On-Site Detection of Explosives inGroundwater with a Fiber OpticBiosensorS A S K I A K . V A N B E R G E N , ‡

I R I N A B . B A K A L T C H E V A , §

J E F F R E Y S . L U N D G R E N , † A N DL I S A C . S H R I V E R - L A K E * , †

Center for Bio/Molecular Science and Engineering,Naval Research Laboratory, Washington, DC 20375-5348,George Mason University, 4400 University Drive, Fairfax,Virginia 22030-4444, and Geo-Centers, Inc., 1801 RockvillePike, Suite 405, Rockville, Maryland 20852-1633

Two primary explosives involved in groundwater con-tamination, 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), were detected on-site at lowppb levels with a semiautomated fiber optic biosensor.Validation of the Analyte 2000 (manufactured by ResearchInternational Inc., Woodinville, WA) for TNT and RDXdetection was performed at two Superfund sites, UmatillaArmy Depot and Naval Surface Weapons Center Crane.Samples from monitoring wells were split for analysis usingthe fiber optic biosensor on-site and using U.S. EPA SW-846 Method 8330 (reverse-phase high performanceliquid chromatography) in an offsite laboratory. The Analyte2000, a multifiber probe fluorimeter, was coupled to afluidics unit for semiautomated operation. The fiber opticbiosensor assay is based on a competitive fluorescentimmunoassay performed on the silica core of a fiber probe.From these studies, the limit of detection was determinedto be 5 µg/L for both TNT and RDX. In addition to thefield samples, extensive laboratory analyses were performedto determine cross-reactivity, matrix effects, and falsepositive/negative rates.

IntroductionPrior to the 1960s, wastewater from explosives manufacturingand demilitarization plants was typically disposed intounlined lagoons. As a result, the groundwater and soil nearthese lagoons have been contaminated with the two majorexplosives, 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). Since groundwater in areassurrounding contaminated sites is often tapped for bothdrinking water and irrigation, serious problems have arisenas both TNT and RDX have been found to be toxic to humansabove 2.0 µg/L TNT and RDX “Lifetime Health Advisory”level designated by the U.S. EPA (1-5).

Many of these contaminated sites are in the process ofcharacterization and/or remediation. It has been estimatedthat explosive remediation at each site will take between 10and 30 years for cleanup (6). Frequent sampling is required

to perform site characterization and monitor the remediationprocess. Current protocols for groundwater characterizationand monitoring remediation involve reverse phase highperformance liquid chromatography (6, 7). The disadvantagesof this method are off-site laboratory analysis and a cost of$200-350 per sample with a 30-day turnaround (8). Theability to perform TNT and RDX analysis of groundwatersamples on-site has many benefits. These advantages includereduced cost for both sample analysis and labor, analysis ofmore samples for better characterization, more effective useof robust analytical methods (i.e., HPLC) on select positivesamples, and the ability to redesign sampling methods dueto real-time feedback (6, 8). For these reasons, the develop-ment of test kits and biosensors for on-site detection ofexplosives has increased over the past few years.

Several test kits are commercially available for on-sitedetection of TNT and RDX in water including the Ensyscolorimetric method and three kits for TNT based on enzyme-linked immunosorbent assays (ELISA): EnvironGard, RaPID,and D Tech (3, 6, 8). The Ensys colorimetric method, originallydesigned for soil analysis, requires the filtration of severalliters of sample water over membranes to remove andconcentrate the explosive (2, 3, 6, 8). The TNT and RDX arethen removed from the membranes with acetone andanalyzed with the soil test kit. The EnviroGard was developedas a quantitative laboratory analysis kit; therefore, on-sitedetection with this method is not always practical. The RaPIDtest kit is effective with a large number of samples but iscostly if only a few samples need to be analyzed at one time.The D Tech kit is employed as a screening tool. The resultsfrom the D Tech kit are given as a range with the lowest levelof detection listed as 5 µg/L. One study showed a large numberof false positives using the D Tech test kit (3).

