ENVIRONMENTAL RELEVANCE OF CL-20: PRELIMINARY FINDINGS A. KOUTSOSPYROS1 , C. CHRISTODOULATOS2 , N. PANIKOV3 , O. MALCHEVA3 , P. KARAKAYA2 and S. NICOLICH4 1 Department of Civil & Environmental Engineering, University of New Haven, 300 Orange Avenue, West Haven, CT 06516, U.S.A.; 2 Center for Environmental Engineering, Stevens Institute of Technology, Hoboken, NJ, U.S.A.; 3 Department of Chemistry & Biology, Stevens Institute of Technology, Hoboken, NJ, U.S.A.; 4 TACOM-ARDEC, Picatinny Arsenal, Dover, NJ, U.S.A. ∗ ( author for correspondence, e-mail: akoutsospyros@newhaven.edu; Phone: +(203) 932-7398; Fax: +(203) 932-7158) Abstract. CL-20 is a recently synthesized component of energetic propellant formulations. Although energetic aspects of CL-20 have attracted considerable attention, its environmental behavior is unknown. A multi-disciplinary study covering a variety of fate, transport, and toxicity issues of CL-20 is currently under way in the Center for Environmental Engineering at Stevens Institute of Technology. Preliminary results on water solubility, biodegradability, hydrolytic reactivity, thermal decomposition and soil microbial and plant toxicity are reported in this article. Keywords: biodegradation, CL-20, fate and transport, microbial toxicity, nitramine, plant toxicity, solubility, thermal decomposition 1. Introduction Hexanitrohexaazaisowurtzitane (2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazatetracyclo [5.5.0.0.5,9 .03,11 ] dodecane ) is a recently (Nielsen, 1997) synthesized compound intended for use as an energetic component of propellant formulations. CL-20, currently produced in limited quantities for development purposes, is praised as tomorrow’s high energetic material because of its superior detonation, and ballistic performance. Synthesis, detonation, and safety aspects of CL-20 have attracted considerable attention worldwide and a fair amount of literature has been published on a variety of pertinent issues. Environmental relevance of this compound must be investigated thoroughly prior to full-production and wide application. Much of the popularity of CL-20 emerges partly from its acclaimed energetic performance (specific impulse, burn rate, ballistics and detonation velocity), and partly from meeting stringent sensitivity requirements (Russell et al., 1993; Kim et al., 1998; Mueller, 1999; Ou et al., 2000; Nedelko et al., 2000). An additional feature, that makes CL-20 more desirable for military applications over its currently used counterparts, is production of minimum exhaust in the visible ultraviolet or infrared wavelengths (minimum signature) characterized by the absence of lead, hydrogen chloride and aluminum oxide emissions that adversely impact human Water, Air, and Soil Pollution: Focus 4: 459–470, 2004. 2004 Kluwer Academic Publishers. Printed in the Netherlands. C  460 A. KOUTSOSPYROS ET AL. Figure 1. Structural formulas of energetic nitramines: (a) CL-20, (b) HMX and (c) RDX. health and the environment and are typical exhaust products of most conventional rocket propellants. CL-20 is a polycyclic nitramine conforming to the molecular formula C6 H6 N12 O12 . The molecular structure of CL-20 consists of a basic isowurtzitane cage with one –NO2 group appended to each of six nitrogen atoms as shown in Figure 1(a). In terms of crystalline structure, several polymorphs (α-ζ ) of HNIW have been identified and confirmed (Russell et al., 1993; Kim et al., 1998; Nedelko et al., 2000) with the ε-polymorph preferred for its higher crystal density, highest thermodynamic stability at ambient conditions, and overall superior performance properties. As a nitramine, CL-20 draws comparisons with other similar energetic compounds such as cyclotetramethylenetetranitramine (HMX) and cyclotrimethylenetrinitramine (RDX) whose structural formulas are also shown in Figures 1(b) and 1(c). Although