PICRIC ACID DEGRADATION IN SEDIMENTS FROM THE LOUISIANA ARMY AMMUNITION PLANT YONGQIANG TAN1 , GREGG R. DAVIDSON2,∗ , CHUN HWA SEE3 , D. CHUCK DUNBAR4 , JOHN H. O’HAVER5 , STEPHANIE RICE2 , DANNY W. HARRELSON6 , and MANSOUR ZAKIKHANI6 1
Department of Chemical Engineering, University of Mississippi, University, MS; Present address: Chemical, Biological, and Materials Engineering, University of Oklahoma, 100 E. Boyd St., EC T-335, Norman, OK 73019; 2 Department of Geology and Geological Engineering, University of Mississippi, MS; 3 Department of Chemical Engineering, University of Mississippi, University, MS; Present address: Assembly Technology Development, Intel Corporation, Penang 11900, Malaysia; 4 National Center for Natural Products Research, University of Mississippi, MS; 5 Department of Chemical Engineering, University of Mississippi, University, MS; 6 U.S. Army Engineer Research and Development Center (ERDC), Vicksburg, MS (∗ author for correspondence, e-mail:
[email protected])
(Received 9 November 2005; accepted 5 March 2006)
Abstract. Picric acid is an explosive historically produced and disposed at the Louisiana Army Ammunition Plant (LAAP) in northern Louisiana. The potential for natural degradation of picric acid was investigated by creating picric-acid slurries with four LAAP sediments of variable composition and monitoring for up to 98 days. The concentrations of picric acid decreased rapidly in all slurries during the first day, attributed to adsorption, followed by slower decreases in some samples due to degradation. Degradation in unsterilized slurries was nearly complete within 80 days for two of the four sediments. Increases in nitrite and nitrate concentration over time were proportional to the loss of picric acid and indicate that at least two of the three nitrite groups were removed from the picric acid molecule. The absence of significant concentrations of compounds with a mass greater than 100 amu in the final solutions suggests that all three nitrite groups were removed. No correlation was found between the degree of degradation and grain size, clay content, organic content, carbonate content, or a suite of element concentrations in the sediment. Degradation in sterilized samples was minimal for all sediment slurries, indicating microbial activity as the primary mechanism of degradation. Keywords: 2,4,6-trinitrophenol, degradation, LAAP, natural attenuation, picric acid
1. Introduction The Louisiana Army Ammunition Plant (LAAP), located approximately 35 km east of Shreveport, LA, was used for ammunition production for more than fifty years before its operations were ceased in 1994. The primary waste products from explosives production and handling were TNT and RDX (2,4,6-trinitrotoluene and hexahydro-1,3,5-trinitro-1,3,5-triazine, respectively), with smaller scale production of other nitro-organic compounds including picric acid (2,4,6-trinitrophenol), HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine), and N,2,4,6-tetranitro-Nmethylaniline. Waste disposal into unlined ponds and ditches was common in the Water, Air, and Soil Pollution (2006) 177: 169–181 DOI: 10.1007/s11270-006-9133-y
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World War II era, with subsequent contamination of underlying soils and groundwater. Remediation of the surface ponds and soils was carried out in the late 1980’s, and long term monitoring of contaminants in the subsurface was begun to determine if contaminants would naturally degrade over time (Pennington et al., 2001; Harrelson et al., 1997). Where serious harm to humans or ecosystems is not imminent, monitored natural attenuation (MNA) is an attractive remediation option due to its lower cost and environmental impact (Cattaneo et al., 2000). Most of the field and laboratory studies investigating natural attenuation at LAAP and at other sites have focused on TNT and RDX and their degradation products (Pennington et al., 2001; Zakikhani et al., 2002; Beller and Tiemeier, 2002; Ringelberg et al., 2003). There are fewer published studies on picric acid, in part because of its comparatively low concentration at most sites with explosives contamination. Picric acid has the potential to be highly mobile and persistent in groundwater systems. A low octanol-water partition coefficient (Kow = 40) suggests that picric acid is not readily adsorbed onto mineral or organic particles (Layton et al., 1987; Goodfellow et al., 1983), and a 30-day degradation study reported resistance to hydrolysis, biodegradation and photolysis (Dave et al., 2000). Different pathways of picric acid degradation have been studied, though most studies have been conducted under conditions not typically found in the subsurface environment. Examples include degradation by UV radiation with or without various catalysts (Kavitha and Palanivelu, 2005, Joshi et al., 2003; Ksibi et al., 2003; Tanaka et al., 1997), and degradation by bacteria or fungi that may use picric acid as an energy source (Hofmann et al., 2004; Gazdaru et al., 1996; Heiss et al., 2002; Lenke and Knackmuss, 1992; Rajan et al., 1996; Takeo et al., 2003; Rieger and Knackmuss 1995; Rieger et al., 1999). Degradation or biotransformation compounds identified in the later studies included 2,4-dinitrophenol and 4,6-dinitrohexanoic acid. More recently, Nipper et al. (2004) conducted a study on the potential for natural degradation of picric acid in marine sediments. Degradation was found to be nearly complete, and was attributed primarily to native bacterial activity. Biotransformation products including 3,4-diaminophenol, amino nitrophenol and nitro diaminophenol were identified. In the present study, the potential for natural degradation of picric acid in a fresh-water aquifer was investigated using batch experiments simulating natural aquifer conditions at LAAP.