Another alternative for on-site detection is the use ofbiosensors (9, 10). The NRL fiber optic biosensor is based ona competitive fluoroimmunoassay performed on the surfaceof an optical fiber probe (11-14). Other fiber optic biosensorsemploying competitive immunoassays have been developedfor nonenvironmental applications (9, 10, 15-18). The NRLsensor employs the Analyte 2000 as a multiprobe fluorimeter(12). A TNT assay was originally developed with this sensor(11, 12), and an assay for RDX was recently established (19).With the NRL biosensor, antibodies specific to either TNTor RDX are immobilized onto the surface of an optical fiber.The groundwater sample is mixed with a solution containinga fluorescent analogue of either TNT or RDX in buffer andpassed over the probe for 5 min. When either TNT or RDXis present in the sample, a decrease in the maximumfluorescent signal proportional to the concentration of theexplosive is observed. Four optical probes can be connectedthrough “jumpers” (up to 50 m in length) to a single Analyte2000. The ability to implement long jumpers provides apotential use for down-hole measurements with a conepenetrometer or similar device during site characterization.

The purpose of this study was to validate a method forthe detection of TNT or RDX in groundwater using the fiberoptic biosensor. On-site analysis of groundwater, surfacewater, and leachate samples was performed at two militarysites: Umatilla Army Depot in Hermiston, OR, and the NavalSurface Weapons Center (NSWC) in Crane, IN. All sampleswere split on-site for biosensor analysis and SW846 Method8330 (HPLC) analysis at an off-site laboratory. In addition tothe on-site analysis, cross-reactivity, false positive/falsenegative rates, and matrix effects studies were performed inthe laboratory using the biosensor.

* Corresponding author phone: (202)404-6045; fax: (202)404-8897;e-mail: lcs@cbmse.nrl.navy.mil.

† Center for Bio/Molecular Science and Engineering, NavalResearch Laboratory.

‡George Mason University.§ Geo-Centers, Inc.

Environ. Sci. Technol. 2000, 34, 704-708

704 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 34, NO. 4, 2000 10.1021/es990815b CCC: $19.00 2000 American Chemical SocietyPublished on Web 01/06/2000

Experimental SectionPortable Fiber Optic Biosensor. The Analyte 2000 portablefiber optic biosensor, built by Research International (Wood-inville, WA), is capable of monitoring four optical fiber probessimultaneously using evanescent wave immunosensing. TheAnalyte 2000 has been previously described in detail (12).During the field trials, a fluidics module built at NRL, wasused to introduce the samples and reagents to the antibody-coated fibers. This semiautomated fluidics system consistedof three-way solenoid valves and a peristaltic pump asdescribed by Ligler et al. (20).

Preparation of the Fiber Probes. The preparation of thefiber probes has previously been reported by Shriver-Lakeet al. (21). To summarize, the polymer cladding was removedfrom the last 7 cm of a 10 cm, 600 µm diameter plastic-clad,silica optical fiber to form the sensing region. This regionwas tapered with hydrofluoric acid and rinsed with RO water.The antibodies were covalently coupled to the fiber surfaceusing the thiol-terminal silane, mercaptopropyltrimethoxy-silane, and a heterobifunctional cross-linker, N-succinimidyl4-malemidobutyrate (GMBS). The anti-TNT antibody, mono-clonal IgG 50591, and the anti-RDX antibody, monoclonalIgG 50359, were purchased from Strategic Diagnostics, Inc.(Newark, DE).

The sensing portion of the fiber probe was secured in acapillary chamber. To stabilize the antibody-coated probesfor field trials, the fibers were incubated for 30 min in asolution composed of 100 mM trehalose and 10 mg/mLbovine serum albumin (BSA) (Sigma) in 15 mM phosphatebuffer (pH 7.4) and dried with nitrogen. The capillary chamberwas then capped and stored at room temperature until use.

