Impact of Environmental Conditions on the Enzymatic

Jul 6, 2013 - National Homeland Security Research Center, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle ...
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Impact of Environmental Conditions on the Enzymatic Decontamination of a Material Surface Contaminated with Chemical Warfare Agent Simulants Lukas Oudejans,*,† Barbara Wyrzykowska-Ceradini,‡ Craig Williams,‡ Dennis Tabor,§ and Jeanelle Martinez∥ †

National Homeland Security Research Center, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711, United States ‡ ARCADIS US, Inc., Durham, North Carolina 27713, United States § National Risk Management Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711, United States ∥ Office of Solid Waste and Emergency Response, Office of Emergency Management, U.S. Environmental Protection Agency, Cincinnati, Ohio 45268, United States ABSTRACT: The efficacies of enzyme-based technologies developed for the decontamination of surfaces contaminated with chemical warfare agent simulants were measured as a function of operating and environmental conditions mimicking possible outdoor conditions. Bench-scale decontamination testing was used to evaluate two enzyme-containing products for their ability to neutralize paraoxon and 2-chloroethyl phenyl sulfide (2-CEPS) selected as simulants of G-, V-series chemical agents and sulfur mustard, respectively. Residual amounts of the simulant on galvanized metal coupons were measured following application of the enzyme. Both enzymatic decontamination methods performed fairly well against the simulants, with higher efficacies at higher temperature against paraoxon and the opposite trend for the second enzyme solution against 2-CEPS. A longer interaction period resulted in better efficacy. Reapplication of fresh enzyme solution yielded a major improvement in efficacy. The neutral pH of both enzyme solutions makes this decontamination approach a useful decontamination tool for surfaces on which harsher decontamination products cannot be applied.

1. INTRODUCTION

as well as diffusion-rate-limited interactions between the chemical and decontaminant at the surface. The detrimental effects (e.g., corrosion) that highly efficacious reactive surface decontamination methods and some fumigants have on various surface types are well-known. Other decontamination technologies themselves can be harmful to the environment or end user.5 Enzymatic decontamination would be a safe and environmentally benign decontamination option. Enzymes are less hazardous, less corrosive, and more environmentally compatible than other alternatives and would lower the logistical and operational burden related to decontamination. External stressors, such as elevated temperatures, extreme pH values, and oxidative environments, might cause disruption of the enzymatic activity and possibly result in complete deactivation. Various research groups have been developing (mutagenized) enzymes for material decontamination purposes against CWAs.6−8 Recent reviews of various enzyme types that are being considered and their mechanisms of enzymatic detoxification of CWAs can be found in refs 5 and 9 and references therein. Most of the current chemical decontamination enzyme research is focused on improvements in agent specific activity, stability of the enzyme that would

Remediation of infrastructure following a chemical release is a critical part of any recovery process, especially in the case of more persistent chemicals or those with a very high toxicity that would pose a risk even in small amounts. Chemical warfare agents (CWAs) are considered to be the most toxic and deadliest chemicals, and hence, their decontamination might require an action plan using multiple decontamination technologies. One mission of the U.S. Environmental Protection Agency’s (U.S. EPA’s) Homeland Security Research Program (HSRP) is to provide scientifically established and practical remediation options following chemical contamination incidents. The systematic laboratory testing of decontamination methods using rigorous quality assurance measures and controlled test conditions increases the likelihood that the remediation strategy will be successful in the field. Proprietary decontamination products have been designed to decontaminate areas either by direct surface decontamination or through volumetric decontamination (fumigation). In general, decontamination efficacy values are obtained from bench-scale decontamination testing1−4 or are provided by the vendor. These latter values are, in most cases, solutionchemistry values derived from reactor studies without the presence of a substrate and, therefore, provide only the best possible efficacy values that can be obtained. Such studies ignore physicochemical bonding of the chemical to the surface, © 2013 American Chemical Society

Received: Revised: Accepted: Published: 10072

April 2, 2013 June 26, 2013 July 6, 2013 July 6, 2013 dx.doi.org/10.1021/ie401052z | Ind. Eng. Chem. Res. 2013, 52, 10072−10079