the stereochemistry of CL-20 is far more complex than those of RDX and HMX, it has the same structural components. Notable differences include higher molecular mass (438), heat of formation, number of N–NO2 bonds (6), and density (2040 kg/m3 for the ε-polymorph). Unlike HMX and RDX that have no C–C bonds in their molecules, CL-20 bears three slightly elongated C–C bonds. Moreover, since CL-20 is more symmetrical and significantly less polar than RDX (Qasim et al., 2000), its aqueous solubility is expected to be relatively small. Very little is known about the chemical and biological reactivity of CL-20. At this point, certain inferences can be made regarding CL-20 reactivity based on behavior of other chemically similar compounds. With respect to chemical reactivity, RDX is known to be prone to alkaline hydrolysis via elimination (E2) ENVIRONMENTAL RELEVANCE OF CL-20 461 TABLE I Properties of hexanitrohexaazaisowurtzitane Property Descriptor Molecular formula C6 H6 N12 O12 Molecular mass 438 Appearance Melting point White crystalline solid in several (a–z) polymorph forms 230–260 ◦ C Heat of formation Polarity Density Carbon content Hydrogen content Nitrogen content Oxygen content + 228 cal/g; + 410.4 kcal/mol 3.61 Debye 1.97– 2.04 g/cm3 16.44% 1.37% 38.36% 43.84% mechanism (Qasim et al., 2000). Similar destabilizing behavior is suspected and needs to be proved for CL-20. Table I summarizes certain known properties of CL-20. As an anthropogenic material, CL-20 is not anticipated to be readily biodegradable. However, certain microbial cultures are known to utilize nitrated energetic compounds (TNT, RDX) as the sole nitrogen source and some strains are capable of using nitroaromatic compounds (2,4-DNP) as both sole carbon and nitrogen sources (Binks et al., 1995). If CL-20 proves to be highly resistant to biodegradation, a combination of chemical treatment methods (i.e., alkaline hydrolysis to destabilize the CL-20 molecule and produce degradable intermediates) with biological methods may prove to be an effective remedial strategy. This strategy has proved effective for a number of other energetic nitro-compounds (Christodoulatos et al., 1996, 2001). The energetic properties of CL-20 have been thoroughly studied and full production is anticipated in the near future, however, very little is known on the environmental fate and potential impacts of the neat compound which will unavoidably find its way into the environment either from accidental spills or industrial production activities. From an environmental standpoint, the toxicological effects on humans, plants and microorganisms and the interactions of CL-20 with soil, air, surface waters and groundwater are of paramount importance for developing sound environmental regulations and emergency response when full scale production and use of the compound are realized. The objective of the proposed work is to evaluate the mobility of neat CL-20 in the environment (soil, groundwater, air interactions), study its bioavailability and degradation (both biotic and abiotic), and assess its toxicity. The results presented in 462 A. KOUTSOSPYROS ET AL. this article are part of on-going, multi-disciplinary efforts and although preliminary, they shed some light on certain aspects of environmental relevance of CL-20. 