Environ. Sci. Technol. 2005, 39, 8776-8783
Effect of Slow Desorption on the Kinetics of Biodegradation of Polycyclic Aromatic Hydrocarbons CESAR GOMEZ-LAHOZ† AND J O S EÄ - J U L I O O R T E G A - C A L V O * Instituto de Recursos Naturales y Agrobiologı´a, C.S.I.C., Apartado 1052, E-41080-Seville, Spain
The bioavailability to bacteria of 14C-labeled polycyclic aromatic hydrocarbons (PAHs) sorbed onto lake sediments was assessed using a mathematical model and three experimental series. The experiments were performed under similar conditions and included: (1) abiotic desorption of PAHs from sediments by Tenax extraction, (2) mineralization of dissolved PAHs with no sediment present, and (3) mineralization of PAHs sorbed onto sediments. Results obtained from the first two series were used to obtain the parameter values for the model, and the experimental results of the third series were compared to model results. We found that microorganisms were able to promote desorption of the more-labile fractions, but were unable to increase the desorption rate of the slow- and very slowdesorbing fractions. Also, our model predictions indicate that, after very long contact times, and in the concurrence of biodegradation, sorbed PAHs remain not under equilibrium conditions, but rather in a steady state. The net rates of PAH desorption from the three sediment domains considered (fast, slow, and very slow) become similar, and the ratio between the aqueous and the sediment concentration remains constant with time.
Introduction Biodegradation is a key process controlling the environmental fate of polycyclic aromatic hydrocarbons (PAHs). However, due to their hydrophobic nature, these chemicals tend to remain sorbed to the solids present in soils and sediments. In this state, PAHs are not available to microorganisms and need to desorb prior to microbial uptake. Substantial advances have been made during the last 30 years in understanding the mechanisms that control sorption-desorption of PAHs in the environment (1), and a number of studies link this process experimentally to biodegradation. For example, White and Alexander (2) examined the biodegradability in soil and aquifer solids of desorption-resistant phenanthrene and naphthalene and found that biodegradation was clearly reduced, as compared to the freshly added compounds. Carmichael et al. (3) determined the rates of desorption, as measured by dilution with water, and biodegradation of PAHs in naturally contaminated soils. They found that the desorption rates of the native compounds were equal to or slower than mineraliza* Corresponding author phone: +34 95-2644711; fax: +34 954624002; e-mail:
[email protected]. † Present address: Departamento de Ingenierı ´a Quı´mica, Facultad de Ciencias, Universidad de Ma´laga (Campus de Teatinos), Ma´laga, Spain. 8776
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tion rates of 14C-PAH measured in short-term experiments (10 h), suggesting that desorption controlled the biodegradation of aged contaminants during this period. Later, Cornelissen et al. (4) introduced into these studies the solidphase extraction method using Tenax beads and determined the kinetics of desorption of 15 PAHs from sediments before and after bioremediation. That study found a nearly direct relationship between the rapidly desorbing fractions (desorption rate constants > 0.1 h-1) and the amounts removed by bioremediation. Other recent studies have also compared, with PAHs, biodegradation rates and Tenax-assisted desorption rates, identifying the clear dependence of biodegradation upon intra-aggregate mass transport limitations (5), and determining, for 15 different soils over a 30-day period, the correlation between the fraction that resists desorption and the fraction that resists biotransformation (6). Finally, Wells et al. (7) examined the relationship between rapidly desorbing PAHs and bioaccessibility, on the basis of experimental results of microbial uptake and Tenax-driven release from model polymer systems. They found that the bioaccessibility is in some cases well correlated with the fitting parameters of desorption, but it cannot be predicted from this information alone. Despite these advancements in the field, there is still a clear need for precise measurements of biodegradation rates during the last, slow phase of biodegradation. The poor prediction capacity for biodegradation rates of slowly desorbing PAHs is today a strong limitation in the management strategies of polluted sites, including bioremediation and natural attenuation, because a high proportion of native PAHs is present in this form. One possible cause for this gap of knowledge is the analytical uncertainty associated with measurements of biodegradation rates of native compounds during the last, slow phase of biodegradation (4, 7). This research constitutes a study on the kinetics of biodegradation of sorbed PAHs. We employed two lake sediments, differing in their content of organic matter, and 14C-phenanthrene (14C-PHE) and 14C-pyrene (14C-PYR) as model PAHs. The assessment of the bioavailability to bacteria of PAHs sorbed onto sediments relied on three experimental series designed to quantify the biodegradation of the fast-, slow-, and very slow-desorbing fractions, and a model aimed at elucidating to what point the biodegradation of the studied contaminants was limited by the desorption kinetics. The use of radiorespirometry allowed us to circumvent some of the problems associated with measurements of biodegradation of slowly- and very slowly-desorbing PAHs. To our knowledge, this is the first attempt to provide net desorption rates for the different kinetic compartments. The resulting model allows new insight of the fate of sorbed PAHs in the environment.