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2. EXPERIMENTAL SECTION 2.1. CWA Simulants and Application. The two CWA simulants used in this study were selected based on chemical structural similarities and known enzymatic reaction pathways. As a simulant of nerve agents, paraoxon (Sigma-Aldrich, St. Louis, MO, 98.6% purity) was used, whereas 2-CEPS (SigmaAldrich, St. Louis, MO, 98% purity) was used as a simulant of HD. The neat chemical (1.0 μL) was applied as a single droplet by 1.0 μL microsyringe (SGE Analytical Science, Victoria, Australia) to clean galvanized steel ductwork test coupons (4.0 cm × 2.5 cm surface dimensions). The 1.0 μL application corresponds to surface concentrations of ∼1 g/m2 for both simulants. The precision with which the 1.0 μL droplets were applied was verified prior to the decontamination testing as part of the extraction efficiency verification and was found to be better than 6% for paraoxon and 11% for 2-CEPS (five replicates). Chemicals were in contact with the coupon surface for 30 min to allow the chemical to reach thermal equilibrium with the environment before the enzyme solution was applied. 2.2. Decontamination Chamber. Decontamination research was conducted in a ∼285 L internal volume modified glovebox (0.9 × 0.9 × 0.9 m3 dimensions). A side port entrance allowed for coupons to be introduced and pulled from the chamber without significant change in temperature and relative humidity (RH). The exterior was insulated to enable control of temperature and RH. Temperature control was accomplished through the use of a thermocouple output in a feedback loop to control a fan that blows the glovebox atmosphere through a small radiator mounted inside the glovebox. A temperaturecontrolled fluid flows through the radiator and returns to a recirculator. This setup has both heating and cooling capabilities that span the experimental temperature range of 5−35 °C. Typically, the required environmental conditions were established on the day before each experiment to ensure equilibrium in temperature and RH during the decontamination testing. RH in the chamber was controlled through the use of a gas humidity bottle. This gas humidity bottle is a heated vessel containing Nafion tubing submerged in water. Water vapor passes through the walls of the Nafion tubing and is picked up by the passing air (∼1−2 L/min). This air leaves the gas humidity bottle saturated with water vapor (90−100% RH). Personal computer software controlled the airflow through the humidity bottle and, thereby, the RH of the chamber. Condensation against the inside walls of the glovebox occurred at 35 °C and RH values larger than 60% (corresponding to dew point temperatures above 26 °C). Therefore, the high-humidity condition was limited to 60% RH at 35 °C, whereas 80% RH was reached at lower temperatures. The air exchange through the chamber was set to one air exchange per hour. 2.3. Enzyme Products. Two enzyme products designed for the decontamination of CWAs were obtained from DuPont Genencor Science as DEFENZ VX-G12 and DEFENZ B-HD. They were developed for decontamination of VX and G-agents and sulfur mustard (HD), respectively, as well as decontamination of specific CWA simulants. The DEFENZ VX-G product contains two enzyme types, the OPAA enzyme DEFENZ 120 and the OPH enzyme DEFENZ 130 in a 1:10 (granulated) mass ratio, so the two enzyme types coexist in the enzyme solution. Consequently, the measured decontamination efficacy cannot be attributed to the specific enzyme type. OPH and OPAA enzymes have both shown activity against the CWA simulant paraoxon (diethyl 4-

