant species growing near the sources of contamination contained higher perchlorate and plant perchlorate concentration, which decreased with distance from the sources of contamination. Most of the fraction of perchlorate taken up by plants was accumulated over time and measurable at the end of the growing season when the plants senesce because phytodegradation is a slow process. Consequently, leaves collected later in their growing season and senesced leaves contained significantly higher perchlorate concentrations than the young and live leaves. Senescent leaves and litter fall contained higher perchlorate concentration than live leaves, implying that plants recycled and released perchlorate back into the ecosystem. Soil collected from wetland areas had little or no perchlorate. Our investigation suggests that harvest and subsequent bioremediation of the plant biomass may be required in the design of a phytoremediation at some sites.
Use of Dissolved Organic Carbon for Biostimulation and Enhancement of Perchlorate Rhizodegradation in Soil and Water Media
The main limitation to biodegradation of perchlorate in most soils and groundwater tends to be the availability of an adequate supply of organic carbon or electron donors. This study investigated the use of electron sources provided as dissolved organic carbon (DOC) from chicken litter/manure extract, mushroom, cow manure, acetate, and ethanol to enhance and sustain rhizodegradation of ClO4ˉ in contaminated soils and groundwater. This approach reduced the residence time of perchlorate in soils and groundwater and reduced the well-documented plant uptake of perchlorate from contaminated field sites. The total perchlorate mass recovered from leaves of willow plants augumented with DOC was an order of magnitude lower than from plants’ leaves grown in media without DOC augumention; thus suggesting that biostimulation of rhizodegradation has reduced largely the potential for uptake of perchlorate by willow plants. Under unlimited supply of electron donors, however, the plants may play a secondary role to ClO4ˉ perchlorate reducing microbes in terms of fast and instant rhizodegradation of perchlorate in the soil. Under this condition, plants play the role of a hydraulic pump, which is responsible for mobilizing the pollutant plume. On the contrary, when DOC is limited (commonly under natural conditions) and on a long-term basis, plants have been reported to play more primary roles in controlling the pollutant plume but also providing the source of organic carbon through root exudates, of which a lag time of a few weeks would be expected. There was a clear distinction between hydroponic bioreactors with and without DOC amendment.
Nonsterile DOC from animal waste, like chicken and cow manure, could be a potential source of perchlorate degrading bacteria, providing the benefits of bioaugmentation that highlights a double-advantage in speeding up the microbial activity in perchlorate contaminated sites. 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 (without DOC). 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 the soil and agricultural waste products used as electron sources confirms the documented ubiquity of perchlorate-degrading microbes.
Analytical Method and Potential Phytoremediation of NDMA as Single and Co-contaminants by Willow Plants
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 UV detector. The 1.2 mL aqueous samples collected from the bioreactors were filtered through 0.2 μm syringe filters (Fisher Scientific), and 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 and changing to 50 percent B in 6 minutes and returning to 98 percent A at 7 minutes and held for further 3 minutes. 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.
Investigation of potential phytoremediation of NDMA by willow plants was done. Hydroponic experiments conducted with S. nigra showed a linear correlation between the mass of NDMA removed from solution and the incremental volume of transpired water. The rate of NDMA removal was described by zero-order kinetics. The linear relationship between rate of NDMA removal and rate of transpiration indicated that the decontamination process mainly was passive plant uptake and phytovolatilization. Radiolabeled studies are needed to determine the fate of NDMA taken up into the plant. To address potential ecotoxicological concerns, it is important to know whether NDMA is phytoaccumulated, phytodegraded, and/or phytovolatilized to the atmosphere. Willow plants grown in NDMA-contaminated water did not show any signs of phytotoxicity at 1,000 μg L-1 NDMA concentration.
Microbial mediated rhizodegradation did not play a role in the decontamination process. In the unplanted bioreactors (control), the concentration of NDMA remained constant for 67 days of the experiment. The constant concentration of NDMA in the unplanted control bioreactors during the duration of the experiment suggests that neither sorption to the glass nor microbial degradation of NDMA in the bioreactors took place.