Materials and Methods Chemicals. Phenanthrene-9-14C and pyrene-4,5,9,10-14C were obtained from Sigma-Aldrich, with a radiochemical purity >98% and with 13.1 and 58.7 mCi/mmol, respectively. Nonlabeled PAHs and sodium azide were purchased from Sigma. Solvents were analysis quality, from Panreac or Scharlau. Tenax (60-80 mesh, 177-250 µm) was supplied by Chrompack. Bacteria, Media, and Cultivation. The bacteria used in this study were able to grow with phenanthrene (Sphingomonas sp. LH128) or pyrene (Mycobacterium sp. VM 552) as the sole source of carbon and energy. Both strains were kindly supplied by D. Springael (Vlaamse Instelling voor Technologisch Onderzoek, Mol. Belgium). The characteristics 10.1021/es050850k CCC: $30.25
2005 American Chemical Society Published on Web 10/13/2005
of the strains and maintenance protocols can be found elsewhere (8). Sphingomonas sp. LH128 was used for 14 C-PHE mineralization experiments, and Mycobacterium sp. VM 552 was used for the 14C-PYR ones. Mineralization experiments were performed with an autoclaved aqueous solution denominated mineralization medium (MM), containing KH2PO4 (0.9 g/L), K2HPO4 (0.1 g/L), NH4NO3 (0.1 g/L), MgSO4‚7H2O (0.1 g/L), CaCl2 (0.080 g/L), FeCl3‚6H2O (0.01 g/L), and 1 mL/L of a microelements stock to obtain the final concentrations of 0.0014 g/L for Na2MoO4‚2H2O and 0.002 g/L for each of the following: Na2B4O7‚10H2O, ZnSO4‚H2O, MnSO4‚H2O, and CuSO4‚5H2O. This MM solution presents a pH value of 5.8. Sediments. Ketelmeer (KET) and Varparanta (VAR) sediments were kindly supplied by Dr. G. Cornelissen (ITM, Sweden). They have been characterized previously (9, 10). Ketelmeer is a lake in the sedimentation area of the River Rhine (52°36′ N, 5°45′ E) and can be considered representative of polluted rivers. Its most important characteristics are dry weight 47.2%, total organic carbon (TOC) 5.51%, and total native PAH content ∼40 mg/kg dry weight. The Varparanta sample is a sandy sediment obtained at 0.3 m depth from a shallow shore area of Lake Ho¨ytia¨inen (Finland, 62°51′59′′ N, 29°46′15′′ E) and presents a TOC of 0.120%, and a native PAH content of 0.007 mg/kg (dw). Sediment samples were air-dried, sieved ( 0.99). The results with the two compounds and two sediments were successfully fitted, and the parameter values are shown in Table 1. The kinetic analysis of desorption showed, in all cases, a similar distribution between the fast-, slow-, and very slow-desorbing fractions. The fast-desorbing fraction accounted for 52-62% of the total amount of sorbed 14 C-compound. However, kfast and kslow were somewhat higher with the VAR than with the KET sediment. There were no significant differences between the kveryslow coefficients, which were approximately 10-3 h-1. These results agree with those obtained by Kukkonen et al. (11), showing that the rate of desorption of PAHs from sediments is not correlated with the amount of organic matter in the sediment. The rate of 14CO2 evolution from dissolved 14C-PHE and 14C-PYR was measured, in the absence of sediment, for a range of initial concentrations (7.75-30.37 µg/L for 14C-PHE, and 0.17-4.46 µg/L for 14C-PYR). This gave a total mass of labeled contaminant in the system similar to that in the mineralization experiments with sediment. Results indicate that the relative rate of mineralization was not dependent on the initial concentration, so first-order kinetics should be considered. When the logarithm of the relative mineralization was plotted versus time, two linear regions were observed. 8780
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The good fit of mineralization results with eq 6 (Figure 2B shows representative results with 14C-PHE, where the model results are for the mean values of the parameters) is consistent with the proposed mechanism of mineralization, given by expressions 4 and 5, which differentiates 14CO2 production from 14C-labeled phenanthrene and biomass. The values of the parameters obtained for 14C-PHE were a ) 0.484 ( 0.044, k1 ) (5.55 ( 0.62) × 10-2 h-1, and k2 ) (1.1 ( 0.4) × 10-4 h-1, and for 14C-PYR a ) 0.295 ( 0.040, k1 ) (9.32 ( 0.75) × 10-2 h-1, and k2 ) (2.6 ( 1.1) × 10-4 h-1. Kinetics of Biodegradation of Sorbed PAHs. Figure 2B presents results for mineralization of 14C-PHE initially sorbed to the KET sediment. The figure shows that, during the early stages (first 24 h), mineralization was slower than in the control with no sediment, but still much faster than predicted by the model. On the other hand, after the first 50 h, the predictions for mineralization progressively approached the experimental 14CO2 evolution, leading to similar extents of mineralization after 500 h. A similar situation was observed with other concentrations of 14C-PHE sorbed to KET and VAR sediments, and with 14C-PYR sorbed to KET sediment (Table 2). These results indicate that microorganisms were able to promote desorption from the fast-desorbing fraction, but not from the slow- and very slow-desorbing fractions. In most cases, the experimental and predicted values of final