2. Materials and methods 2.1. S AMPLE
COLLECTION AND CHARACTERIZATION
Groundwater and contaminants in the subsurface at LAAP occur in unconsolidated Pleistocene-age, terraced fluvial sediments deposited in a generally fining
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TABLE I Stratigraphic zone and depth of sediment collected at LAAP Zone
Sample ID
Depth
Upper Terrace
U1 U2 U3 L1
1.2 m 2.4 m 3.4 m 5.5 m
Lower Terrace
upwards sequence. Previous studies have divided the deposits into the Lower Terrace, consisting of fine sands and trace gravels, and the Upper Terrace, consisting of fine-grained silts, clays and silty clays (Pennington et al., 1999). For this investigation, four sediment types were selected representing the range of sediments present beneath LAAP. Samples were collected at an uncontaminated site at LAAP from pits dug using a backhoe to a maximum depth of 6 m. Three samples were collected from different depths within the Upper Terrace deposits (identified as U1, U2 and U3), and one from the Lower Terrace deposits (L1) (Table 1). Approximately 30 kg of each sediment was collected and homogenized before sub-sampling. Samples of each sediment were characterized for grain size distribution, organic and carbonate content, and elemental composition. Grain size distribution was determined by wet sieving for the larger fraction (down to 75 μm), and by laser diffraction using a multi-wavelength particle size analyzer for the silt and clay size fractions. Organic and carbonate content were determined using a thermogravimetric analyzer (TGA) (Heiri et al., 2004). Samples for metal analysis were digested using Aqua Regia and analyzed using an inductively coupled plasma optical emission spectrophotometer (ICP-OES). 2.2. SAMPLE
PREPARATION
Batch experiments with each sediment type were set up to approximately simulate natural conditions found at LAAP. Sub-samples of each sediment were dried at room temperature for 14 days and disaggregated to pass through a 0.6 mm sieve. Sediment slurries were created in 1.8 L glass roller bottles using 600 g of dried sediment and 800 mL of 100 mg/L picric acid solution. Picrate acid solutions were made from 98% purity solid picric acid. Solutions of 100 mg/L picric acid are bright yellow due to the formation of picrate ions (Fig. 1). Unsterilized slurries were run in duplicate for each of the four sediments. Sterilized slurries, used as controls to differentiate biotic and abiotic processes, were created using sediments baked at 105◦ C overnight, and using picrate acid solutions made with water that was first boiled for a minimum of 15 minutes. All slurries were kept in the dark and continuously rolled at 2 rpm at ambient temperature for up to 98 days.
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Figure 1. Picric acid and picrate ion structure
For each sediment, a blank slurry was prepared using 600 g of sediment and 800 mL of deionized water (>18 megohm/cm). The blank slurries were rolled and sampled at the same frequency as the picric acid slurries. An additional solution containing 100 mg/L picric acid with no sediment was also stored in the dark at ambient temperature and analyzed for picric acid loss in the absence of sediment. 2.3. CHEMICAL
ANALYSES
Picric acid concentration was measured daily for the first 5 days, and then weekly for up to 98 days. Each slurry was removed from the rollers, allowed to settle for 6 hours, and 2 mL of solution was removed, filtered and stored in sealed 1.8 mL autosampler vials under refrigeration until analysis. Picric acid concentration was determined using a high performance liquid chromatography (HPLC) system with an Alltech Adsorbosphere XL-C18 column (250 × 4.6 mm) and a UV detector operated at 365 nm. The mobile phase was a 40/60 (v/v) methanol/buffer solution at a flow rate of 1.5 mL/min. The buffer was 6.8 g/L KH2 PO4 acidified to pH 3.5 with acetic acid (Thorne and Jenkins, 1995). The detection limit for picric acid, based on 3 times the background noise, was 0.01 mg/L. The pH of one set of unsterilized sediments (run 1, with and without picric acid) was measured at the same time each picric acid sample was collected. Solutions were analyzed for nitrite and nitrate in addition to picric acid in the second set of unsterilized sediments (run 2). Nitrite and nitrate analyses were performed using an ion chromatograph (IC) with a Dionex IonPac ASI4A column (4 × 250 mm) at a flow rate of 1 mL/min. Identification of degradation products was attempted using a high resolution time-of-flight liquid chromatography/mass spectrometer system (Agilent MSD/TOF mass spectrometer interfaced with and Agilent 1100 HPLC). Samples were introduced into the mass spectrometer through electrospray ionization (ESI) in negative ion mode. The HPLC separation was carried out at 0.6 mL/min beginning with a water:acetonitrile ratio of 9:1 with a linear increase to a ratio of 1:1 over 50 min. A Phenomonex Luna C-18 column (150 × 4.6 mm) with 5 μm packing was used.