increase shelflife and potlife (defined as the period of time that the enzyme is active in aqueous solution) through immobilization of the enzyme, and enhanced thermostability.10,11 Organophosphorus acid anhydrolase (OPAA) and organophosphorus hydrolase (OPH) enzymes have drawn particular attention and can be generated in larger amounts using various biological sources. Both types of enzymes are able to detoxify organophosphorus nerve agents through hydrolysis of the organophosphate with preferred substrate specificities.5,9 A scaleup program by Genencor for the OPAA and OPH enzymes developed by Edgewood’s Chemical Biological Center (ECBC) resulted in a commercially available granulated form of the enzymes, which is the basis of one of the enzyme products tested here.12 It is, however, unknown whether further modifications were made between this initial scaleup production and the evaluated product. Two enzyme products were recently evaluated for their ability to decontaminate building material surfaces such as carpet, wood, laminate, tile, and galvanized metal contaminated with CWAs [thickened soman (GD), VX, and sulfur mustard (HD)].13 These tests were conducted under normal laboratory conditions and did not address the impact that environmental conditions might have on the reported decontamination efficiencies. The main objective of this study was to determine this impact, mimicking possible outdoor decontamination scenarios when applied to building materials. The enzyme activity defined as the number of moles of agent converted per time unit, kcat, is generally derived from enzyme reactor tests in which the enzyme and substrate interact in a batch reactor containing a stirrer to produce a highly dynamic and reactive environment. Here, decontamination occurs at the interface of a chemical with the enzyme solution, which is different from the conditions in batch reactor tests. Additional experiments were, therefore, conducted to relate the coupon decontamination results obtained from interaction of the enzyme solution with the chemical on the surface to the more dynamic enzyme reactor test conditions. CWA simulants are less toxic and are used in conventional chemical research laboratories, thus increasing the feasibility of performing systematic decontamination studies. Paraoxon, chosen as a model organophosphate compound, is considered a simulant for VX for the evaluation of OPH-based decontamination.15 Paraoxon is also a simulant for G-agents when considering OPAA decontamination.16 With both enzymes present in the DEFENZ VX-G product, paraoxon should be considered a nonspecific nerve agent simulant. The detoxification of paraoxon occurs through hydrolysis of the phosphotriester P−O bond, resulting in the formation of pnitrophenol and diethyl phosphate.15 Oxidation of HD has been investigated as a detoxification path, although the oxidation products generated still exhibit some vesicant toxicity.17,18 Further hydrolysis of the HD oxidation products results in nontoxic substances. 2-Chloroethyl phenyl sulfide (2-CEPS) was chosen as a simulant based on its chemical similarity to HD and the presence of sulfur as the oxidation site. The more frequently used simulant 2chloroethyl ethyl sulfide (2-CEES) has a significantly higher vapor pressure19 and would have resulted in appreciable evaporation on the time scale of the decontamination interaction times, possibly leading to the inability to detect this simulant even without decontamination. 10073

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were always verified 20 min after preparation of every enzyme solution. Peracetic acid test strips (EM Quant Peracetic Acid Test, EMD Millipore, Billerica, MA) were used for this (semiquantitative) determination of PAA concentration in the working solution; their response was verified against a known PAA concentration. In this study, the enzyme products were applied as a liquid with the volume per surface area representative of a backpack (agricultural) sprayer-type application. The applied enzyme solution volume (100 μL) was derived from the measured increase in weight of a galvanized metal surface following application of an aqueous solution using a backpack-type pressurized sprayer under representative operating conditions.3 This weight increase was converted to applied volume using the measured density of the enzyme solution (1.022 g/mL) and normalized to coupon surface area. Coupons were sufficiently large in size to avoid runoff of the 100 μL volume of enzyme solution that was applied to each coupon. 2.4. Quenching of Decontamination Reaction and Extraction Procedure. To measure the decontamination efficacy of either enzyme product for a specific decontamination/interaction time, the enzyme reaction must be quenched without affecting the remaining amount of chemical on the coupon surface. Changing the pH through addition of an acid, for example, would presumably end enzymatic activity but could result in the potential degradation of paraoxon or 2CEPS, thereby creating a bias in the efficacy results. In this study, quenching was accomplished as a part of the extraction process by placing the whole coupon in 50 mL of dichloromethane (DCM) and then applying sonication for 10 min.2 Successful quenching of the enzymatic reaction was demonstrated by measurement of the recovered amount of simulant (1 μL volume) as added to a solution containing 50 mL of DCM and 100 μL enzyme solution (the “quenched enzyme solution”) and the recovered amount of simulant (1 μL volume) as added to a solution consisting of 50 mL of DCM and 100 μL of buffer solution without the enzyme present. For paraoxon, the ratio between these two amounts was 0.98, whereas for 2-CEPS this ratio was 0.94. With both ratios close to 1.00, it was concluded that, on the time scale between experiment and gas chromatography/mass spectrometry (GC/ MS) analysis (typically 1 day), no additional enzymatic reaction occurred following extraction of the coupon. This can be explained by a combination of the actual loss in activity of the enzyme in an organic solvent environment such as DCM, the preferable phase transfer of remaining chemical from the aqueous (enzyme-containing) layer to the solvent phase (octanol−water partition coefficient of log Kow = 1.98 and 3.58 for paraoxon and 2-CEPS, respectively), and the lower reaction rate between chemical and enzyme in a highly diluted environment. The mean extraction efficiencies of (74 ± 4)% for paraoxon and (86 ± 10)% for 2-CEPS were observed for immediate extraction from five replicate galvanized metal coupons. 2.5. Analysis. Samples were analyzed for paraoxon by gas chromatography/mass spectrometry (GC/MS) in selected-ionmonitoring (SIM) mode. The m/z 109 ion was used to quantify the amount of paraoxon in the extract. The gas chromatograph was equipped with a 60-m-long DB-5 (0.25 mm diameter × 0.25 μm coating thickness) column. A volume of 1 μL of the extract was injected using splitless mode. For the paraoxon analysis, the GC temperature program rose from an initial temperature of 75 °C (2-min hold) to a final temperature