2. Materials and Methods 2.1. A NALYTICAL METHODS FOR D ETERMINATION OF CL-20 Two analytical methods were used for determination of CL-20 namely, UV spectrophotometry and HPLC. A stock solution of unground 1 g/l CL-20 in HPLC-grade acetonitrile was prepared. Several dilutions ranging from 0.5 to 100 mg/l were subsequently prepared. Absorption was determined using a Hewlett-Packard 8452A diode array spectrophotometer. A strong absorption of CL-20 was observed at λ = 226–228 nm. These results are in agreement with CL-20 spectra recorded by other researchers (Pace, 1991). A highly linear calibration curve (r 2 > 0.99) for absorption at 228 nm versus concentration of CL-20 in acetonitrile was produced. The HPLC method was based on U.S. EPA Standard Method 8330, which was originally developed for analysis of nitroaromatic and nitramine explosives (Pace, 1991; Oehrle, 1994; Larson et al., 2001). A Varian 9095 autosampler, and Varian 9065 photodiode array detector were used. The mobile phase was a mix of 50% acetonitrile and 50% of water at a flow rate of 1.5 ml/min and 50 µl injection volume. The column used was a Supelcosil LC-8 (4.6 mm × 250 mm) obtained from Supelco. CL-20 eluted as a symmetrical peak (retention time 8.4 min). 2.2. A QUEOUS SOLUBILITY OF CL-20 Two sets of solubility experiments were carried out according to EPA OPPTS 830.7840 recommendations, using unground form of CL-20 (ID No. PCL69, Thiokol Propulsion). A constant temperature shaker was used in the first set while a constant-temperature water bath that allowed a more refined temperature control (±0.5 ◦ C) and extended equilibration times (3 days) was used for the second set of experiments. Several temperatures were used in the 15–50 ◦ C range. The soluble concentration of CL-20 was measured using a UV Spectrophotometer (HewlettPackard 8452A diode array) at λ = 226–228 nm. Solubility experiments above 50 ◦ C, were not performed as thermal decomposition, causing significant alteration in the sample spectra, was suspected. 2.3. PRELIMINARY BIODEGRADATION STUDIES Mycobacterium cultures were supplemented with external carbon (1 g/l of glucose or succinate) and/or nitrogen (0.5 g/l of ammonium sulfate) sources. Unground CL-20 (ID No. PCL69, Thiokol Propulsion) was used as a substrate. Cultures were incubated under aerobic (constant temperature shaker at 30 ◦ C◦ ) conditions. An ENVIRONMENTAL RELEVANCE OF CL-20 463 ample number of controls were used appropriately for the purpose of establishing background values (quality control) and/or abiotic losses. 2.4. A LKALINE H YDROLYSIS PRELIMINARY EXPERIMENTS Batch alkaline hydrolysis experiments were carried out using 5 ml of 1 M NaOH and 0.021 g of crystalline CL-20. Complete disappearance of CL-20 crystals was observed after 1 h of incubation at room temperature. Absorbance of the resulting bright yellow solution diluted 10-fold with water was measured at λmax = 306 nm. Nitrite and nitrate ions were analyzed using a Dionex IC25 Ion Chromatograph equipped with Dionex Ionpac AS14 column. The sample was split into two 25 ml aliquots, which were subsequently incubated for 1 week at room temperature, and at 46 ◦ C respectively. Samples, taken daily, were analyzed by ion chromatography to monitor the nitrite and nitrate ions. Thin-layer chromatography experiments using several different eluents are currently under way to identify the product(s) of alkaline hydrolysis of CL-20. 2.5. T HERMAL DECOMPOSITION STUDY Thermal decomposition experiments were carried out in a Brill Cell connected to a CDS 2000 Pyrolyzer equipped with a CDS FTIR probe for near real time analysis. Samples (2 mg each) were heated at a rate of 50,000 ◦ C/s to 1000 ◦ C and held at that temperature for 30 s. Four different atmospheres were used: (1) pure N2 (pyrolysis), (2) 15% O2 , 85% N2 (sub-atmospheric combustion), (3) 21% O2 , 79% N2 (atmospheric combustion), (4) 25% O2 , 79% N2 (over-atmospheric combustion). Four types of samples were used: (a) pure CL-20 (white crystalline powder), (b) pure HMX (white crystalline powder), (c) CL-20 explosive 94.5%, estane polymer binder 5.5% (yellow balls, 2–3 mm), (d) HMX explosive 95.5%, estane polymer binder 4.5% (white and purple balls, 0.5–1 mm). This study was carried out by the U.S. Army Aberdeen Test Center at Aberdeen Proving Grounds, MD. 2.6. S OIL MICROBIAL TOXICITY Toxicity experiments on soil microbial communities were carried out using two types of soil. A