Using Nitrogen Isotope Fractionation To Assess Abiotic Reduction of

Institute of Biogeochemistry and Pollutant Dynamics (IBP), ETH Zurich, Universitätsstrasse 16, CH 8092 Zurich, Switzerland, and Eawag, Swiss Federal ...
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Environ. Sci. Technol. 2006, 40, 7710-7716

Using Nitrogen Isotope Fractionation To Assess Abiotic Reduction of Nitroaromatic Compounds A K A N EÄ H A R T E N B A C H , T H O M A S B . H O F S T E T T E R , * ,† MICHAEL BERG, JAKOV BOLOTIN, AND R E N EÄ P . S C H W A R Z E N B A C H Institute of Biogeochemistry and Pollutant Dynamics (IBP), ETH Zurich, Universita¨tsstrasse 16, CH 8092 Zurich, Switzerland, and Eawag, Swiss Federal Institute of Aquatic Science and Technology, 8600 Dubendorf, Switzerland

l k KIEE ) h k

where lk and hk are the rate constants for a molecule exhibiting the light isotope (e.g., 12C, 14N) and heavy isotope (e.g., 13C, 15 N), respectively, at the reactive position. One generally observes a normal isotope effect, that is, the molecule carrying the light isotope reacts somewhat faster than the one with the heavy isotope (lk > hk and therefore KIEE > 1). This means that during the course of the reaction, the parent compound is enriched in the heavier isotope. If this enrichment is independent of the concentration of the compound, that is, constant during the course of a reaction, the linearized Rayleigh-equation (eq 2) can be applied to derive a bulk isotopic enrichment factor, E, which is commonly reported in per mil (‰)

(

ln

Compound-specific isotope analysis (CSIA) is an increasingly important tool for the qualitative and quantitative assessment of transformations of organic compounds in contaminated environments. To date, the use of CSIA has been mainly restricted to the elements C and H, although N constitutes a very important reactive center for many priority contaminants. To evaluate the potential use of N isotope effects in the fate assessment of organic contaminants, we investigated the N isotope enrichment during the abiotic reduction of 4 substituted nitroaromatic compounds (NACs), using two abiotic model reductants, namely Fe(II) sorbed to goethite (R-FeOOH) and juglone (8-hydroxy-1,4naphthoquinone) in the presence of H2S. Substantial and virtually identical isotope enrichment factors, N, of about -30‰, indicative of the breaking of one N-O bond, were found for all NACs, regardless of the reductant involved and the substitution of the NAC. These results indicate that the N-values determined in our study could be representative for the reduction of aromatic NO2-groups and thus be used to assess the abiotic transformation of NACs qualitatively and quantitatively in complex anoxic environments.

Introduction Compound-specific isotope analysis (CSIA) is an increasingly important tool for the identification of contaminant sources and for a qualitative and quantitative assessment of abiotic and enzymatic transformations of organic contaminants in the environment (1). In addition, CSIA may provide information on specific reaction mechanisms, particularly if isotope signatures (i.e., the relative isotopic compositions of a given element within a compound) are studied on more than one element (2-4). Indeed, the breaking of a bond is commonly associated with a characteristic intrinsic kinetic isotope effect (KIEE) for the elements E involved in the bond breaking * Corresponding author phone: +41 44 632 83 28; fax: +41 44 633 11 22; e-mail: [email protected]. † Institute of Biogeochemistry and Pollutant Dynamics. 7710

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(1)

)

E 1000 + δhE ) ln f h 1000 1000 + δ E0

(2)

where f is the fraction of compound that has not reacted, and δhE and δhE0 are the isotopic signatures of the compound for the element E at times t and zero, respectively. Note that E has a negative value for normal KIEE. In principle, these enrichment factors can then be used to quantify the extent of (bio)transformation, which corresponds to the fractional amount of substrate conversion F (5) or 1-f (see eq 3), based on measurements of isotopic signatures in field samples, for example along the path of a plume (3, 6)

F)1-f)1-

(

)

1000 + δhE 1000 + δhE0

1000/E

(3)

where δhE and δhE0 are the isotopic signatures of the compound at two different locations, such as at a given distance from the source and at the source, respectively. Since E-values can depend on the prevailing environmental conditions, the extent of their variability must be known when using eq 3 for a given type of transformation in the field. This information can be obtained by considering the actual reaction mechanism leading to the observed isotope effect and converting bulk E into bond-specific KIEs (2). If only one atom of the element E is present in the molecule, and if this element is involved in the reaction, then the conversion is straightforward, and an “apparent” kinetic isotope effect (AKIEE) can be derived directly from observed E (eq 4).