nitrophenyl phosphate), with OPH having a higher activity against paraoxon than OPAA.14 Enzyme activities are known to be highly agent-specific, including no observed activity of the OPAA enzyme against VX. It is unknown whether the individual enzyme activities can be affected by the presence of both enzyme types. Such evaluation is beyond the scope of this study. Therefore, this DEFENZ VX-G enzyme product is identified as an organophosphate-degrading (OPD) product. Information on the actual enzyme amount per granule is considered proprietary information. The OPD enzyme-containing solution was prepared according to the vendor’s instructions (at room temperature) by mixing the granulated DEFENZ VX-G enzyme product with the buffer (predominantly sodium bicarbonate) and then dissolving it in water. The product, as received, is packaged to generate 10 L of enzyme solution. Smaller quantities of the granulated enzyme/buffer product and deionized water were used through proportional reduction in granulated enzyme and buffer product weights and water volume to reach the default enzyme solution concentration in 100 mL volume sizes, hereafter described as “enzyme solution”. Following preparation of this enzyme solution, the beaker containing the enzyme solution was transferred to the decontamination chamber and placed on an aluminum block to shorten the time to reach (temperature) equilibrium. A 2-h waiting period was found to be sufficient to reach temperature equilibrium for all environmental conditions tested. This delay between preparation and application is well within the limited 24-h potlife of this enzyme solution. Variations in this delay were kept as small as possible and were less than 10 min, ensuring reproducible conditions for the prepared enzyme solution at the time of application. The second enzyme product, DEFENZ B-HD, contains an arylesterase enzyme that, when mixed with sodium percarbonate, propylene glycol diacetate, and water as the activation step, produces (among other products) peracetic acid (PAA) as the active ingredient. The arylesterase enzyme is not involved in the catalytic mechanism that leads to oxidation of the agent but rather serves as a catalyst for the PAA-generating reaction. On-site generation of this strong oxidant is preferable, as special handling is otherwise required during shipping and transportation. The arylesterase enzyme was prepared according to the vendor’s directions. The DEFENZ B-HD product was received as a premixed slurry (∼1 L total volume) of granulated arylesterase enzyme, sodium percarbonate, and propylene glycol diacetate. According to the vendor, addition of 37.9 L (10 gal) of water results in the formation of a 0.36% (w/v) PAA-containing solution. PAA generation was expected to be complete after 20 min. Smaller quantities of this enzyme product were obtained by taking a “core” sample from the slurry and sediment using a 5 mL pipet and transferring its contents into a graduated cylinder. Deviations from the original ratio of sediment height to solution height above the sediment were corrected through addition or removal of solution above the sediment from this smaller volume. A proportionately smaller amount of (deionized) water was added to reach the same PAA concentration. This water was brought into equilibrium with the decontamination chamber test temperature conditions for at least 12 h prior to mixing with the enzyme slurry to reduce the actual time to reach temperature equilibrium between the chamber temperature and the enzyme solution. With the PAA concentration as a measurable quality control variable for this enzyme solution, PAA concentrations 10074

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Figure 1. Means of recovered amounts of paraoxon from positive controls and test coupons following the application of the DEFENZ VX-G enzyme product for two interaction times (15 and 30 min) under different environmental conditions. Error bars represent one standard deviation from the mean. Marked (*) positive control amounts recovered were statistically different from (lower than) expected values assuming no impact of temperature or RH on amounts recovered.