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3. Results The three samples collected from different depths within the Upper Terrace (U1, U2, and U3) were thought to represent more of a range in grain size when collected in the field, but were found to have similar grain size distributions once analyzed (Fig. 2). Grain size distributions for these three samples were 53–63% fine sand, 35–44% silt, and 3–4% clay. Sediment from the Lower Terrace (L1) was 88% fine sand, 11% silt, and less than 1% clay. The deepest of the Upper Terrace samples (U3) contained the highest fraction of combined silt and clay (47%), followed by U2, U1 and L1. The organic content ranged from 1.2 to 3.2% and the carbonate content from 0.07 to 0.15% by weight. Organic and carbonate content were higher in the finer grained sediments (U3 > U1 > U2 > L1) (Fig. 3). The elemental composition of the sediments (Table 2) did not show a consistent trend with grain size. Of the elements found above 10 ppm, only Fe, Al, Mn and Ba stand out as being significantly lower for the more coarse grained Lower Terrace sediment. The blank slurries contained no measurable picric acid (detection limit of 0.01 mg/L) throughout the period of investigation. The concentration of a 100 mg/L picric acid solution with no sediment, stored in the dark at ambient temperature, decreased by 7.5% to 92.5 mg/L after 98 days. Changes in picric acid concentration in the sediment-picric acid slurries are shown in Fig. 4. Initial decreases in concentration ranging from 10 to 20% were observed in all slurries, both sterilized
Figure 2. Grain size distribution. Silt and clay sizes determined by laser diffraction (% by volume). Sand sizes determined by wet sieving (% by weight). Sediments with high picric acid degradation in batch study are shown with solid data markers
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TABLE II Elemental concentration in the solid phase. Concentrations are in mg/kg. High and Low degradation refers to the degree of picric acid removal in the batch experiments. Elements are ordered from high to low concentration in U1 High degradation U1 Fe Al Ca Mg K Na Mn Ba Zn V Cu Pb Cr Sr Ti As Ni Co Li Sb Cd Be Mo
9009 5717 1104 509 385 262 172 55 33 16 15 14 8.9 7.6 7.0 4.6 4.4 3.9 3.2 1.6 1.2 0.4 0.3
U2 7251 5330 857 311 339 233 180 67 12 15 6.4 8.3 8.1 6.7 5.0 4.5 3.3 3.6 3.0 0.7 1.0 0.4 1.4
Low degradation U3 9793 6720 823 729 474 332 74 58 18 14 9.7 6.8 7.6 7.8 6.3 2.6 5.4 3.9 4.1 1.1 0.7 0.4 0.2
L1 2249 2041 722 320 249 235 39 16 17 2.8 6.2 2.3 2.5 5.4 3.9 0.6 3.3 3.1 2.0 1.3 1.0 0.1 0.5
Figure 3. Organic and carbonate content. Sediments with high picric acid degradation in batch study are shown as solid bars
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Figure 4. Picric acid concentrations over time in sterilized and unsterilized sediment slurries
and unsterilized, within the first day. Significant loss of picric acid following the first day was observed in only two of the four sediments, (U1 and U2). Picric acid concentrations in all the unsterilized U1 and U2 slurries decreased to less than 1 mg/L within 56 to 84 days. The yellow color of the solution faded over time as picric acid concentrations decreased. Picric acid concentrations did not decrease significantly after the first day in slurries made with the deeper sediments (U3 and L1), nor in any of the sterilized slurries using all four sediments. The yellow color of these solutions remained vivid throughout the study. The pH of unsterilized slurries containing picric acid was initially 0.2 to 0.3 pH units lower than the corresponding blank slurries (Fig. 5). In the two slurries with little degradation (U3 and L1), the pH remained lower than the blank throughout the experimental period. In the two slurries with nearly complete degradation (U1 and U2), the pH of the blank and picric acid slurries approached the same value as the picric acid concentration neared zero. Once the picric acid concentration dropped below 1 mg/L (>99% degradation), the pH of the spiked slurries remained higher than the corresponding blanks. Nitrite and nitrate concentrations were measured in one unsterilized slurry for each sediment (Fig. 6). Nitrite and nitrate remained relatively constant for approximately 10 days in all slurries, and then began to increase in the two slurries
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Figure 5. pH of blank and picric-acid spiked, unsterilized sediment slurries over time (run 1 from Fig. 4)
exhibiting significant loss of picric acid (U1 and U2). Nitrite concentrations peaked and began to decrease while nitrate concentrations continued to rise. None of the degradation products discussed in the Introduction were observed in any of the sediment slurries. In solutions drawn from slurries with greater than 99% loss of picric acid, the picric acid mass spectra dominated all other peaks above 100 amu. Only trace amounts of one apparent biotransformation product, O-picrylhydroxylamine, were observed. 4. Discussion 4.1. FACTORS
