The overall objective was to improve the understanding of RDX transformation in plant tissues and the subsequent cycling of tissue-associated RDX and RDX daughter products among soil mineral and humic fractions following plant senescence. The hypothesis was that environmental risks from RDX at military training ranges can be reduced, and possibly eliminated, through a series of coupled processes involving plant uptake, plant enzyme mediated transformation, photodegradation in the plant, and finally humification of plant-tissue-associated RDX conjugates into soil organic matter after plant senescence and leaf drop. Although the effect of each individual process may be small, the combined effects of the processes taken as a system for sustainability may have a significant impact on RDX residues on surface soils. If so, they may lead to feasible range sustainability management practices. RDX is found in the soils and groundwater of bombing ranges and manufacturing sites. Plants of the family Lamiaceae were used to determine if either their enzymatic activities could accelerate the degradation of RDX once taken up from an aqueous solution. Plant tissue with higher chlorophyll content was found to contain higher concentrations of RDX, while the presence of anthocyanin appeared to have no impact. Of the four varieties of mint tested, chocolate mint, a variety of spearmint [Mentha spicata], had significantly lower RDX concentrations in its leaf tissues. Further research is needed to determine what processes are responsible for the reduced RDX content. Ascorbate, pH, and glutathione (GSH) were found to be statistically significant factors in the photodegradation of 2,4,6-trinitrotoluene (TNT), a process applicable to RDX. Ascorbate and pH increased the rate of TNT degradation, whereas GSH inhibited it. Photo-induced degradation of TNT occurs at approximately the same rate in extract-based solution. The results indicate that ascorbate and pH increase the rate of photolysis of TNT, whereas glutathione decreases it. In sufficiently reduced systems, RDX has been shown to attenuate, but the specific reactions and characterization of the residues that are produced have not been completely determined. Recent studies have demonstrated that both bacteria and fungi can also mineralize RDX, but, again, the pathways and intermediates formed are poorly understood. Because precedence has been established for RDX transformation, and explosives have been shown to bind covalently to soil humic fractions or organic material in compost, a humification approach may have significant utility in treating surface soils on impact and training ranges. DISCLAIMER: The contents of this report are not to be used for advertising, publication, or promotional purposes. Citation of trade names does not constitute an official endorsement or approval of the use of such commercial products. All product names and trademarks cited are the property of their respective owners. The findings of this report are not to be construed as an official Department of the Army position unless so designated by other authorized documents. DESTROY THIS REPORT WHEN NO LONGER NEEDED. DO NOT RETURN IT TO THE ORIGINATOR. ERDC/CRREL TR-13-4 iii
34 Figures and Tables
Table 1. Experimental conditions for the multivariate photolysis experiments in which the effects of pH, glutathione, and ascorbate on TNT photodegradation were examined. (*Indicates center point experiments).
Figure 2. Volume of stock RDX-water solution taken up by each plant species.
Table 2. Five concentration levels matching the coded factor levels for the three-factor central composite design (n = 3 for axial and factorial point experiments; n = 6 for center point experiments).
Figure 3. Pigmentation patterns of all coleus used: a) all-green; b) tri-color; c) green-white; d) dark red-green; e) green-red; f) dissection pattern of all-green coleus leaves.
Table 3. Experimental conditions (factor levels) and corresponding kobs values for TNT photodegradation.
Figure 4. Location of RDX in leaf tissue sections of all-green coleus. RDX concentrated disproportionately in margins of leaves.
Table 4. β-values and corresponding t-test comparisons for the full factor model at the 95% confidence level. The model has a correlation coefficient (r2) of 0.821.
Figure 5. Location of RDX by mass in each corresponding section of tri-color coleus. The RDX is located primarily in the green leaf margins. However, no significant difference exists in RDX concentration between the red and white leaf sections (n = 4).
Table 5. Percentage of each factor’s contribution to the overall photolysis rate.
Figure 6. Location of RDX in leaf tissues of green-white coleus. The RDX is located primarily in the green, leaf margins (n = 4).
Table 6. Degradation rates of TNT from the extract photolysis experiments.
Figure 7. RDX concentration in all plant tissues for two varieties. Concentration of RDX in each tissue standardized by mL of RDX-water solution taken-up by each plant (n = 4, tri-color coleus; n = 4, green-white coleus).
Table 7. RDX uptake and accumulation in three plants.
Figure 8. RDX distribution within the dark red-green coleus. The RDX is located primarily in the marginal green leaf tissue (n = 5, RDX mass; n = 1, leaf biomass).
Table 8. Differences in RDX update in tricolored leaves of coleus.
Figure 9. RDX distribution in the green-red coleus. The RDX is distributed proportionately throughout the plant (n = 4).
Figure 10. RDX distribution in the leaf tissue of green-red coleus. The RDX is distributed proportionately to the tissue mass and does not correspond to pigmentation pattern (n = 4, RDX mass; n = 1, leaf biomass).
Figure 11. RDX and chlorophyll concentrations in all coleus green leaf tissues. There does appear to be a positive correlation between RDX concentration and chlorophyll concentration.
Figure 12. RDX and chlorophyll concentrations in mint plants. There is a positive relationship between RDX concentration and chlorophyll concentration.
Figure 13. RDX and chlorophyll concentrations in all coleus and Mentha plants. There is an overall positive relationship between chlorophyll concentration and RDX concentration.
Figure 14. Distribution of RDX within tissues of Mentha plants.
Figure 15. RDX-exposed spearmint plants vs. control plants prior to takedown. Note brown lesions on exposed plants.
Figure 16. RDX concentration and anthocyanin absorbance in all plants used. There is no relationship between RDX concentration and anthocyanin absorbance.
Figure 17. Direct photolysis of TNT occurs rapidly (t1/2 = 1.46 hours) in water. T = 26 ± 1°C; n = 3; pH = 7.25; [TNT] initial = 101.8 ± 3.3
Figure 18. Three-dimensional plot of kobs as a function of ascorbate (0–50 mM) and glutathione (0−10 mM) in water at pH = 7.25 was derived from the model.
Figure 20. RDX photolysis does not correlate with the presence of several different cytosol components.
Figure 21. Photodegradation of nitrobenzene correlates strongly with the presence of glutathione. Ascorbate and cysteine may also be significant.
Figure 22. RDX and the reductive transformation products, MNX, DNX and TNX.
Figure 23. Approach to track and identify fate of 14C-RDX added either directly to soil or from plant tissue.
Figure 24. Soil organic matter fractionation scheme.
Figure 25. Color changes in the organic fractions following soil extraction.
Figure 26. Humic/fulvic ratios in high and low OM soils treated with either 14C-labelled RDX applied directly to the soil or 14C-labelled RDX that was incorporated into plant tissue by growing plants in 14C-labelled RDX in soil (+ Leaf).
Figure 28. Summary of significant pathways in RDX fate in soil–plant– microbial systems. Numbers associated with each labile pool of RDCderived C are discussed below.
Figure 29. Treatments that were tested using a modified leachate toxicity test based on the growth inhibition of the algae Selenastrum capricornutum.
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