I D A H O N A T I O N A L L A B O R A T O R Y Use of Enzyme Probes for Estimation of Trichloroethylene Degradation Rates and Acceptance of Monitored Natural Attenuation M. Hope Lee D. Rogers, H. Silverman, A. O’Hagan, and D. Swift Estimating degradation rates is often a high priority for evaluating the potential of selecting monitored natural attenuation (MNA) as a remedy at field sites. Molecular biological tools (MBTs) can contribute significantly in directly estimating or documenting the extent and rate of microbial degradation. A demonstration to develop the relationship between enzyme activity probes (EAP) for aerobic cometabolic enzymes, and TCE degradation rates is presently underway for contaminant plumes located at: (1) Building 3001, Tinker Air Force Base (AFB), Oklahoma and (2) Installation Restoration Program Site 2, El Toro, California. The primary objectives of this demonstration are to validate methods for (a) directly assessing the activity and contribution of targeted enzymes for cometabolism, and (b) determining TCE degradation rates for aerobic chlorinated solvent plumes. Tinker AFB is the site for several large aerobic contaminant plumes; one such plume is a mixed chromium, chlorinated solvent plume located beneath Building Contaminant trend data suggest that the chlorinated solvents present in situ are attenuating and regions of the plume are aerobic. Initial EAP analysis of groundwater from the site revealed that the in the upper zone or shallow aquifer, there was a significant response to at least one of the EAPs at 4 of the 5 wells analyzed. Other aerobic geochemical indicators, including elevated alkalinity, elevated chloride concentrations and overall lower total organic carbon concentrations, were present in this zone as well. The middle zone of the aquifer also showed significant response with the EAPs at 3 of 5 wells. The three lower zone groundwater wells evaluated also exhibited activity with at least one of the EAP. In general, groundwater wells which exhibited conditions appropriate for aerobic microbial growth also showed significant activity with EAPs. As such, a microcosm study to evaluate TCE degradation rates is planned for early August. Currently, the first round of screening for aerobic cometabolic activity is underway for the IRP Site 2 in El Toro, California. Based on historical groundwater geochemical conditions, including aerobic conditions, the presence of organic carbon, this site is considered a good candidate site for validation of the methods. To date, the MBTs described have not been applied in shallow, tidally influenced groundwater systems. Specific work includes microbiological sampling for EAP analysis and collection of contaminant and geochemical data, providing control assays for groundwater and soil analyses, employing molecular techniques independently of the EAP, and executing a microcosm study to obtain a degradation rate. Figure 2. Example micrographs following application of enzyme activity probes to groundwater. A: Micrograph of total cells DAPI-stained. B: Positive Response, and C: Negative Response. ABC Geochemistry Samples Reason for Analysis: VOCsQuantify contaminants and identify if any anaerobic degradation products are present TOCIndicates the amount of available organic material for biodegradation Anions – chloride, sulfate, carbonate, and bicarbonate Indicative of conditions that may affect microbial growth (either inhibitory or beneficial) Cations – calcium, magnesium, potassium, and sodium CopperPresence of copper inhibits microbial growth Methane/ethane/ethene Indicative of anaerobic processes, low levels serve as cometabolic substrate Phosphate and nitrateUsed as an electron acceptor Sample NameAquifer Zone Sample Depth (ft) TCE (µg L -1 ) DAPI cells ml -1 PA cells ml -1 3HPA cells ml -1 CINN cells ml BRUSZ E E E E BUSZ E E E E BUSZ E E E E+03 M-2BRUSZ E E E E+02 M-4BUSZ E E E E AR 300U-LSZ E E E E AR 299U-LSZ E E E E AUM-LSZ E E E E+03 M-4ARUM-LSZ E E E E ARM-LSZ E E E E AM-LSZ E E E E CRL-LSZ E E E E AL-LSZ E E E E+02 M-1CRL-LSZ E E E E+00 Located approximately 8 miles S of Oklahoma City 1942, Mission: international repair depot for aircraft, engines, & weapons systems. These activities used TCE, PCE, and metals; have resulted in on-site disposal of industrial wastewater and solvents into waste pits & trenches ( ) The aquifer under 3001 is divided into five discrete units Highest concentration of TCE is shallow (35,000 mg/L, < 75ft bgs) 3001 has been listed on NPL since 1987; ROD directs P&T P&T was shut down in April 2004, followed by rebound test; allowing for the opportunity to assess contributions of natural attenuation processes Dissolved Oxygen (DO) has been evaluated in plume and range from < 5 mg/L in the upper and middle zones to greater than 5 mg/L in the lower. Other geochemical parameters support these findings (low methane, ferrous iron) Tinker Air Force Base: Microcosm Upper Saturated Zone: Showed a significant response to the EAP PA at all but one of the wells sampled (Table 2), The least aerobic zone with only one well within the plume exhibiting aerobic conditions, Indications of anaerobic conditions : lower redox within the plume than other zones, elevated ferrous iron elevated alkalinity, elevated chloride presence, overall lower TOC. Exhibits the highest average contaminant concentrations (both TCE and cis-DCE; Figure 4). Upper Lower Saturated Zone: Well 2-444A, which is outside the plume showed significant activity with two of the EAP (Table 2) A exhibits low TCE concentrations (3 µg L -1 ). Wells outside contaminant plume are aerobic – 1- 13AR, 1-1A and 34B. Average TCE concentrations in this zone; vinyl chloride concentration higher than other zones. Nutrients essential for microbial growth are present (phosphorus, potassium). Alkalinity as CaCO3 and HCO3 lower than other zones indicating depressed rate of biodegradation. Middle - Lower Saturated Zone: One of the wells had a significant response to one of the EAP, and the other wells did not (Table 2). Lower-Lower Saturated Zone: All three of the wells sampled in this zone exhibited activity with one of the EAP (Table 2); The wells sampled for EAP varied in TCE concentrations (< µg L -1 ; Figure 4) The well in this zone assumed to be outside the plume exhibited: reduced sulfate, all iron present is ferrous (reduced state), depleted oxygen, likely impacted by contaminant. The samples within the plume exhibit higher DO concentrations than outside plume. Time Control: PA Control: Na Azide1-12BR1-13AR Location Most Recent TCE Concentration (ug/L) 1 Aquifer Condition 2 Location Description 02NEW29>100UnknownHigh-range TCE concentration area. 02NEW Unknown Low-range TCE concentration area; down and crossgradient from highest concentration area. 02NEW UnknownDowngradient, plume fringe. 02PZ04*5 - 50Unknown Low-range TCE concentration area; shallow; crossgradient from highest concentrations area. 02NEW19<5UnknownPlume fringe. 02NEW28<5UnknownPlume fringe. 02NEW16<5AerobicUp and crossgradient from highest concentration area. 02NEW15ND (2005)AnaerobicBackground; upgradient of plume. 02NEW07NDUnknownDowngradient of plume. Abstract Background Objectives Demonstration Site 1: Tinker Air Force Base Tinker Air Force Base: EAP screening Based on the initial screening EAP, three groundwater samples were collected and analyzed for: EAP (activity; Figure 5) DNA (metabolism of interest) TCE degradation (Figure 5) Oxygen availability (Table 3) Objective 1: Determine the importance of natural attenuation mechanisms at DoD sites. Combine a suite of innovative techniques including enzyme activity probes (Figure 2-3) and FISH along with historical geochemical data (Table 1) to provide a comprehensive assessment of active degradation mechanisms. Objective 2: Determine an aerobic biological degradation rate for the contaminant TCE, based on laboratory microcosms. Normalize the microcosm degradation rate based on enzyme probe activity, a direct measurement of degradation, such that a realistic rate for the field can be obtained. The primary objective is to develop a method for measuring in situ aerobic biological degradation rates using enzyme probes. Well NameZoneReasons for Selection 1-12BRUSZAerobic, high EAP activity, and high-range TCE concentration (~6000  g/L) 1-13ARU LSZAerobic, low EAP activity, and high-range TCE concentration (~3000  g/L) 2-444UM LSZ Aerobic, high EAP activity, and low-range TCE concentration (~100  g/L) O2O2 O2O2 Demonstration Site 1: El Toro, IRP Site 2 IPR Site 2, Former Marine Corps Air Station Shallow and Deep Aquifer ( bgs) TCE < 5µg L -1 – 160 µg L -1 Potential carbon source (humics- aromatic) No co-contaminants MNA proposed remedial strategy Tinker AFB: EAP screening & microcosm studies provide convincing evidence that aerobic cometabolism is an active mechanism in situ that is likely to be contributing to the attenuation of TCE. Rapid metabolism of oxygen in microcosm studies suggest that enhancing the aerobic processes in situ would be useful to (a) sustain the aerobic processes and (b) increase the biodegradation of TCE Paducah Gaseous Diffusion Plant(PGDP): EAP screening of groundwater from the North West plume was positive for activity (aromatic oxygenases) Significant activity in plume has resulted in a proposed microcosm study to determine attenuation rates for the distal portion of the plume Savannah River National Laboratory: Four distinct groundwater plumes were positive for activity (with EAP), A microcosm study was completed for a single groundwater sample,T-Area ; the laboratory determined half life for the gw sample analyzed was approximately 30 years from TCE. Over the past two years …EAP screening efforts have been conducted at over 15 sites; microcosm studies have been conducted at 8 DoD or DOE sites. 81 Figure 3. Cartoon schematic of EAP, oxygenase enzymes. Table 1. List of relevant geochemical parameters evaluated for aerobic processes. Figure 4. Contaminant plume maps for discrete aquifer units at Tinker AFB, Building Overlay, qualitative EAP data is included on each figure. Figure 5. Microcosm data for Tinker AFB; three independent wells were chosen for examination. Each treatment is evaluated for both activity with EAP and contaminant degradation over time. Table 3. Oxygen availability (mg L -1 ) data for microcosm study (Figure 5). Table 2. EAP and biomass (DAPI) data for the screening of groundwater from Building 3001, Tinker AFB. Figure 6. Contaminant contour maps for El Toro IRP Site 2, California. Table 4. List of groundwater well locations chosen for screening with EAP at IRP Site 2. Conclusions Figure 1. Toluene oxygenase pathways. * EAP cinnamonitrile * Methane Probe TCE Cell Labeled cell Probe (EAP) Carbon Source TCE Conclusions, Tinker AFB EAP determined activity of in situ populations from gw at Building 3001 Microcosm study provided evidence that in situ populations could degrade the contaminant TCE Rapid rate of degradation as evidenced by depletion of oxygen in microcosm (Figure 5; Table 3) TCE half-life estimated at years (95% confidence interval), assuming best conditions.