virgin (not arable) soil under a mixed deciduous forest from the National Interstate Park (New Jersey), and a meadow urban soil from the Stevens Institute of Technology campus, were used. Soil samples were collected from the A horizon (1–10 cm deep topsoil). Sample pretreatment included removal of big plant roots, screening through a 1 mm sieve to remove bulky material, and mixing. Pretreated soil (40 g) at field moisture content (30%) was well mixed with variable amounts of CL-20 and the mixture was subsequently packed into plastic containers placed in a constant-temperature water bath at 20 ◦ C. During incubation, at 40-min intervals, the soil column was flushed for 2 min (flushing rates 260–280 ml/min) 464 A. KOUTSOSPYROS ET AL. and the CO2 accumulated between flashings was determined using a continuous flow IR gas analyzer LiCor 800 (Lincoln, USA). 2.7. PLANT TOXICITY STUDIES Plant toxicity experiments were carried out using the same types of soil and the same pretreatment protocol as in soil microbial toxicity. CL-20 was added at application rates of 500, 1000, 2000 mg/kg dry soil. Mixtures of soil at field moisture content (50 and 30% for virgin and meadow soil, respectively) and CL-20 were planted with seeds (rye-grass or beans) and were placed inside a plant growth incubator (Precision Inc.) at a constant temperature of 20 ◦ C. Several times during the plant growth cycle, each plant was transferred into a special plastic container for measurement of respiration and photosynthesis rates. The soil–plant system was continuously flushed at a rate of 400–500 ml/min (electronic flowmeter) and the CO2 level determined with IR gas analyzer LiCor 800. 3. Results and Discussion 3.1. AQUEOUS SOLUBILITY OF CL-20 The solubility data for CL-20 obtained from the two separate runs described above are shown in Figure 2. Each data point shown represents the mean soluble concentration of CL-20 of two discrete and two replicate samples for each temperature. The relative errors were found to be within 5%. Evidently, differences in the temperature control method (water bath or shaker flasks) and equilibration times did not play Figure 2. Aqueous solubility of CL-20 as a function of temperature. ENVIRONMENTAL RELEVANCE OF CL-20 465 Figure 3. Degradation of CL-20 by Mycobacterium sp. strain HL4-NT1. a significant role. At 25 ◦ C, the solubility of CL-20 in water was measured to be 4.8 mg/l. In the range of 15–50 ◦ C, the solubility of CL-20 increases significantly (from a little over 2 mg/l at 15 ◦ C to slightly under 16.0 mg/l at 50 ◦ C). However, CL-20 is a substance of limited water solubility over the entire temperature range (15–50 ◦ C). 3.2. B IODEGRADABILITY Batch biodegradation experiments with a pure culture of Mycobacterium sp. strain HL4-NT1 are still in progress. Up to date results are shown in Figure 3. Accordingly, CL-20 depletion in 63 days is approximately 25% for samples supplemented with succinate as a co-substrate. CL-20 depletion in a non-inoculated control-containing mineral medium is about 20% for the same period. This indicates that a significant portion of the substance undergoes some kind of abiotic transformation most likely of hydrolytic nature. This abiotic transformation appears to be amplified by the mineral medium as controls containing de-ionized water show smaller depletions. Batch biodegradation experiments inoculated with a mixed activated sludge culture, currently on their fourth week, have not produced any statistically significant CL-20 depletion. 3.3. C HEMICAL STABILITY Preliminary chemical stability results have indicated hydrolytic action of a strong alkali solution on CL-20. Addition of 1 M NaOH resulted in complete solubilization of the crystalline CL-20 accompanied by rapid development of yellow color. Accordingly, alkaline treatment yields a nitrite ion to initial CL-20 molar ratio of 4:1. This behavior is in good agreement with the hydrolytic behavior of other nitramines (Qasim et al., 2001). The average nitrite production over a period of 170 h was about 0.14 mM (the values around 120 h are more than 3 S.D. away from the mean and are treated as outliers). The results are shown in Figure 4. 