of 310 °C (5-min final hold) at a ramp of 20 °C/min. The carrier gas was helium at a 1.0 mL/min flow rate. The temperature of the injection port was optimized for the paraoxon analysis and set at 175 °C. All extraction samples except for those from procedural and laboratory blanks were diluted by a factor of 20 to be in range of the 0−2000 pg/μL paraoxon calibration curve. The internal standard (parathiond10) spiked into an aliquot of the 20× diluted extraction samples was used for quantification of the target analyte. The instrument detection limit (IDL) was determined using U.S. EPA guidelines20 and calculated to be 39 pg/μL. This allowed for the detection of paraoxon remaining on a coupon down to 39 ng (assuming 100% extraction efficiency). Samples were analyzed for 2-CEPS by GC/MS in SIM mode using the same column specifications as for paraoxon analysis, and 1 μL of the extract was injected using splitless mode. The m/z 123 ion was used to quantify the amount of 2-CEPS in the extract. The GC temperature program rose from an initial temperature of 55 °C (2-min hold) to a final temperature of 300 °C (5-min final hold) at a ramp of 4 °C/min. The carrier gas was helium at a 1.0 mL/min flow rate. The temperature of the injection port was optimized for the 2-CEPS analysis and set at 200 °C. All extraction samples except for procedural and laboratory blanks were diluted by a factor of 20 to be within the range of the 0−1500 pg/μL calibration curve. Quantification of the target analyte occurred through use of an internal standard (1,4-dichlorobenzene-d4) spiked into an aliquot of the 20× diluted extraction samples. The IDL was determined using U.S. EPA guidelines20 and calculated to be 26 pg/μL. Assuming 100% extraction efficiency, this IDL allowed for the detection of 2-CEPS remaining on a coupon down to 26 ng.

Poor chromatographic behavior of the internal standard midway through the analysis of the 2-CEPS samples (spread across a four-month period) led to a switch in GC/MS analysis equipment with changes to the GC column (30 m BPX5 column), temperature program (from 40 to 290 °C at a 10 °C/ min ramp with a 10-min final hold time), and injection port temperature (250 °C), resulting in an IDL of 5 pg/μL. Continuity across the two GC/MS systems was verified and confirmed by reanalysis of two batches of data (10 samples total), indicating a 6% higher response for the second GC/MS system compared to the first GC/MS system, well within the expected accuracy of either GC/MS system. Note that such a change in response has an effect on the measured amounts recovered from both the test coupons and the positive controls, canceling each other in the determination of the decontamination efficiency. 2.6. Test Matrix. Decontamination efficacy tests were conducted for six environmental conditions consisting of three temperatures (5, 20, and 35 °C) associated with either low (30% RH) or high (60−80% RH) humidity. These temperature and RH conditions were considered to be extreme weatherrelated conditions under which aqueous-based decontamination might occur. With water as the main constituent of the enzyme solution, it was expected that application below 0 °C would not be feasible. Each test point consisted of five test coupons, five positive control coupons (contaminated with paraoxon or 2-CEPS but not decontaminated), two procedural blank coupons (not contaminated but decontaminated), and two laboratory blank coupons (not contaminated and not decontaminated). Positive controls are relevant in decontamination studies, as they allow differentiation between possible 10075

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applies to both positive controls and test coupons and therefore does not impact the calculated decontamination efficacies. Paraoxon is highly persistent (vapor pressure of 1.1 × 10−6 mmHg at 20 °C19) and did not evaporate appreciably from these coupons during the (up to) 30 min of exposure, even at the highest applied temperature. The amounts recovered from the test coupons decreased with increasing temperature. This trend was observed for all RH conditions, as well as for both interaction times. The absolute amounts recovered from the test coupons also varied with decontamination time and RH. This variation is more readily visible following the calculation of the associated decontamination efficacy for each test condition using eq 1. Figure 2 shows that these efficacies are dependent on

natural attenuation or degradation and the actual enzymatic decontamination process. Two interaction times were used, namely, 15 and 30 min, with the shorter interaction time selected as the interaction time recommended by the vendor of the enzyme products.21 The use of an adaptive test design allowed for revision of the initial test plan if the impact of an environmental parameter was found to be negligible. In such a case, additional interaction times up to 60 min and repeated applications were evaluated. This occurred during the decontamination testing against 2-CEPS. Galvanized metal was selected as a nonporous, hard, and nonreactive surface that is inert to environmental conditions. Simulated enzyme reactor tests were conducted (at room temperature) by applying a 1 μL droplet of 2-CEPS (2 μL for paraoxon) to six 1 mL vials and adding 100 μL of enzyme solution (200 μL for the paraoxon reactor test) to three vials. Next, the vials were sonicated for 15 min. The contents of all six vials were subsequently extracted using the same procedure as employed for extraction from galvanized metal coupons, except that twice the extraction volume (100 mL of DCM) was used for paraoxon-containing vials to bring the expected concentration in line with the calibration range of the GC/MS instrument. Analysis of the amounts recovered from the test vials and positive control vials allowed for the calculation of the decontamination efficiency in a simulated enzyme reactor environment. 2.7. Calculation of Decontamination Efficacy and Associated Statistics. The amount of paraoxon or 2-CEPS remaining after enzymatic decontamination and the amount of paraoxon or 2-CEPS remaining on the positive control coupon was used to calculate the efficacy of the decontamination product. This efficacy (E, %) is described using the equation ⎞ ⎛ M m on test coupon E = ⎜1 − ⎟ × 100% M m on positive control coupon ⎠ ⎝