AKIEE )

1 1 + E/1000

(4)

Otherwise, the AKIE determined with eq 4 needs further correction for additional effects (5). If several atoms of the same element are present in a contaminant, those not taking part in the reaction will decrease the observable isotope fractionation and the corresponding E-value due to isotopic dilution. Such corrections also have to take into account that several atoms of the same element can react in a concerted way at different positions in the molecule thus making corrections for isotopic dilution less straightforward. Furthermore, intramolecular isotopic competition may occur if competing reactions take place at the reactive center (for details see ref 2). Finally, intrinsic KIEE-values might be masked by nonfractionating, rate-limiting steps such as binding to an enzyme or transport to a reactive surface site 10.1021/es061074z CCC: $33.50

 2006 American Chemical Society Published on Web 11/01/2006

as well as by commitment to catalysis (7). Hence, AKIEE values must be compared with intrinsic KIEE-values reported in the literature or derived from theoretical calculations to assess the degree of E-variability for a given type of contaminant and its transformation under different environmental conditions. In contaminant hydrology, most applications of CSIA for the assessment of organic contaminant transformations have been carried out with the elements C and H measuring δ13Cand δ2H-signatures, respectively (8-11). However, organic molecules usually have nonreactive C and H atoms, and both elements are subject to significant isotopic dilution, thus making the determination of accurate AKIEE from bulk E more difficult. To the best of our knowledge, no N-values have been reported for organic contaminants, although many priority contaminants exhibit nitrogen containing functional groups that are frequently the sites of abiotic and/or enzymatic reactions. Common examples include hydrolysis of amides, carbamates or ureas, or redox reactions involving nitro-, azo-, or amino groups. In such hydrolysis reactions, N values could be a more sensitive parameter than Cseven though a carbon-nitrogen bond is brokenssince the latter may be very small due to isotopic dilution. In order to evaluate the potential use of N isotope fractionation in contaminant hydrology, we investigated Nvalues associated with the abiotic reductive transformation of nitroaromatic compounds (NACs). Due to their widespread use as agrochemicals, explosives, and dyes and due to their considerable toxicity, NACs are important soil and groundwater contaminants worldwide (12, 13). For the purpose of this study, NAC reduction to the corresponding anilines was chosen as the model reaction due to the lack of N isotope dilution and because two N-O (but no C-N bonds) are broken in the course of the reaction (Scheme 1).

SCHEME 1

Previous studies involving inorganic nitrogen species have shown that AKIEN values associated with the reductive cleavage of a N-O bond are significant. In the case of nitrite (NO2-) reduction to hydroxylamine (NH2OH) as well as hydroxylamine reduction to ammonia (NH3) by Fe(II) in alkaline media AKIEN values of the order of 1.03 have been observed for both reactions (14, 15). As with other contaminants, NACs are quite persistent in the presence of oxygen, but they are susceptible to enzymatic and abiotic reduction under anoxic conditions. This process may lead to products that can be further (bio)degraded in the contaminated subsurface or to products that are of equal or greater (eco)toxicity than the parent compound (16). Knowledge of rates and extent of NAC transformation in anoxic soils and groundwaters is therefore essential to assess the risks of subsurface contamination of drinking water resources. In previous work, we have investigated the kinetics of the reduction of a series of substituted NACs covering a wide range of one-electron reduction potentials, E1h, in various model systems including two hydroquinones (HQs) as well as natural organic matter (NOM) in the presence of hydrogen sulfide as electron source, a ferrous iron porphyrin (FeP), Fe(II) adsorbed to goethite (R-FeOOH, Fe(II)/goethite), and in two-column systems run under iron-reducing conditions (17-20). We found that, depending on the rate-limiting step, the relative reduction rates of the investigated NACs varied by 5 orders of magnitude (HQs/H2S; NOM/H2S), 2 orders of magnitude (FeP, Fe(II)/goethite), or they were all