FIGURE 3. Model predictions of kinetic fractions of 14C-phenanthrene sorbed to Ketelmeer sediment in the presence of active biodegradation. (A) Time course of the relative distribution of 14C-phenanthrene carbon between external (14CO2, biomass, and aqueous-phase) and sediment (fast-, slow-, and very slow-desorbing fractions) compartments. (B) ξj versus time. (C) Relative distribution of 14C-phenanthrene with respect to the actual 14C-phenanthrene sediment content versus time. (D) Kinetic fractions of phenanthrene in Ketelmeer sediment: (i) experimental, abiotic desorption of 14C-spiked sediment, as obtained with Tenax; (ii) model prediction for steady state with biodegradation; and (iii) abiotic desorption of native phenanthrene, as obtained with Tenax. mineralization extent were similar. For 14C-PYR-sorbed VAR sediment, composed mainly of sand particles, the good agreement with model predictions of mineralization rates indicates that the possible enhancement of desorption, if any, was negligible. We considered three hypotheses that could affect these conclusions, based on mineralization measurements: (i) interference caused by 14CO2 production from 14C-labeled biomass, (ii) overestimation of Tenax capacity to act, in our system, as an infinite sink for desorbed PAH, and (iii) possible effects of DOM on PAH bioavailability. The first hypothesis affects conclusions drawn for the later stages of desorption (after the first 50 h); the second for earlier stages (first 24 h); and the third for all desorption stages. (i) To investigate whether the microorganisms were able to exceed the model predictions during later periods, another series of experiments was performed in which the labile fraction was removed by Tenax extraction (for 27 h) before inoculation. These experiments were performed using the same procedure as described for the Tenax desorption experiments, except that the MM used for the centrifugation cycles did not contain NaN3. After 27 h of extraction, the Tenax was removed, the aqueous phase and the sediment were transferred to an Erlenmeyer flask, and from that point the procedure was the same as the one described for the mineralization experiments. Only the results for the KET sediment are presented. Figure 2C shows the results,
calculated with respect to the labeled contaminant remaining after Tenax extraction (approximately 30%), obtained for mineralization of 14C-PHE. This represents 96.5% of the very slow-desorbing fraction, 64.4% of the slow-desorbing one, and only 0.04% of the fast desorbing fraction (Table 1). As can be seen in Table 2, there is very good agreement between the experimental results and the model. Similar results were obtained with 14C-PYR. (ii) We estimated the maximum errors introduced by the perfect sink assumption. For this aim, we performed experiments to obtain the rate of decrease of the aqueous concentration of dissolved PAHs due to adsorption to Tenax (kT) under the same experimental conditions as for the desorption experiments. The rate of decrease from the aqueous phase was kT ) 24.0 ( 4.8 h-1 for 14C-PHE, and kT ) 25.8 ( 0.0 h-1 for 14C-PYR. We then calculated, for phenanthrene and pyrene, the amount of substrate desorbed from the fast fraction during the first hour, using the values for kfast given in Table 1 for the KET sediment, and considering a permanent zero value for the aqueous concentration: kfast
∞
sediment fraction (Sfast) 98 aqueous 98 Tenax
(14)
We compared these values to the theoretical amounts of compound desorbed in a system where the aqueous phase concentration could be replenished: VOL. 39, NO. 22, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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kfast
kT
sediment fraction (Sfast) {\ } aqueous {\ } Tenax k′ k′ fast
T
(15)
The theoretical amounts of compound desorbed, calculated in these two different ways, were only slightly different (