Synthesis of Fluorescently Labeled Analogues. The assaysfor both TNT and RDX are competitive immunoassays wherethe analyte and the fluorophore-labeled analogue competefor the limited number of available antibody binding sites.The synthesis of cyanine-5-ethylendiamine trinitrobenzene(Cy5-EDA-TNB), the fluorescently labeled analogue of TNT,was similar to the method described by Shriver-Lake et al.(11) with the following modifications. The pH of dissolvedEDA solution was lowered to 3.5 with acetic acid prior topurification by HPLC using a C18 column (µBondapak, 19 ×300 mm, Waters). The intermediate product (Cy5-EDA) waspurified with a gradient consisting of H2O and MeOH. Thegradient program for the Waters column was as follows: 0-8min, 0% MeOH; 8-12 min, 0-50% MeOH; 12-20 min, 50%MeOH; 20-40 min, 50-0% MeOH, at a flow rate of 5 mL/min. The final product (Cy5-EDA-TNB) was purified with agradient consisting of solvents H2O and MeOH. The gradientprogram for the Waters column was as follows: 0-6 min,35% MeOH; 6-14 min, 35-50% MeOH; 14-20 min, 50-100% MeOH; 20-24 min, 100% MeOH; 24-40 min, 35%MeOH at a flow rate of 5 mL/min. The synthesis of cyanine5-RDX-Hapten (Cy5-EDA-RDH), the fluorescently labeledanalogue of RDX, was performed as reported in Bakaltchevaet al. (19).

Competitive Immunoassay on the Fiber Probe. Shriver-Lake et al. (21) provides protocols for both the TNT and RDXassays. The concentration of the reference solution used was7.5 µg/L Cy5-EDA-(TNB or RDH) in PBAT buffer which iscomposed of phosphate buffered saline (PBS), 2.0 mg/mLBSA, 5% acetone, and 0.1% Tween 20 (Sigma). The ground-water samples were filtered, and stock fluorescent analoguewas added so that the final concentrations of componentswere 7.5 µg/L Cy5-EDA-(TNB or RDH) in PBAT buffer.

Multiple analyses were performed on each fiber due tothe ability to “regenerate” the probes by exposure to 50%EtOH in a buffer composed of 8.3 mM phosphate buffer,10% acetone, 0.2% Tween 20 for either 5 min for the TNTassay or 10 min for the RDX assay (11, 19, 22). This permits

a reference sample (no explosives) to be run before and aftera test sample to determine the maximum fluorescence fromthe bound Cy5 analogue. Equation 1 was

used to determine the percent inhibition from the maximumsignal for the test sample. Use of both the pre- and post-reference signals slightly reduces variability between anal-yses. The percent inhibition was proportional to concen-tration of TNT or RDX in solution. The unknowns werequantified by comparison to best-fit standard curves thathad been established from spiked concentrations of TNT orRDX (Figure 1). More than 10 samples were analyzed on asingle fiber probe with regeneration.

Field Trials at Umatilla Army Depot and Naval SurfaceWeapons Center Crane. Groundwater and well effluentsamples were collected at both sites by Black and Veatch,Special Projects Corp. (Takoma, WA) and were kept cool (4˚C) and stored in amber glass bottles. Testing at the two sitesallowed the opportunity to evaluate the sensors with waterfrom different geochemical areas. Samples from Umatillagenerally had high concentrations of nitrates, whereas somesamples from Crane sites were acidic and contained highlevels of trichloroethane. At Umatilla, each of the 21 unknownsamples was tested on a minimum of seven fibers. Highlycontaminated samples were diluted in order to get less than70% inhibition results for quantitation (7 RDX, 2 TNT).Fourteen unknown samples were tested at Crane on aminimum of seven fibers.

Laboratory Experiments. Additional tests were performedto determine cross-reactivity, matrix effects, and false signals.Competitive assays were carried out using compounds thatcould potentially react with the immobilized antibody. Theimmunoassays were performed as previously stated with thepotentially cross-reactive compound being spiked into thebuffer sample. The limit of detection and the concentrationat which 50% inhibition of the maximum signal (IC50)occurred were determined for each possible cross-reactant.

The effects of different matrices on the assays were studiedusing blank water samples from various sites that were spikedwith high and low concentrations of TNT or RDX. The assayswere performed as previously stated. A minimum of sevenreplicates were performed for each spiked sample. Thesedata demonstrated precision and accuracy of the assays withreal world samples.