There was no competitive removal of NDMA and perchlorate observed in bioreactors dosed with both NDMA (0.7-1.0 mg L-1) and perchlorate (27 mg L-1). Although NDMA was removed primarily from the solution by plant uptake, perchlorate was removed predominantly by rhizodegradation. In the presence of NDMA, a slower rate of rhizodegradation of perchlorate was observed but still significantly faster than the rate of NDMA uptake. Experiments conducted with radiolabeled NDMA revealed that 46.4 (± 1.1) percent of the total 14C-activity recovered was in the plant tissues and 47.5 percent was phytovolatilized. The 46.4 (± 1.1) percent in the plants was distributed as follows: 18.8 (± 1.4) percent in leaves, 15.9 (± 5.9) percent in stems, 7.6 ± 3.2 percent in branches, and 3.5 ± 3.3 percent in roots. The poor extractability of NDMA with methanol-water (1:1 v/v) from stem and leaf tissues suggested that NDMA was assimilated by plants. The calculated transpiration stream concentration factor of 0.28 ± 0.06 further suggests that NDMA was taken up passively by the phreatophytes and mainly phytovolatilized.
Effect of Perchlorate Concentration on Phytoremediation
The effect of perchlorate concentration on rhizodegradation and plant uptake using hydroponic and soil bioreactors planted with willows (Salix babylonica) was studied under greenhouse conditions. In both hydroponic and soil bioreactors, the water and soil used were spiked with DOC from cow manure extract at approximately 300 mg L-1. The study was conducted at three perchlorate concentration ranges: low (50-150 mg L-1); high (100-700 mg L-1); and very high (1,000-10,000 mg L-1) with DOC nonlimiting. To determine the role of plants, the first two experiments (low and high concentrations) were accompanied with similar nonplanted bioreactors. A planted set of soil bioreactors filled with sandy loam soil having a perchlorate concentration range of 1,000-10,000 mg L-1 was used to confirm some of the findings obtained in the earlier high concentration hydroponic bioreactor experiment.
Perchlorate degradation from the low-concentration experiments showed that regardless of concentration within the 50-150 mg L-1 range, complete perchlorate removal was reached within 5-8 days for both planted and nonplanted bioreactors. Experiments with high perchlorate concentration (100-700 mg L-1) showed significant difference in perchlorate removal rate between planted and nonplanted bioreactors. The high perchlorate concentration planted bioreactors experiment had similar results to the low-concentration experiment in which all of the perchlorate was removed within 6-11 days. For nonplanted high perchlorate concentration bioreactors, a substantial amount of perchlorate was still in solution when the experiment was terminated at day 11 (4.52%, 9.68%, 17.89%, and 61.07% of the initial concentrations for 100, 300, 500, and 700 mg L-1, respectively).
Planted hydroponic bioreactors spiked with very high perchlorate concentration (1,000-10,000 mg L-1) were not able to remove completely perchlorate in 15 experimental days despite the abundance of electron donors in form of cow manure extract. Signs of phytotoxicity in the hydroponic studies were evident 2 days after application of perchlorate. Willow plants in bioreactors containing 2,000 mg L-1 of perchlorate or more had shed all their leaves by the fourth day. All willow plants planted in bioreactors containing 1,000 – 10,000 mg L-1 perchlorate suffered phytotoxic effects from day 2 and totally dried up by day 8. When the experiment was stopped on day 15, approximately 46.02 percent, 66.24 percent, 55.99 percent, 90.61 percent, and 92.18 percent of the initial perchlorate doses were still in solution for 2,000; 4,000; 6,000; 8,000 and 10,000 mg L-1 perchlorate, respectively. It was interesting to note that bioreactors augumented with DOC showed more severe phytotoxicity than bioreactors without DOC augumentation.