466 A. KOUTSOSPYROS ET AL. Figure 4. Dynamics of nitrite ion concentration during prolonged incubation of alkaline hydrolysis products of CL-20 at 25 and 46 ◦ C. 3.4. T HERMAL DECOMPOSITION Thermal decomposition products generated under various atmospheric conditions, for two CL-20 and two HMX propellant formulations, are shown in Table II. Data reported on this table represent only the two extreme atmospheric conditions namely, pyrolysis and combustion. It should be noted that no parent compound residuals were present in pyrolysis or combustion product spectra, meaning that each explosive is completely consumed. Apparently, in CL-20-containing formulations, under pyrolysis conditions, binders make no difference in the composition of end products however, significant compositional differences exist between initial and final products. In addition, N2 dominates initial and CO2 dominates the final products whereas small amounts of HNO2 and HCN seen in initial products are not present in final products. Thus, it can be stated that CL-20 generates much less toxic products than HMX under pyrolysis conditions. The opposite is true under combustion conditions where HMX is favored. 3.5. M ICROBIAL TOXICITY Investigation of the microbial toxicity of CL-20 was assayed by using four amendment rates on soil samples (0, 500, 1000, and 2000 ppm), the first serving as a control. Soil microbial community activity was monitored respirometrically. Net respiration rates for the non-zero application rates are obtained by subtracting the respiration rate of the control. A plot of the net respiration rate for each amendment rate is presented in Figure 5(a). The rates of CO2 formation decline exponentially over the incubation period. This decline is attributed to depletion of the limited amount of readily available organic compounds and is a typical pattern for freshly collected soil samples. Evidently, CL-20 enhances soil microbial activity. This effect becomes more pronounced as the CL-20 amendment rate increases in the range of 500–2000 ppm. This finding, verified by plotting the cumulative CO2 release TABLE II Exhaust pyrolysis and combustion gases of HMX and CL-20 based propellants PYROLYSIS HMX LX-14-0 CL-20 PAX-11 COMBUSTION HMX I F I F • ◦ • ◦ • ◦ • ◦

 I F • • 

 I F I F LX-14-0 • ◦ • ◦ • ◦ CL-20

PAX-11

 I • F I F I F I F I • ◦ • ◦ • ◦ • ◦ • ◦ • ◦ • •



  



I F I F I F I • • F I F I F • • • ◦ • ◦ 

F I
• ◦ • ◦ • ◦ F 
 I F • ◦ • ◦











 ENVIRONMENTAL RELEVANCE OF CL-20 Carbon Carbon Hydrogen Isocyanic Nitric Nitrogen Nitrous Nitrous Dioxide Monoxide Formaldehyde Cyanide Acid Oxide Dioxide Acid Oxide I Initial products: • HMX based (initial); ◦ HMX based (final). F Final products:
CL-20 based (initial);  CL-20 based (final). 467 468 A. KOUTSOSPYROS ET AL. Figure 5. (a) Dynamics of CL-20 oxidation as a function of the soil amendment rate; (b) cumulative CO2 output from the soil resulted from CL-20 oxidation. as a function of incubation time in Figure 5(b), signifies the absence of toxicity of CL-20 on soil microbial communities. 3.6. PLANT TOXICITY Plant toxicity of CL-20 was assayed using two types of plants (rye-grass and beans) and produced a similar effect as that reported above for soil microbial ENVIRONMENTAL RELEVANCE OF CL-20 469 Figure 6. (a) Effect of CL-20 on plant photosynthetic and respirometric activity, (b) setup for respirometric measurements of bean plants [left: soil amended with CL-20; right: soil without CL-20 (control)]. toxicity. Plant respiration and photosynthesis data are presented on Figure 6. Plants amended with CL-20 appeared to be growing at faster rates than the unamended controls. 