Experiments were conducted to determine the false-positive and false-negative rates for TNT and RDX. Buffer

FIGURE 1. TNT and RDX standard curves. The percent inhibitionwas obtained from spiked buffer samples to generate standardcurves for TNT (9) and RDX ([). A minimum of seven replicateswere performed for each spiked concentration.

% inhibition ) [1 - [(test signal)/((referencepre + referencepost)/2)]]*100 (1)

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was spiked with 2× and 0.5× the mean detection limitconcentration, and the assays were performed as describedabove.

SW 846-Method 8330. Confirmatory analysis employingMethod 8330 was performed on all the field water samplesby the EPA-certified contract laboratory QST, EnvironmentalScience & Engineering, Inc. (Gainesville, FL) under contractto U.S. EPA Region 10. A salting-out preconcentration stepwas done on all test water samples by QST.

Results and DiscussionThe fiber optic biosensor was used to measure the concen-tration of TNT and/or RDX in water samples from UmatillaArmy Depot and NSWC Crane. Four devices were operatedat each test site for 3-4 days. Two different instruments wereemployed per sample to obtain the more than seven analyses.Inter- and intra-probe variability for each instrument werenot separately identified, and both are included in thestandard deviation value.

Analysis of Groundwater for TNT. Table 1 shows the TNTresults for HPLC (SW 846 Method 8330) and the fiber opticdata from Umatilla. TNT analysis was not performed on thefield samples from Crane due to laboratory QA/QC issuesregarding the silane used for immobilization and TNT holdingtimes. All biosensor data reported are signal averages fromseven or more analyses including four sample values minus

outliers as determined by the Q-test at 95% confidence. Basedon the standard curve, the limit of detection, calculated fromthe lowest TNT level producing a signal ( 3 SD from allreference samples or zero inhibition (3 SD ) 9% inhibition),for the fiber optic biosensor was determined to be 5 µg/L forTNT. The HPLC data is from a single analysis. The salting-out procedure combined with HPLC in Method 8330 is listedas having a detection limit of 0.11 µg/L TNT, while the limitof the direct injection method is 20 µg/L (7). The fiber opticbiosensor identified all but two field samples containinggreater than 5 µg/L TNT as positives. The two samples hadstandard deviations greater or equal to the averages and wereconsidered “negative”. In both cases there were almost evensplits of replicates between very low and higher values.

A regression analysis of the fiber optic biosensor resultsfrom Umatilla versus those measured by HPLC for all positivesamples gives the equation, Y ) 0.54X + 14.19 (r 2 ) 0.79).If the two techniques were in perfect agreement, the linearregression would have a slope of 1.0, a y-intercept of 0, anda correlation coefficient of 1.0. The regression analysisindicates that the fiber optic assay overestimated the resultsat low analyte concentrations and underestimated the resultsof TNT at high concentrations as compared to the HPLCmethod.

Cross-reactivity studies were performed with a numberof compounds closely related to TNT or those potentiallyfound in contaminated groundwater. The results of thesestudies are shown in Table 2. Limit of detection (LOD) valuesgreater than 1000 µg/L indicate no detectable inhibition. Therelative cross-reactivity is determined by the IC50. Only 1,3,5-trinitrobenzene (TNB) showed any appreciable level of cross-reactivity with the anti-TNT antibody. TNB was detected at10 µg/L and gave 50% inhibition of the maximum signal(IC50) at 500 µg/L. This cross-reactivity was expected as theantibody was raised against a TNB conjugate. Other com-pounds were detected at significant concentrations, but thecross-reactivity at the IC50 was less than 4% (IC50 for testedcompound divided by IC50 for TNT).