identical (column systems). From these findings, we concluded that in the HQs/H2S and NOM/H2S system, the actual electron-transfer step was rate-determining (a difference in E1h of 59 mV corresponded to an increase in the rate constant by a factor of 10), whereas in the FeP and Fe(II)/goethite systems, adsorption to reactive surface sites was postulated to influence the observed reduction rate. Finally, in the twocolumn systems, regeneration of reactive Fe(II) sites was assumed to be rate-limiting. Obviously, such great variability in rate constants renders it very difficult, if not impossible, to make any sound predictions of reduction rates of nitroaromatic contaminants, particularly in complex field systems. Consequently, besides establishing mass balances of reactants and the various reduction products, the use of nitrogen and/or oxygen isotope fractionation is the only alternative to attempt quantification of such processes in the environment. In this paper, we present results on N isotope fractionation occurring during the abiotic reduction of a series of NACs in two of our model systems, namely in solutions of hydroquinone/H2S and in suspensions of Fe(II)/goethite. The goals of this study were (i) to provide first insights into the extent of N isotope fractionation of these reactions, (ii) to determine the influence of compound properties (e.g., different substituents) and reductant characteristics on the N isotope enrichment factor, N, and (iii) to evaluate whether AKIEN values derived from N-values provide mechanistic insight into the reactions in the two model systems. To the best of our knowledge, this is the first study on the use of N isotope fractionation to assess organic contaminant transformation under conditions typical for environmental systems.