One set of soil bioreactors was dosed with 300 mg L-1 DOC as cow manure extract and perchlorate concentrations of 1,000; 5,000; and 10,000 mg L-1, whereas a second set with similar perchlorate treatments was not amended with DOC. Results from this experiment showed that the willow plants in the DOC-treated bioreactors with 5,000 and 10,000 mg L-1 perchlorate started to dry up within 12 hours of perchlorate dosing. By day 2, the plants were dead and had shed all their leaves. The corresponding no-DOC treatments of 5,000 and 10,000 mg L-1 started to show signs of phytotoxicity approximately 24 hours after application. Four days later all plants without DOC started to shed their leaves. Treatments that received 1000 mg L-1 perchlorate had less intense and slow phytotoxic reactions. All other plants had died and dried up by day 8 except the 1000 mg L-1 perchlorate dosed plants. There was still high concentration of perchlorate in solution (46.02%, 66.24%, 55.99%, 90.62%, and 92.18% for 2,000; 4,000; 6,000; 8,000 and 10,000 mg L-1, respectively) at the time of termination of the experiment.
Phytotoxicity for young willow plants was evident at 1000 mg L-1 perchlorate, whereas plant mortality started at 2,000 mg L-1 perchlorate. Phytotoxicity and mortality could have been caused by either perchlorate, sodium, or both.
Conclusions:- A significant fraction of perchlorate taken up into leaves is not phytodegraded by the onset of senescence, resulting in phytoaccumulation of perchlorate in above-ground plant tissues mainly in plant leaves.
- Plants potentially recycle phytoaccumulated perchlorate through senescence and leaf fall, hence recontaminating previously phytoremediated soil and groundwater. This phenomenon negates the effort of phytoremediation.
- Use of DOC as an electron donor from synthetic and natural sources such as animal waste and compost extract (chicken litter/manure, cow manure, mushroom) biostimulates and enhances rhizodegradation of perchlorate.
- Enhanced rhizodegradation reduces the amount of perchlorate uptake by plants by an order of magnitude, thus minimizing perchlorate recycling through senescent leaf fall and decomposition.
- NDMA is absorbed passively (taken up) by willow plants and mainly phytovolatilized; there is yet no evidence of rhizodegradation.
- There is a linear correlation between the mass of NDMA removed from solution and the incremental volume of transpired water.
- There is no preferential phytoremediation of perchlorate and NDMA as co-contaminants.
- Young willow plants exhibited signs of phytotoxicity at 1,000 mg L-1 of perchlorate concentration. Plants shed their leaves and eventually die at perchlorate concentrations above 2,000 mg L-1.
- Enhanced rhizoremediation is a feasible and viable technology to degrade perchlorate in contaminated/polluted soil and groundwater.
Journal Articles on this Report: 1 Displayed | Download in RIS Format
| Other project views: | All 17 publications | 2 publications in selected types | All 2 journal articles |
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Mwegoha W, Mbuya OS, Jain A, Ugochukwu NH, Abazinge MD. Use of chicken manure extract for biostimulation and enhancement of perchlorate rhizodegradation in soil and water media. Bioremediation Journal 2007;11(2):61-70. |
R831090 (Final) |
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phytotoxicity, phytoremediation, ecotoxicology, phreatophyte, in situ, dissolved organic carbon, DOC, perchlorate, NDMA, co-contaminants, manure, polymerase chain reaction, PCR, ubiquitous, carcinogen, emergent contaminants,
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INTERNATIONAL COOPERATION, Scientific Discipline, Waste, RFA, Molecular Biology/Genetics, Hazardous Waste, Environmental Microbiology, Contaminated Sediments, Hazardous, Bioremediation, bioavailability, industrial waste, biodegradation, microbial degradation, phytoremediation, perchlorate, degradation, contaminated sediment, Superfund site remediation, N-Nitrosodimethylamine, contaminants in soil, contaminated soils, microbiology, contaminated soil, bioremediation of soils, biochemistry
Progress and Final Reports:
2004 Progress Report
2005 Progress Report
Original Abstract