Several water samples from various sites, which did notcontain TNT, were tested for matrix effects. The blank watersamples were spiked with 25 and 250 µg/L TNT, the

TABLE 1. Fiber Optic Biosensor Analysis on Site Samples

RDX (µg/L) TNT (µg/L)

site sampleAnalyte

2000Method

8330Analyte

2000Method

8330

Umatilla Army Depot4-24 77 ( 18 39 BDLb BDLb

combine 2c,d 72 ( 37 109 67 ( 19 3combine 1c,d 60 ( 12 118 37 ( 26 34-102c,d 31 ( 7 40 18 ( 4 37WO-21c 28 ( 6 39 BDLb BDLb

4-25 27 ( 9 21 9 ( 8 BDLb

4-117c 22 ( 11 21 BDLb BDLb

4-112d 21 ( 6 15 37 ( 9 164-3c 17 ( 4 13 BDLb 0.074-7c 15 ( 4 13 BDLb BDLb

EW-1c 14 ( 4 9 >100 126WO-24c 9 ( 5 9 19 ( 11 BDLb

009c,d 9 ( 6 4 28 ( 11 23EW-4c,e BDLb 20 BDLb 0.54-114e BDLb 16 58 ( 43 94WO22d,e BDLb 14 BDLb 0.02SB-3e BDLb 14 BDLb BDLb

4-114Dc-e BDLb 0.34 56 ( 15 2.04-113d,e BDLb 9 BDLb 1EW-3c,d BDLb 2 17 ( 16 94-111c BDLb 0.4 76 ( 20 94

NSWC Crane10C55 84 ( 44 184NAa NAa NAa

03C08AP2 57 ( 32 126 NAa NAa

03C10 50 ( 25 121 NAa NAa

03C09P2 44 ( 9 146 NAa NAa

03C03P2d 40 ( 9 68 NAa NAa

spring 36 ( 19 119 NAa NAa

10-07 13 ( 6 29 NAa NAa

10C55P2 12 ( 9 51 NAa NAa

10-17e BDLb 35 NAa NAa

03-34 BDLb 41 NAa NAa

10-08 BDLb 24 NAa NAa

03C04 BDLb BDLb NAa NAa

10C37 BDLb BDLb NAa NAa

10C57 BDLb BDLb NAa NAa

a NA - not analyzed. bBDL - below detection limit. c Dilutionperformed to determine RDX values. d Dilution performed to determineTNT values. e Considered “negative”.

TABLE 2: Cross-Reactivity of Immobilized Anti-RDX Antibodyand Anti-TNT Antibodya

50% inhibition(IC50) µg/L

limit of detectionµg/L

sample RDX TNT RDX TNT

RDX 33 >1000 5 >1000HMX >1000 >1000 100 >1000TNT >1000 46 >1000 51,3,5-trinitrobenzene >1000 500 1000 102-amino-4,6-

dinitrotoluene>1000 1500 >1000 50

2,4-dinitrotoluene >1000 >1500 >1000 50-100tetryl >1000 >1500 1000 1501,3-dinitroglycerin >1000 >1000 1000 2501,2-dinitroglycerin >1000 >1000 >1000 3504-amino-2,6-

dinitrotoluene>1000 >1500 1000 500

dinitroethylene glycol >1000 >1000 1000 5001,3-dinitrobenzene >1000 >1500 1000 750trinitroglycerin >1000 >1000 1000 >10002,6-dinitrotoluene >1000 >1500 >1000 1500nitrobenzene >1000 >1500 >1000 >15002-nitrotoluene >1000 >1500 >1000 >15003-nitrotoluene >1000 >1500 >1000 >15004-nitrotoluene >1000 >1500 >1000 >1500

a Limit of detection: lowest concentration to give more than 9%inhibition of the reference. Signal IC50: concentration that gives 50%inhibition of the reference signal.

