Transformation of TNT by Aquatic Plants and Plant Tissue Cultures JOSEPH B. HUGHES,* JACQUELINE SHANKS, † MINDY VANDERFORD, JOHN LAURITZEN, AND RAJIV BHADRA George R. Brown School of Engineering, Rice University, 6100 Main, Houston, Texas 77005-1892 The ability of plants to uptake and transform 2,4,6- trinitrotoluene (TNT) was investigated using the aquatic plant Myriophyllum spicatum, axenic Myriophyllum aquaticum, and Catharanthus roseus hairy root cultures. Studies dem- onstrate that Myriophyllum, with or without its periphyton, and C. roseus transform TNT. Low concentrations of aminated nitrotoluenes (2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene) were observed in the extracellular medium and tissue extracts. Primary products of transformation were not identified, and mineralization was not observed. Mass balances demonstrate that a large percentage of the unknown TNT transformation products were associated with the plant. This fraction could be at least partially recovered from the plant tissue with methanol extraction. A soluble fraction was also present in the medium. The absence of periphyton had little impact on the results observed. Medium concentrations of 4-amino- 2,6-dinitrotoluene were greater in systems in which the periphyton was not removed. For the first time, the intrinsic ability of plants to transform TNT has been confirmed. The formation of soluble, uncharacterized transformation products is a concern for potential phytoremediation applications. Introduction Munitions production and handling over the past 90 years has led to widespread and persistent 2,4,6-trinitrotoluene (TNT) contamination at a number of government facilities (1). Since TNT is toxic to many aquatic (2-4) and terrestrial species (5),the remediation ofcontaminated soilsisnecessary. Excavation and thermal processes are currently the most common remedial actions (1), but are costly. Recent efforts have sought to reduce the cost ofremediation with alternative technologies such as bioremediation (6) and phytoremedia- tion (7, 8). Phytoremediation processes rely on the ability of plants to uptake and, in some cases, to metabolize pollutants. Information regarding the potential for TNT uptake or transformation in plants is cited in onlya few studies. Palazzo and Leggett (9)reported TNTuptake from soilbybush beans and identified traces of several reduction products (e.g., aminonitrotoluenes)in leafextracts. Wolfe et al.(7)reported rapid TNT disappearance from an aqueous medium in the presence ofStonewort (Nitella ), also identifying low levels of aminonitrotoluenes in the aqueous medium. In both these studies,however,the experimentalsystems contained plant- associated microflora, so the ability of plants to transform TNT without the participation of associated microbes re- mained arguable. Moreover, because mass balances were not performed, it was impossible to evaluate the final distribution of TNT and/or transformation products. In this paper, we report studies on the fate of [U- 14 C]TNT in two plant systems: (1)native Myriophyllum spicatum with its associated microflora and (2) aseptic plant systems using axenic Myriophyllum aquaticum and Catharanthus roseus hairyroot tissue cultures. M.spicatum was selected because of its availability at a munitions-contaminated facility (Ala- bama Army Ammunition Plant, Childersburg, AL) and the commercialavailabilityofaxenic Myriophyllum plant cultures. C. roseus hairy root cultures were employed for their rapid growth rates and as a microbe-free surrogate for plant-root activity. Allsystems were sediment-free to avoid complicating results with sorption or irreversible binding processes. Followingincubations,massbalanceswereperformed. These studies demonstrate the ability of plants to transform TNT rapidly and, interestingly, without the stoichiometric ac- cumulation of aminonitrotoluenes or azoxy dimers. Experimental Section Chemicals. 2,4,6-Trinitrotoluene, trinitrobenzene, 2,6-dini- trotoluene,2,4-dinitrotoluene,nitrobenzene, o-nitrotoluene, p -nitrotoluene,and toluene (ChemService,Westchester,PA); [ring-U- 14 C]-2,4,6-trinitrotoluene (NEN Research Products, Boston, MA), specific activity of 1.3 mCi/mmol and purity of 98%; 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitro- toluene (AccuStandard Inc., New Haven, CT); 2-hydrox- ylamino-4,6-dinitrotoluene, 4-hydroxylamino-2,6-dinitrotol- uene, and 2,4-diamino-6-nitrotoluene (Dr. Ron Spanggord, SRI International, Menlo Park, CA); 2,6-diamino-4-nitrotolu- ene, 2,4,6-triaminotoluene, 2,2′,6,6′-tetranitro-4,4′-azoxytolu- ene,and 4,4′,6,6′-tetranitro-2,2′-azoxytoluene (Prof.Deborah Roberts,UniversityofHouston,Houston,TX);NaH 14 CO3 (New England Nuclear, Boston MA) specific activity of 3.0 mCi/ mmol; 2-propanol (EM Science, Gibbstown, NJ) were used. Plants and Tissue Cultures. Native samples of M. spicatum were collected from ponds located at the Alabama Army Ammunition Plant, Childersburg, AL. Plants were cultured outdoors in 20-gal containers recharged naturally with rainwater. The water depth in these systemswastypically 40 cm. Sediments collected from ponds located at the Ala b a m a Army Ammunition Plant were added to these containers with limestone gravelas a source oftrace nutrients and carbonates and to provide for a rooting matrix. Axen ic M. aquaticum plants were purchased from Ameri- can Type Culture Collection (ATCC, Rockville, MD) and propagated vegetativelyon NH 4 + -free Murashige Skoog(MS) medium (Sigma Plant Culture, St. Louis, MO) supplemented with agar. Axenic plants were maintained in magenta boxes at room temperature under light from cool white and broad spectrum bulbs for a 14-h photoperiod at a light intensity of 100 μEinstein/m 2 . Axenic plants were grown for 3 months to obtain sufficient biomass for harvesting. Hairyroot cultures of C. roseu s LBE-6-1 were grown in the dark at 26 °C on an orbital shaker (110 rpm) in 250-mL Erlenmeyerflaskscontaining50mLhalf-strength Gamborg’s B5 salts (Sigma Plant Culture, St. Louis, MO) and 30 g/L sucrose. Medium pH was adjusted to 5.7 with HCl prior to filter sterilization (0.22 μm). Roots were subcultured every 3-4weeksbytransferringfive tips,approximately35-40mm in length and 0.04 g total, into a flask with fresh medium. Fresh weight ofthe root biomass was determined bymedium displacement as in Bhadra et al. (10). *To whom correspondance should be addressed: telephone: (713)285-5903; fax: (713)285-5203; e-mail address: hughes@ owlnet.rice.edu. † Department ofChemicalEngineering,Rice University,Houston, TX, 77005. Environ. Sci. Technol. 1997, 31, 266-271 266 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 31, NO. 1, 1997 S0013-936X(96)00409-9 CCC: $14.00  1996 American Chemical Society