Figure 2. Efficacy of DEFENZ VX-G enzyme against paraoxon as applied to galvanized metal surfaces at (A) 60−80% and (B) 30% RH. Data points in panel A are not connected as different RH conditions were used.

temperature with the highest efficacy observed at the highest temperature. Note that the higher efficacy values at 35 °C might be biased somewhat by the lower-RH testing condition (60% at 35 °C versus 80% RH at 5 and 20 °C). The temperature at which this OPD enzyme product becomes less active due to enzyme unfolding was apparently not reached. A longer interaction time between paraoxon and the OPD enzymes resulted in a moderately higher efficacy (factor of 1.1− 1.5 improvement), suggesting that most of the enzyme activity occurred in the first 15 min. Statistical analysis using a Student’s t test of the efficacy value for the 15-min against the 30-min interaction time showed that statistically significant higher efficacy values were observed for only two of the six tested conditions, namely, 20 °C/80% RH (p = 0.046) and 35 °C/ 60% RH (p = 0.047). No interaction times longer than 30 min were studied. An unexpected dependence of efficacy on RH was also detected; that is, a higher efficacy of the enzyme solution against paraoxon was observed under higher RH conditions. More research would be required to identify whether this latter observation is of scientific significance. Note that this latter observation is not due to an additional decontamination reaction (e.g., enhanced hydrolysis of paraoxon at high RH) because the paraoxon amounts recovered from the positive control coupons were found to be independent of RH. Finally, the galvanized metal surface was not visually impacted by the slightly basic enzyme solution (pH 8.1), even after being left to dry on the surface over a multiday period. 3.2. Simulated Enzyme Reactor Decontamination of Paraoxon. The amounts recovered from positive control vial

(1)

where Mm represents the mean of the measured mass of paraoxon or 2-CEPS (μg). The same equation was used to calculate the simulated enzyme reactor test decontamination efficiency by entering the amounts recovered from the test and positive control vials. A two-tailed Student’s t test22 was used to determine whether efficacy values associated with specific test conditions (pairwise) were considered to be statistically significantly different (p ≤ 0.05).

3. RESULTS AND DISCUSSION 3.1. Enzyme Decontamination of Paraoxon on Surface. The mean amounts of paraoxon recovered from the positive control and test coupons are shown in Figure 1. Amounts recovered from positive controls were found to vary slightly with temperature and RH without a clear dependence on either variable. A mean recovery of paraoxon across all conditions of (73 ± 5)% from the theoretical amount of paraoxon applied to the coupon surface is consistent with the percentage recovered during the extraction efficiency evaluation (74%). Minor deviations might be due to less paraoxon being spiked and do not appreciably impact the calculated efficacy values. The somewhat low recovery can be explained by the selection of DCM as the extraction solvent of paraoxon from the galvanized metal coupons. No further efforts were made in this study to improve the amounts recovered, for example, by using a different extraction solvent. Note that this low recovery 10076