706 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 34, NO. 4, 2000

concentrations suggested by the EPA (5 and 50 times thedetection limit). Figure 2 shows the percent inhibition resultsfrom this study. At the lower concentration, all the spikedsamples were lower than the theoretical value for 25 µg/L,while the percent inhibitions for the higher spiked concen-trations were closer to the theoretical value. From theseassays, bias (accuracy) and precision were determined usingthe following equations (eq 2 and 3)

where Xh is the mean value for seven or more replicatedeterminations, X is the spiked concentration, and s is thestandard deviation. Ideal bias is 100% and precision is 0%.The accuracy is close to ideal for the 250 µg/L spiked samplesfrom distilled water (104%), Umatilla (115%), and LAAP (97%)but is only 77% for the SUBASE Bangor samples. Three of thefour 25 µg/L spiked samples had bias values greater than75% with the exception being SUBASE Bangor at 54%. Exceptfor the 25 µg/L SUBASE Bangor sample, the precision wasless than 15%. This study indicates that there are matrix effectsfrom groundwater especially from SUBASE Bangor. There-fore, it is recommended that prior to full scale analysis at atest site, uncontaminated water from that site be spiked atseveral concentrations and analyzed for matrix effects.Further study needs to be performed to determine if theseeffects could be reduced or eliminated with simple samplepreparation, i.e., solid-phase extraction.

The goal of any field analysis is to identify all samplescontaining TNT greater than the stated detection limit (i.e.,no false negatives). The detection limit of 5 µg/L wasdetermined to be the lowest concentration that can bedefinitely and consistently identified by the fiber opticbiosensor assay. Laboratory experiments conducted withsamples spiked with TNT at 1/2 (2.5 µg/L) and 2 times (10µg/L) the detection limit gave 62% positives for the 2.5 µg/Lsamples and 0% negatives for the 10 µg/L samples. The goalof having no false negatives was achieved with the 10 µg/Lsamples. The high level of positives at 2.5 µg/L can be partiallyexplained by the standard curve and variability. The standardcurve for TNT (Figure 1) is an asymmetric sigmoid which islinear in the middle range and gradually levels off at thelower and upper ends of detection. The variability betweenanalyses and fibers can also effect the number of positives.The high number of positives below 5 µg/L suggests that thesensor detection limit could possibly be lower upon furtherreduction in probe variability and matrix effects.

Analysis of Groundwater for RDX. On-site analysis forRDX was performed on samples from Umatilla and Crane.Table 1 shows the HPLC (SW 846 Method 8330) and fiberoptic biosensor RDX results. As previously mentioned, thereported data for the fiber optic biosensor are the averageresults and their standard deviations from seven or morefibers including two samples minus outliers (Q-test at 95%confidence limits). As with TNT, the limit of detection,calculated from the lowest RDX level producing a signal (3 SD from all reference samples or zero inhibition (3 SD )9% inhibition), for the fiber optic biosensor was determinedto be 5 µg/L RDX. The salting-out procedure with HPLC inMethod 8330 is listed as having a detection limit of 0.84 µg/LRDX, while the direct injection method is about 20 µg/L (7).The fiber optic biosensor positively identified 20 out of 28samples containing greater than 5 µg/L RDX. Seven samplesthat had standard deviations greater or equal to the resultswere considered “negative”. In all cases, the seven or moreresults were evenly split between high and low values, suchthat no signal value could be tested as a possible outlier.During the trials, the operator could not identify any obviousproblems which would suggest rerunning the samples.

A regression analysis of the fiber optic biosensor resultsversus those measured by HPLC for all positive samples givesthe equation Y ) 0.51X + 13.16 (r 2 ) 0.64) for Umatilla andY ) 0.36X -9.15 (r 2 ) 0.69) for Crane. The results fromUmatilla indicate that the fiber optic assay overestimatedthe results at low RDX concentrations and underestimatedthe results at high concentrations. The fiber optic biosensorunderestimated all the RDX samples from Crane.

A number of compounds closely related to RDX weretested to determine cross-reactivity with the monoclonal IgG50359. The detection limit and the concentration where 50%inhibition of the maximum fluorescence signal occurred foreach compound tested are given in Table 2. Only HMXshowed an appreciable limit of detection level at 100 µg/L.The cross-reactivity of HMX was expected as it is a byproductof RDX manufacture. All the compounds evaluated failed tocause 50% inhibition of the maximum fluorescence signal atthe 1000 µg/L concentration (<3% cross-reactivity).

