Besides their explosive properties, energetic compounds, such as RDX, HMX, and TNT, are environmental pollutants contaminating numerous military sites in North America.  Phytoremediation has been shown to provide a cost-effective alternative to classical technologies for cleaning up nitro-substituted explosive-contaminated sites.  However, although higher plants have been shown to take up energetic pollutants from groundwater and soil, little information is available about the fate of pollutants inside plant tissues.  In the absence of further transformation and detoxification, metabolites will sooner or later return to the soil, resulting in a pollution transfer and a potential biohazard for the environment.

The main objective of the project was to explore further the metabolic routes and the catabolic enzymes involved in the transformation/detoxification of the nitrosubstituted explosives TNT, RDX, and HMX by poplar trees, a model plant for phytoremediation studies.  Particular attention was given to biocatalysts known or suspected in the degradation of nitro-substituted explosives by microbes and having corresponding equivalents in plants.  A secondary objective was to investigate the toxicity and the environmental hazard associated with nitro-substituted explosives and phyto-transformed metabolites once taken up inside poplar tree tissues.

Degradation experiments were performed by incubating TNT, RDX, and HMX in the presence of whole plants (in vivo experiments) and in the presence of cell cultures, tissue cultures, or enzyme crude extracts (in vitro experiments).  Identification and quantification of nitro-substituted pollutants and related metabolites in the different plant fractions allowed us to clarify the metabolic pathways and provide clues for the enzymes potentially involved in the process.  Catabolic enzymes were studied by gene expression and through detection of specific activities in crude plant extracts before their further purification and characterization.  A set of toxicity tests (MicroBioTests) based on organisms from different trophic levels were performed in order to evaluate the efficiency of the phytoremediation process and the environmental hazard associated with contaminated plants.

Three explosives were taken-up by hybrid poplar without interacting effects.  TNT was bound and immobilized in root tissues, but RDX and HMX were translocated into leaves.  HMX was leached from leaf litter more easily than RDX and TNT, mostly as parent compound.  RDX and TNT that were taken up by plants were released mostly as transformed products from leaf tissues, but leaching of TNT and its metabolites was not significant.  The leached explosives (from plant tissues) and their transformed products could pose potential hazards in the environment.  However, Microtox® tests, used as a preliminary screening indicator for ecotoxicity, showed that HMX was not significantly toxic and that RDX and TNT toxicity was removed from hydroponic solution after 13 days by poplar cell tissue cultures.

A method was developed from which to pursue the involvement of several detoxification/transformation enzymes up-regulated upon exposure to explosive compounds using gene expression.  Besides fundamental insights about the metabolism of RDX and TNT by plants, the identification of genes potentially involved in their degradation could have several practical applications, such as to assess the toxicity associated with residual explosive compounds and metabolites inside plant tissues; to monitor soil, groundwater, or plant contamination by energetic compounds; or to develop transgenic plants with enhanced phytoremediation capabilities.  These results will continue to support and potentially benefit the use of phytoremediation for the treatment of groundwater contaminated with nitro-substituted explosives.

The project accomplished both of its main objectives, gaining insight in the metabolic routes and catabolic enzymes involved in detoxification/transformation of explosive compounds, as well as determining phytotoxicity and ecotoxicity of the compounds and their transformed products.

According to the high level of contamination of target areas and because periodic inputs of energetic materials are expected, one of the only reliable in situ biological treatments seems to be phytoremediation.  The proposed research will provide a better understanding of the metabolic pathways and catabolic enzymes underlying phytotransformation of nitro-substituted explosives.  The study also will determine the toxicity of stored or bound pollutants and related metabolites inside the plant tissues.  The expected results will allow us to evaluate the relevance and the biological hazard potentially associated with the phytoremediation process.

Results from this study were used as the basis for additional research under SERDP Project ER-1499, where studies were undertaken to understand the mechanisms by which toxic energetic compounds are detoxified in contaminated subsurface soils at DoD ranges by plants native to the site, either by direct uptake and transformation in plant tissues or by microbial activity in the rhizosphere. Results from this follow-on effort are available within the ER-1499 project overview.