Hexanitrohexaazaisowurtzitane (CL-20) is being considered as a potential replacement for existing high explosive and propellant materials. In the past, chemicals similar in nature to CL-20 were released into the environment without knowing the fate and effects of these compounds. Multi-millions of dollars and corresponding man-hours have been spent on risk assessment and remediation of previously released energetic compounds. The potential impact of releases of CL-20 must be assessed prior to adoption of CL-20 as a commonly used energetic material. 

The objective of this project was to investigate the fate, transport through the soil vadose zone, and environmental effects of CL-20 on terrestrial plants, soil organisms, and aquatic species.

Technical Approach

Environmental effects of CL-20 were determined using both standardized single-species toxicity assays for soil invertebrates, plants, and aquatic species and an approach designed to assess both community- and ecosystem-level effects. Toxicity endpoints (i.e., survival, growth, reproduction) were correlated with concentrations of acetonitrile-extractable CL-20 to establish ecotoxicological benchmarks based on concentration-response relationships that describe CL-20’s bioavailability for ecologically relevant soil and aquatic biota. Transport and fate were assessed using a system of standardized intact soil-core microcosms. Data from the soil-core microcosms were used to determine CL-20 transport through the soil vadose zone, potential toxicity in groundwater, and fate endpoints such as persistence, mobility, and plant uptake.


Results of the phytotoxicity studies showed that CL-20 did not adversely affect terrestrial plant growth or seedling emergence of Medicago sativa L. (alfalfa; dicot), Echinochloa crusgalli L. (Japanese millet; monocot), and Lolium perenne L. (perennial ryegrass; monocot) up to and including 7,856 mg/kg. These results were obtained in a soil that supports relatively high bioavailability of CL-20 with species that represent a wide variety of plant genera and habitats. Alfalfa and perennial ryegrass are crop plants; whereas, Japanese millet grows naturally and can tolerate relatively wet habitats. These factors in combination with the test results suggest that CL-20 is relatively nontoxic to the majority of terrestrial plants.

Results from soil invertebrate testing showed that reproduction measurement endpoints were more sensitive indicators of ecotoxicity than was adult survival. The reproduction endpoints for earthworms, potworms, and collembola were production of juveniles, plus cocoon production for earthworms. The results of standardized single-species toxicity tests showed that toxicity of CL-20 to the earthworm E. fetida (ISO 11268-2), enchytraeid worm E. crypticus (ISO 16387), and collembolan F. candida (ISO 11267) in natural sassafras sandy loam (SSL) soil were orders of magnitude greater than that of the explosives RDX, HMX, or TNT. Results of toxicity tests with CL-20 weathered and aged in SSL soils showed significantly increased toxicity for potworm E. crypticus, but decreased toxicity for collembolan F. candida. These changes in toxicity strongly indicate that the soil chemical environment was altered during the 20-week weathering and aging period, similar to changes that can occur in soil vadose zone environments in the field. The findings of increased toxicity to E. crypticus indicate that additional studies are required to investigate the toxicity of the CL-20 degradation products. Further investigations of the degradation of CL-20 in soil and the resulting soil toxicity should have a weathering and aging component, so that the level of persistence and long-term impact on the ecotoxicity of these degradation products may be assessed. Resolving CL-20 degradation pathways that lead to formation of toxic products, their fate in aerobic soils, and assessment of the individual toxicities of degradation products to soil receptors require further investigation.

The results of the microcosm study showed that indigenous microarthropod and nematode communities exhibited different sensitivities to CL-20 in soil. Total numbers of nematodes were either unaffected, or increased, through the highest CL-20 treatments tested. In contrast, CL-20 significantly adversely affected the microarthropod community after 4 and 8 weeks of exposure, but the total number of microarthropods was not significantly different from controls at the end of the 12-week exposure. Analysis of community structure revealed greater sensitivities to CL-20 of predatory mesostigmatid mites and predatory nematodes. The observed decreases in respective predatory groups of the microinvertebrate community can potentially result in disruption of the soil food web structure. Effects of weathering and aging of CL-20 in soil on the exposure of soil receptors were investigated in this study by chronosequential harvesting of subset replicate-units to determine potential alterations in bioavailability and resulting toxicity of CL-20 to microinvertebrates. Toxicity of CL-20 decreased overtime for Collembola, prostigmatid mites, and predatory nematodes, but briefly increased for oribatid mites after 8 weeks before returning to approximately initial level after 12 weeks. These results showed the complexity of possible interactions among the physicochemical fate processes during weathering and aging of CL-20 in soil, changes in bioavailability of parent material and its possible degradation products, and the soil receptor-specific sensitivities of diverse groups of the soil invertebrate community. Results of the concurrent litter decomposition investigation indicated indirectly that soil biotic activity controlling the rate of litter decomposition was either unaffected or stimulated by exposure to CL-20 in SSL soil up to and including 10,300 mg/kg initial (8238 mg/kg final after 8 months). The two litter grazing groups, oribatid mites and collembola, jointly comprised approximately 40% of the microarthropod community throughout the study. Furthermore, dominance of bacterivore and fungivore nematodes among the nematode community and the increases in their absolute numbers that were sustained throughout the study suggest indirectly that availability of their respective food sources, bacteria and fungi, were also unaffected, or increased in soil CL-20 treatments.

