Environ. Sci. Technol. 1999, 33, 3761-3767
Formation of Nonextractable Soil Residues: A Stable Isotope Approach H A N S H . R I C H N O W , * ,† ANNETTE ESCHENBACH,‡ B E R N D M A H R O , ‡ M A T T H I A S K A¨ S T N E R , § EVA ANNWEILER,† RICHARD SEIFERT,† AND WALTER MICHAELIS† Institut fu ¨ r Biogeochemie und Meereschemie, Universita¨t Hamburg, Bundesstrasse 55, D-20146 Hamburg, Germany, Institut fu ¨ r Technischen Umweltschutz, Hochschule Bremen, Neustadtwall 30, 28199 Bremen, Germany, and Department of Remediation Research, Umweltforschungszentrum Leipzig-Halle GmbH, Permoserstrasse 15, 04318 Leipzig, Germany
Stable carbon isotopic measurements were employed to characterize the transformation of a 13C-labeled polycyclic aromatic hydrocarbon (PAH), anthracene, in a closed soil bioreactor system. The 13C-label was used to calculate a carbon mass balance including mineralization and the formation of nonextractable soil-bound residues. Similar results were obtained from 13C-labeled carbon and 14Clabeled carbon mass balance calculations for separate batch experiments with labeled anthracene. In concentration ranges typical for real PAH-contaminated sites, the sensitivity of the 13C tracer method meets the requirements of classical radiotracer experiments. Therefore, our balancing method based on stable isotope-labeled chemicals may supplement or substitute radiotracer experiments under many circumstances. One major advantage of using stable isotopelabeled tracers is the possible application in transformation studies where the use of radioactive substances is of environmental concern. The transformation of 13C-labeled PAH into nonextractable residues clearly depends on the metabolic activity of the soil microflora and occurs during an early phase of biodegradation. Successive contamination of the soil by anthracene leads to a progressive adaptation of the microflora to a complete mineralization of anthracene in the soil. The extent of residue formation is controlled by the capability of the microflora to degrade the contaminant. Results of long-term experiments indicate that nonextractable residues are relatively stable over time.
soil organic matter may be initiated by enzymatic activity (1) or by condensation reactions occurring during natural humification processes in soils (3). Inorganic components of soils such as clay minerals and metal oxides may also mediate certain condensation and polymerization reactions (4-7). One major consequence of the formation of bound residues is a reduced bioavailability of the carbon from the xenobiotic compound that is sequestered within the soil organic matrix. Radioactively labeled model substances have often been used successfully to evaluate biodegradation, decomposition, and flux of pollutants in the environment within a wide range of applications, e.g., to study the decomposition of natural organic matter (8), the decomposition of humic substances and bound residues (9), and the evaluation of the fate of pesticides in the environment (10). This technique was also applied in the case of PAH (e.g., refs 11-15). 14C-Labeled PAH have been used to study their partitioning into nonaqueous-phase liquids and the effects on bioavailabilitity and aging processes (14, 16). They have been applied to trace relevant mineral matrix-catalyzed chemical reactions (17) to investigate the transformation of PAH compounds in soil bioreactor systems (18, 19) and in complex macrocosm experiments (13, 15, 20). The formation and stability of soil nonextractable PAH residues have been evaluated by the immobilization of the radiolabel within soils. Although radiotracers allow precise studies of transformation processes even at low concentrations, work with radioactive substances has been loosing public acceptance continuously in environmental chemistry because of safety considerations. To overcome the limitations and disadvantages of applying radiolabels, the application of stable isotopelabeled substances in elucidating the interaction of xenobiotica with complex natural organic macromolecules may be a promising strategy (21-26). We have developed a method based on tracer compounds labeled with stable isotopes to calculate for the first time a complete carbon inventory of the 13C isotope in a bioreactor system. To investigate the transformation of xenobiotics in soil systems, we used 13Clabeled anthracene stressing on the formation of nonextractable residues. One major advantage of 13C-labeled tracer substances is their applicability to open systems such as field studies, since tracer substances labeled with stable isotopes are not subjected to specific safety requirements. Based on the 13C-label, conventional analytical methods such as gas chromatography-mass spectrometry (GC-MS) and 13C nuclear magnetic resonance spectroscopy (13C NMR) can be applied for structural assignments of metabolic products in the same experiment (21, 26). Anthracene is chosen as a model compound for PAH transformation in soil since it is biodegradable and its microbial metabolism is well documented (27, 28).
Experimental Section Introduction The decontamination of polluted soils through enhanced formation of nonextractable or bound residues derived from xenobiotic compounds has been suggested as an alternative bioremediation strategy (1, 2). Binding of contaminants to * Corresponding author telephone: 0049-40-42838-4987; fax: 0049-40-42838-6347; e-mail:
[email protected]. † Universita ¨ t Hamburg. ‡ Hochschule Bremen. § Umweltforschungszentrum Leipzig-Halle GmbH. 10.1021/es980927n CCC: $18.00 Published on Web 09/24/1999
1999 American Chemical Society
Materials. The soil was obtained from a pristine Ah-horizon of a Luvisol (collected near Hamburg). The slightly loamy soil material consists of clay (6.4 wt %), silt (15.4 wt %), and sand (78.2 wt %) and has a maximal field capacity of 31.1 wt %. The soil was sieved to a grain size fraction