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coupons were kept at the target environmental conditions for 30 min before the decontamination started. The apparently higher volatility of 2-CEPS (estimated vapor pressure of 0.0186 mmHg19) compared to paraoxon resulted in a partial evaporation from all surfaces, including the positive controls. Hydrolysis of 2-CEPS might also have occurred, resulting in a lower recovery from all coupons, including positive controls. It is unclear why the amount of 2-CEPS recovered from the positive control coupons after 30 min at the lowest temperature (5 °C) was lower than the amounts recovered at the two higher temperatures. Because the recovered test coupon amounts were also lower, the low recovery can be attributed to a lower amount of 2-CEPS applied on all coupons related to this test point. Hence, the calculated efficacy should be unaffected, as the impact of a somewhat lower amount applied does not affect the calculated efficacy. Figure 5 shows the calculated efficacies for this enzyme against 2-CEPS. The efficacy value at 35 °C might be biased somewhat by the lower-RH testing condition (60% RH at 35 °C versus 80% RH at 5 and 20 °C). Efficacy values were generally lower than those observed for DEFENZ VX-G against paraoxon and did not improve with temperature. The highest temperature resulted in lower efficacies for both 30% RH and 60% RH conditions. Differences in efficacy values with temperature, however, were not statistically significant (p values of Student’s t test were always >0.05). For the 15-min decontamination time, RH had no impact on the amounts recovered from positive controls or test coupons or the associated calculated efficacies. Hence, the 60−80% RH condition was not evaluated for the longer 30-min decontamination time. Instead, tests were conducted to determine whether improved efficacies were to be expected for longer interaction times and for the repeated application of fresh enzyme solution. Table 2 lists the amounts recovered and the associated efficacies. Efficacy did not improve appreciably with increasing interaction time as the efficacy values for the 15-, 30-, and 60-min interaction times were not significantly different. A statistically significant improvement was observed in efficacy when 100 μL of fresh enzyme solution was reapplied after the first 15 or 30 min of decontamination had passed. The efficacy more than doubled following the repeated application after 15 min (p = 1 × 10−5), whereas the effect of repeated application after 30 min was less prominent but still significant (p = 5 × 10−4). Finally, the DEFENZ B-HD solution did not visually impact the galvanized metal surface. 3.4. Simulated Enzyme Reactor Decontamination of 2-CEPS. Amounts of 2-CEPS recovered from positive control vial contents and test vial contents and associated decontamination efficacy values are reported in Table 3 for two interaction times. The recovered positive control amounts reflect mean extraction efficiencies of 96% and 101% for the 15and 30-min interaction times, respectively, which are both higher than that obtained when extracting 2-CEPS from galvanized metal coupons. This difference can be attributed to either higher evaporation of 2-CEPS from galvanized metal coupons in the decontamination chamber when compared to closed vials or some adherence of 2-CEPS to the galvanized metal surface whereas the amber glass vials are more inert. Both explanations would result in higher extraction efficiency as observed during the simulated enzyme reactor tests. The observed 30% decontamination efficacy against 2-CEPS after 15 min and 26% after 30 min is slightly below the corresponding 35% efficacy for both interaction times for decontamination from galvanized metal. However, in comparing the efficacy

contents and test vial contents and the associated decontamination efficacy values are reported in Table 1. The observed Table 1. Amounts of Paraoxon Recovered from a Simulated Enzyme Reactor Test for Positive Controls and Test Vials (Three Replicates) at an Interaction Time of 15 mina positive control ± SD test vial ± SD efficacy ± SD

1934 ± 266 μg 513 ± 200 μg (74 ± 31)%

a

Reported amounts are, by approximation, twice the amounts recovered during coupon testing, as the spiked paraoxon amount for this test was doubled.

74% efficacy against paraoxon is statistically significantly higher (p = 0.04) than the corresponding 45% efficacy for decontamination from galvanized metal under otherwise similar conditions. A 10-min simulated enzyme reactor interaction time resulted in a decontamination efficacy of 70%, suggesting that most of the reaction had already occurred in the first 10 min. The higher efficacy for the simulated enzyme reactor test can be attributed to better mixing of the paraoxon and the aqueous enzyme-containing environment under sonication and suggests that reported efficacies from enzyme reactor tests might overestimate the actual enzyme activities when used in a static environment during passive decontamination of surfaces. In addition, decontamination of other more porous or permeable materials results in different efficacy values.13 3.3. Enzyme Decontamination of 2-CEPS on Surface. PAA concentrations were recorded during the first 2 h following the addition of water to the enzyme-containing slurry (at t = 0 min). Figure 3 shows a representative PAA

Figure 3. PAA concentration and pH of DEFENZ B-HD solution as functions of time following activation through the addition of water (at t = 0 min).

concentration as a function of time. In agreement with the preparation instructions, the PAA is not present in the first 10 min, and a steep increase in concentration occurs after 10−15 min. Equilibrium at a 2500−3000 ppm concentration level occurs after ∼40 min. Meanwhile, the pH value of the enzyme solution decreased from pH 10 to reach equilibrium at pH 7, indicative of PAA formation. Application of this enzyme solution to the coupon occurred 25 min after water was added to the slurry to account for the delayed PAA formation. Amounts of 2-CEPS recovered from positive control and test coupons are shown in Figure 4. Spiking of the coupons with 2-CEPS occurred prior to the preparation of the enzyme solution. Consequently, all spiked 10077

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Figure 4. Mean amounts of 2-CEPS recovered from positive controls and test coupons following the application of the DEFENZ B-HD enzyme product for two interaction times (15 and 30 min) under various environmental conditions. The 30-min interaction time was not considered at the high-RH condition. Error bars represent one standard deviation from the mean.