CONTROLLING DEGRADATION
The observed degradation in the U1 and U2 slurries is clearly bacterially mediated since no significant degradation was observed in the same sediments when sterilized. Abiotic factors such as grain size, element concentration, and organic content were considered, however, that may facilitate bacterial growth or aid in biotic reaction mechanisms. Finer grained sediments and those with higher organic material may support larger bacterial populations, but no apparent relationship was found between grain
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Figure 6. Concentrations of nitrite, nitrate and picric acid in mmol/L over time in unsterilized sediment slurries (run 2 from Fig. 4)
size distribution or organic content and picrate acid degradation. Only minor degradation was observed in sediments containing both the highest and the lowest proportion of fine grained sediment and organic content (U3 and L1). Redox sensitive elements such as Fe and Mn can influence decomposition reactions (Sahrawat, 2004; Negra et al., 2005). In their oxidized states, Fe and Mn can act as electron acceptors to facilitate the breakdown of complex organic molecules. These elements are much weaker oxidants than O2 , however, so their role in decomposition reactions is generally limited to oxygen-deficient environments. In this investigation, oxygen was not strictly limited. Slurries were opened to the atmosphere each week during sampling. Under aerobic conditions, Fe or Mn oxides may still serve as catalysts in degradation reactions (Nowack & Stone, 2000; Hunter et al., 1999). Iron is abundant in all four sediment samples (Table 2), but here too, only minor degradation was observed in the sediments with both the highest (U3) and the lowest (L1) Fe concentrations. Manganese is the only measured variable that correlates with degradation. The Mn concentration is 2.3 to 4.6 times larger in the two sediments with nearly complete degradation of picric acid. Other elements such as Zn and Ti may also serve to
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catalyze decomposition reactions under special circumstances (Joshi et al., 2003; Ksibi et al., 2003; Tanaka et al., 1997), but these elements are low in concentration in the LAAP sediments and do not correlate with the degree of degradation observed in the batch studies. 4.2. DEGRADATION
PRODUCTS
The clearest evidence of degradation is provided by the increase in the total nitritenitrate concentrations in the unsterilized U1 and U2 slurries as picric acid concentrations decreased (Fig. 6). As nitrite is removed from the picric acid molecule, oxidation readily converts nitrite to nitrate, resulting in a decrease in nitrite concentrations after an initial peak, and a continuous increase in nitrate concentration. If all three nitrite groups are stripped from the picric acid molecule, the molar ratio of nitrite plus nitrate relative the initial mass of picric acid should be 3. The observed ratio for samples collected after the first 30 days averaged 2.0 for U1, and 2.2 for U2. All sediment samples, sterilized and unsterilized, experienced a rapid decrease in picric acid concentration during the first 1 to 2 days that is attributed to adsorption. If the adsorbed picric acid (15 to 20% of the initial concentration) is subtracted from the starting mass, the molar ratios of nitrogen to picric acid increase to 2.6 and 3.0, respectively. Alternately, if the adsorbed picric acid was degraded, it is possible that a portion of the released nitrite groups were subsequently adsorbed onto the sediment. Loss of aqueous nitrogen could also occur through denitrification, though not likely in these experiments where atmospheric air was periodically introduced into the vessels. Partial adsorption of released nitrite is indirectly supported by the fact that the aqueous nitrite and nitrate concentrations did not increase between days 2 and 10 while picric acid concentrations were gradually declining. The decline in picric acid during this time is attributed to degradation rather than additional adsorption since decreases after the second day were observed only in the unsterilized sediments (Fig. 4). Preliminary efforts to identify degradation products by mass spectrometry focused on masses above 100 amu in samples at the end of the study. This mass range includes decomposition products observed by others in the literature (e.g. Rajan et al., 1996; Rieger and Knackmuss 1995; Rieger et al., 1999), but not the benzene ring stripped of all functional groups, nor smaller degradation products. None of the degradations products reported by others were observed in this study. Only trace amounts of O-picrylhydroxylamine were identified, with mass peaks substantially smaller than the residual picric acid mass peaks (picric acid concentration