Studies were performed on the effects of different samplematrices. In these studies, blanks of groundwater from severalmilitary sites were spiked with 20 and 75 µg/L RDX. Thepercent inhibition results for the matrix effects experimentsare shown in Figure 3. All samples gave values lower than thetheoretical values indicating some level of matrix interference.The higher concentrations of RDX had bias levels (eq 2) ofgreater than 80% for all samples except Umatilla. The

FIGURE 2. Matrix effects on TNT fiber optic assay: Blankgroundwater from several sources are spiked with 25 and 250 µg/LTNT. A minimum of seven replicates were performed for each spikedsample. The theoretical percent inhibition is shown for 25 and 250µg/L TNT as determined from the standard curve.

bias ) (Xh/X) * 100 (2)

precision ) (s/X) * 100 (3)

FIGURE 3. Matrix effects on RDX fiber optic assay: Blankgroundwater from several sources are spiked with 20 and 75 µg/LRDX. A minimum of seven replicates were performed for each spikedsample. The theoretical percent inhibition is shown for 20 and 75µg/L RDX as determined from the standard curve.

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precision values (eq 3) were less than 10% for all the 75 µg/Lsamples. Larger discrepancies were found in the 20 µg/Lsamples. Due to the observation of matrix effects at somesites, it is recommended that blank water be spiked atdifferent concentrations and tested initially at each site todetermine the level of interference. If interference occurs, astandard curve should be generated in that matrix for thatspecific site. As with the TNT, further studies are needed inthe area of sample pretreatment to reduce or eliminate matrixeffects.

As with the TNT analysis, the goal for RDX field analysiswas to identify all the samples containing RDX greater thanthe stated detection limit of 5 µg/L. The rate of positives,determined by spiking water samples at 1/2 the concentrationof the detection limit (2.5 µg/L), was 42%, while 0% negativeswere obtained for samples spiked at 10 µg/L (2 times thedetection limit) achieving the false negative goal of 0%. Aswith the TNT studies, the high level of positives for the 2.5µg/L samples indicates a conservative limit of detection. At1 µg/L, the positive rate drops to 25% and 10% for 0.5 µg/L(data not shown).

Further studies will continue to focus on identifyingsources of variability and their solutions. There are manyprobable causes of variation such as the sample matrix, probemanufacturing differences, and operator and instrumentvariability. As mentioned previously, the effects of differentmatrices will be investigated. Results from calibration curvesfor the individual instruments will be compared to thecombined instrument standard curve currently used to helpaccount for instrument to instrument variability. Variabilitybetween fibers currently cannot be extensively addressed asthey are not commercially available, and, therefore, limitedmeasures of quality control are possible.

The fiber optic biosensor provides a method for perform-ing on-site analysis of explosive contaminated groundwaterfor remediation and/or site characterization. TNT and RDXcan be detected at concentrations greater than or equal to5 µg/L in under 20 min with little sample preparation orwaste generation. Minimal cross-reactivity was observed withthe antibodies employed on the fiber optic probe. At thistime, the method is semiquantitative. It clearly providespositive/negative answers for samples above the detectionlimit with indications of the concentration. In addition, ifthe sensor is consistently underestimating the sampleconcentration at a specific site when compared to Method8330 results (i.e. Crane), a correction factor could be used.Minimal cross-reactivity was observed with the antibodiesemployed on the fiber optic probe. Further studies will beperformed to reduce matrix interference.

AcknowledgmentsThe authors thank David Cuttino, Chris Rowe, Naveed Naz,Paul Charles, Paul Gauger, Anne Kusterbeck, Charles H.Patterson, Jr., and Frances Ligler for their assistance andsuggestions concerning the field demonstrations. The authorswould also like to pay special thanks to Harry Craig of U.S.EPA Region 10 for support and help in the coordination andaccess to the sites and collection of samples. This work was

funded by the Office of the Undersecretary of DefenseEnvironmental Security Technology Certification Program(ESTCP). The views expressed here are those of the authorsand do not represent those of the U.S. Navy, Department ofDefense, or the U.S. Government.

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Received for review July 19, 1999. Revised manuscript re-ceived November 16, 1999. Accepted November 18, 1999.

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