Table 3. Amounts of 2-CEPS Recovered from Simulated Enzyme Reactor Tests for Positive Controls and Test Vials (Three Replicates) interaction time positive control ± SD test vial ± SD efficacy ± SD

15 min

30 min

1130 ± 95 μg 787 ± 140 μg (30 ± 6)%

1184 ± 73 μg 880 ± 70 μg (26 ± 3)%

when used for decontamination of a hard nonporous and nonreactive surface. Figure 5. Efficacy of arylesterase enzyme against 2-CEPS as applied to galvanized metal surfaces at (A) 60−80% and (B) 30% RH. Data points in panel A are not connected as different RH conditions were used.

4. CONCLUSIONS The observed decontamination efficacies of DEFENZ VX-G against paraoxon on galvanized metal increased with increasing temperature, RH, and interaction time. Efficacy values ranged from 24% to 68% for a 15-min interaction time and from 32% to 75% for a 30-min interaction time. Decontamination at 60% RH and 35 °C for 30 min resulted in the highest paraoxon removal from galvanized steel. Results from the simulated enzyme reactor test with the OPD enzyme suggest that enzyme reactor tests might overestimate the expected decontamination efficacy when the enzyme solution is applied for nonporous hard surface decontamination. Decontamination efficacies of DEFENZ B-HD against 2CEPS as a surrogate for HD ranged from 24% to 40% for the 15- and 30-min interaction times, with a tendency for poorer performance at higher temperatures. Improvements in efficacy were observed following a second application of the enzyme solution, resulting in 75% destruction of 2-CEPS. Simulated enzyme reactor efficacy results for 2-CEPS were comparable to the efficacy value (15 min, single application) for decontamination of a hard nonporous surface.

Table 2. Amounts of 2-CEPS Recovered from Positive Controls and Test Coupons interaction timea (min) 15b 15 + 15 30b 30 + 30 60

positive control ± SD (μg) 702 793 725 631 629

± ± ± ± ±

187 47 48 48 45

test coupon ± SD efficacy ± SD (μg) (%) 457 199 473 270 391

± ± ± ± ±

39 25 35 44 77

35 75 35 57 38

± ± ± ± ±

10 10 3 10 8

a

Description 15 + 15 min refers to a reapplication of a fresh enzyme solution after 15 minutes for another 15 minutes; the same for 30 + 30 min. bData from Figure 5.

values, no statistically significant differences were found. For this enzyme product, there is no evidence that enzyme reactor data would overestimate the expected decontamination efficacy 10078

dx.doi.org/10.1021/ie401052z | Ind. Eng. Chem. Res. 2013, 52, 10072−10079

Industrial & Engineering Chemistry Research

Article

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Enzyme activities, most noticeably the OPH and OPAA enzyme activities, are strongly dependent on the formulation of the enzymes. Consequently, results from this study should not be interpreted as an indication that all OPH/OPAA enzymes will behave identically. Activity rates of enzyme are also highly target-compound-specific, and decontamination efficacies against the actual CWAs will differ. A short- and long-term assessment of possible surface damage showed that neither enzyme-based decontamination methods caused any visual changes to the galvanized metal material under any of the test run conditions, making these products suitable for decontamination of sensitive equipment provided that such equipment can handle the impact of aqueous solutions. From this study, enzymes appear to have a great potential to decontaminate surfaces because of their ease of application, negligible damage to surfaces, and relatively effective decontamination under all tested environmental conditions. The addition of a cosolvent, for example, to an enzyme solution (at a sufficiently low concentration to avoid degradation of the enzyme itself) might further enhance the decontamination efficiency through the improved solubility of these types of chemicals.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

Disclaimer. This article has been subject to an administrative review but does not necessarily reflect the views of the U.S. EPA. No official endorsement should be inferred. The U.S. EPA does not endorse the purchase or sale of any commercial products or services mentioned in this article. The authors declare no competing financial interest

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ACKNOWLEDGMENTS We acknowledge David Mickunas for his contributions as a project team member, and Matt Clayton and Andy Stinson for their contributions in the design of the experimental setup and chemical analyses. Additionally, the authors thank Larry Kaelin and Sang Don Lee for review of the original manuscript. The U.S. Environmental Protection Agency, through its Office of Research and Development, funded and directed the research described herein under Contract EP-C-09-027 with ARCADIS US, Inc.

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REFERENCES

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