Risks to aquatic ecosystems from potential direct release into aquatic habitats, contaminated surface soil runoff or erosion of contaminated soil into water bodies, and transport of CL-20 to groundwater were assessed by quantifying the toxicity of CL-20 to ecologically relevant aquatic organisms using standardized single-species toxicity tests. The toxicity of CL-20 to aquatic species was investigated using algae (S. capricornutum), Ceriodaphnia (C. dubia), and fathead minnows (P. promelas) in independent tests. Chemical analyses were done to determine soluble CL-20 in media. Results of aquatic toxicity assays indicated that relevant ecological receptors may be negatively impacted by exposure to CL-20 if the compound was released into the environment. This risk to aquatic species is significant both from potential direct release into aquatic habitats and from contaminated surface soil runoff or erosion of contaminated soil into water bodies. Several factors indicate that the release of CL-20 to aquatic ecosystems can potentially cause significant ecological damage in affected sites. These factors include high toxicity of CL-20 to heterotrophic aquatic species and its potentially stimulating effect on autotrophic algae, which can lead to eutrophycation of aquatic habitats. Ecological impacts of CL-20 on aquatic receptors can be further exacerbated by some of the CL-20 fate characteristics, including its apparent persistence in soil and the long-term mobility of aqueous CL-20. Overall, results indicate that accidental release of CL-20 into the environment can have detrimental effects on resident aquatic ecological receptors.

Assessment of the transport and fate of CL-20 in the soil vadose zone were determined using an improved system, the Controlled Environment Soil-core Microcosm Unit (CESMU), adapted from the USEPA and ASTM standard soil microcosm designs. An important modification to the original design was the application of tension at the bottom of each soil column to mimic field conditions and prevent the build-up of water within columns, which can otherwise change the chemical, physical, and biological properties of soil. Intact soil-cores of the SSL soil type used in toxicity tests were used in the transport and fate study. CL-20 concentrates of 98.4 or 9,555 mg/kg were prepared in SSL soil and amended atop the surface of the intact SSL soil-cores. During the 35-week study, CL-20 concentrations in soil were periodically determined for soil collected from different depths, using the acetonitrile extraction method. Ryegrass plants were grown in each of the soil-cores, harvested at the time of soil sampling, weighed, and placed in a -80°C freezer until they were analyzed. Soil-cores were sectioned incrementally by depth, and the CL-20 concentration in each soil section was analytically determined. The fate of that portion of CL-20 that partitions into water was assessed by analyzing both the leachates throughout the study period and the plant tissues of perennial ryegrass. These investigations showed that potential transport and fate of CL-20 in the soil vadose zone was effectively assessed using the CESMU method. Precipitation (simulated rainfall) caused CL-20 to migrate downward in the soil, with concentrations of CL-20 rapidly decreasing with increasing depth of the soil core. Exceptionally small quantities of CL-20 migrated below the 12-cm soil depth of the intact soil core, indicating that CL-20 is not expected to migrate to substantial depth in solid crystal form. Transport of CL-20 in the soil vadose zone was primarily due to solubilization and subsequent partitioning between soil and pore water. Concentrations of CL-20 in soil leachates never reached the CL-20 limit of solubility but increased over 11 weeks, becoming relatively stable at approximately 0.5 and 2 mg/L in 98.4 and 9,555 mg/kg soil treatments, respectively. These results suggest a reasonably high potential for long-term transport of CL-20 to groundwater. Soil physico-chemical parameters that enhance the carrying or sorptive capacity of soil for CL-20 may retard but not eliminate CL-20 migration. However, soil factors that enhance the rate of dissolution of CL-20 in soil, or its aqueous solubility in soil pore water, will increase its potential for transport into groundwater. The bioaccumulations factor values of 0.09 and 0.02 established in studies with ryegrass grown in 98.4 or 9,555 mg/kg CL-20 treatments for 35 weeks, respectively, suggest low bioaccumulation potential for CL-20 in plants.


Draft ecological soil screening levels (Eco-SSLs) for soil invertebrates were derived for CL-20 freshly amended into SSL soil and for CL-20 weathered and aged in SSL soil. No draft Eco-SSL for terrestrial plants was developed as CL-20 was not phytotoxic up to nominal 10,000 mg/kg, the highest concentration tested with the three plant species. The toxicity benchmark values and reports detailing these studies will be provided to the USEPA Ecological Soil Screening Level Workgroup. Results will undergo quality control review by the Eco-SSL Task Group before inclusion in the Eco-SSL database and acceptance for derivation of Eco-SSLs for CL-20.

It is recommended that this information be considered by those who manufacture CL-20, as well as potential users, risk assessors, site managers, and especially CL-20 project managers, prior to transition of CL-20 to military products that currently use RDX, HMX, or TNT.