4. Conclusions Preliminary results concerning environmental relevance of CL-20 are reported. Accordingly, CL-20 is a compound of limited solubility (4.8 mg/l at 25 ◦ C), with possible implications of limited mobility in aquatic systems as well as limited bioavailability. Moderate CL-20 depletions were observed in pure cultures of Mycobacterium sp. strain HL4-NT1 can not be conclusively attributed to biodegradation as non-inoculated controls exhibited similar behavior, most likely of hydrolytic nature. CL-20 appears to be prone to alkaline hydrolysis as evidenced in preliminary experiments. Contact with solutions of NaOH was accompanied by production of nitrites and various presently unidentified products of yellow color. Thermal decomposition experiments were carried out in various atmospheres and were compared with HMX. Accordingly, CL-20 generates significantly less toxic products than HMX under pyrolysis conditions while the opposite is true under combustion. Toxicity assays carried out in soil microbial communities and plants (rye-grass and beans) indicated absence of CL-20 toxicity in both systems. In fact, significant growth enhancement was observed in plants grown on CL-20-treated soils than in controls. Acknowledgment This research was supported by the DOD US ARMY TACOM/ARDEC Contract No. DAAE30-00-D-1011#7. 470 A. KOUTSOSPYROS ET AL. References Binks, P. R., Nicklin, S. and Bruce, N. C.: 1995, ‘Degradation of hexahydro-1.3.5-trinitro-1,3,5triazine (RDX) by Stenotrophomonas maltophilia PB1’, Appl. Environ. Microbiol. 61 (4), 1318–1322. Christodoulatos, C., Su T.-L. and Koutsospyros, A. D.: 1996, ‘Denitrification kinetics of hydrolyzed solid propellant waste streams’, in E. Diamadopoulos et al. (eds.), Proceedings of Protection and Restoration of the Environment III, Chania, Greece, pp. 278–286. Cristodoulatos, C., Su T.-L. and Koutsospyros, A.: 2001, ‘Kinetics of the alkaline hydrolysis of nitrocellulose’, Wat. Environ. Res. 73, 185–191. Kim, J.-H., Park, Y.-C., Yim, Y.-J. and Han, J.-S.: 1998, ‘Crystallization behavior of hexanitrohexaazaisowurtzitane at 298 K and quantitative analysis of mixtures of its polymorphs by FTIR’, J. Chem. Eng. Jpn. 31 (3), 478–481. Larson, S. L., Felt, D. R., Escalon, L., Davis, J. D. and Hansen, L. D.: 2001, ‘Analysis of CL-20 in environmental matrices: Water and Soil’, US Army Corps of Engineers, ERDC/EL TR-01–21. Mueller, D.: 1999, ‘New gun propellant with CL-20’, Prop. Expl. Pyrotech. 24, 176–181. Nedelko, V. V., Chukanov, N. V., Raevskii, A. V., Korsounskii, B. L., Larikova, T. S., Kolesova, O. I. and Volk, I.: 2000, ‘Comparative investigation of thermal decomposition of various modifications of hexanitrohexaazaisowurtzitane (CL-20)’, Prop. Expl. Pyrotech. 25, 255–259. Nielsen, A. T.: 1997, ‘Caged polynitramine compound’, U.S. Patent 5,693,794. Oehrle, S. A.: 1994, ‘Analysis of CL-20 and TNAZ in the presence of other nitroaromatic and nitramine explosives using HPLC with photodiode array (PDA) detector’, J. Ener. Mater. 12, 221–222. Ou, Y.-X., Xu, Y.-L., Chen, B.-R., Liu, L.-H., Wang, C.: 2000, ‘Synthesis of hexanitrohexaazaisowurtzitane from tetraacetyldiformylhexaazaisowurtzitane’, Chin. J. Org. Chem. 20 (4), 556–559. Pace, M. D.: 1991. ‘EPR spectra of photochemical NO2 formation in monocyclic nitramines and hexanitrohexaisowurzitane’, J. Phys. Chem. 95, 5858–5864. Qasim, M., Flemming, B. and Hansen, L.: 2001, ‘An approximation methods study and comparison of the chemical and physical properties of CL-20 and RDX for prediction of reactivities’, in Proceedings of Current Trends Computers in Chemistry, pp. 184–187. Russell, T. P., Miller, P. J., Piermarini, G. J. and Block, S.: 1993, ‘Pressure/temperature phase diagram of hexanitrohexaazaisowurtzitane’, J. Phys. Chem. 97, 1993–1997.