Experimental Section Preparation of Fe(II)/Goethite Suspensions. Reactors for the kinetic experiments were prepared in an anoxic glovebox (N2 atmosphere) following a modified procedure of ref 8. Goethite (1.5 g L-1) was suspended in 24 mL of a deoxygenated buffer solution (10 mM MOPS, pH 7.0) followed by addition of 93 µL of 0.52 M FeCl2 solution. The 25 mL serum flasks were sealed with Viton stoppers, and the suspensions were equilibrated in the dark on a reciprocating shaker for at least 24 h. A minimum of 6 reactors was prepared for each kinetic experiment. The blank consisted of the same suspension without the addition of the FeCl2 solution. Dissolved Fe(II) and pH were measured after the equilibration period. Preparation of the Juglone/H2S Solutions. Reactors containing H2S and juglone were prepared in the glovebox using aqueous stock solutions of sulfide and methanolic solutions of juglone (20). Sulfide and juglone were added to 19 mL of 50 mM KH2PO4 deoxygenated buffer solution at pH 6.8 and 7.5 to achieve final concentrations of 5 mM and 200 µM, respectively. The 25 mL serum flasks were sealed with Viton stoppers. Solutions were hand-shaken and subsequently equilibrated in the dark (25 °C) for at least 48 h. A minimum of 6 reactors was prepared for each kinetic experiment. The blank consisted of the same matrix without juglone. Kinetic Experiments. Experiments were initiated in the glovebox by the addition of methanolic NAC spike solutions (final NAC concentration 150 µM). Fe(II)/goethite and juglone/H2S reactors were agitated outside the glovebox in the dark at 25 °C with a reciprocating shaker (20 rpm) and a horizontal shaker (240 rpm), respectively. For both experimental systems, assays were sacrificed at given time intervals for concentration and isotope analysis. Heterogeneous reactions (Fe(II)/goethite) were quenched by centrifugation (8 min at 4000 r/min, 4 °C) as described earlier (8). The supernatant was transferred into 20 mL amber flasks, and 0.5 mL was taken for concentration measurement by VOL. 40, NO. 24, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Reduction of 4-chloronitrobenzene (4-Cl-NB) to 4-chloroaniline (4-Cl-An) in 2 mM Fe(II)/1.5 g L-1 goethite at pH 7.0: (a) time course of the concentrations; lines represent model fits for sequential pseudo-first-order reduction of the NAC and formation of the corresponding aniline (see Table 1 for rate constants and text for details on modeling), (b) measured δ15N values for 4-Cl-NB and 4-Cl-An and calculated isotope enrichment using eqs S11-S16 (SI), (c) linearized 15N-enrichment of 4-Cl-NB according to eq 2, and (d) measured δ13C values for 4-Cl-NB and calculated isotope enrichment for 4-Cl-NB and 4-Cl-An (note that δ13C data for 4-Cl-An were not determined) as well as (e) linearized 13C-enrichment of 4-Cl-NB according to eq 2. Note that experimental errors were smaller than symbols for data points in panel e. HPLC. The remaining solution was stored at 4 °C until isotope analysis.Homogeneousreactions(juglone/H2S)werequenched for concentration measurements by extraction of 0.5 mL aliquots with 1.0 mL of ethyl acetate. After sampling for HPLC analysis, 4.8 mL of 1.0 M zinc acetate were added to the remaining solution to precipitate sulfide quantitatively and quench reduction reactions of NACs or partially reduced intermediates. The ZnS precipitate was centrifuged (6 min at 4000 r/min, 4 °C), and the supernatant was subsequently transferred into amber flasks to avoid potential reaction of the NAC with the ZnS phase. The flasks were stored at 4 °C until isotope analysis. Good agreement between isotope signatures of pure compounds and of the NACs measured at the start of kinetic experiments with juglone/H2S indicates that negligible isotope fractionation (δ15N < (0.3‰) occurred due to ZnS precipitation. Concentration Measurements. NAC and their corresponding anilines were quantified by HPLC on a Supelcosil LC-18 reverse phase column (25 cm × 4.6 mm, Supelco, Division of Fluka; Buchs, Switzerland). Eluents consisted of MeOH/H2O mixtures (55%/45% for the chlorinated NACs, 60%/40% for the nitrotoluenes), and UV/vis detection was performed at the absorption maxima of the nitrobenzenes and anilines (18). An injection volume of 30 µL was applied for all aqueous samples, whereas an injection volume of 6 µL was set for ethyl acetate extracts. In both, homogeneous and heterogeneous systems, one intermediate observed in HPLC chromatograms at lower retention times than the substituted aniline could be identified as N-hydroxylamine (21). However, lack of commercially available reference materials compromised its quantitative evaluation. The extent of formation of intermediates was more pronounced in experiments with juglone/H2S than with Fe(II)/goethite as was observed in earlier work (18). 7712

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Isotopic Analysis. NACs were extracted by solid-phase microextraction (SPME) from the aqueous solutions after addition of 4 M NaCl at 40 °C for 45 min using a 85 µm polyacrylate fiber. Desorption of NACs took place at 270 °C for 3 min and was coupled online to a GC-C-IRMS (gas chromatography isotope-ratio mass spectrometry with a combustion interface (22)). Chromatographic separation of the NACs and corresponding anilines was achieved on a Zebron ZB-5-MS (30 m, i.d. 0.32 mm, film thickness 0.45 µm, Phenomenex) column. The δ15N- and δ13C-values are reported relative to air and Vienna PeeDee Belemnite (VPDB), respectively. Isotope signatures of NACs and anilines were derived from triplicate measurements with precisions of (0.6 ((1σ). A detailed description of the SPME-GC-C-IRMS method for NACs developed on the basis of previous work suggested for isotopic analysis of organic compounds (23, 24) is provided in the Supporting Information (SI). Calculation of Reaction Rate Constants and Isotopic Enrichment Factors. Rate constants for the reduction of the NACs were calculated using a simple kinetic model describing the pseudo-first-order transformation of the NACs to reaction intermediates and the corresponding substituted anilines (see ref 25 for modeling details). Isotopic enrichment factors for N and C were determined according to Scott et al. (26) from a linear regression analysis using eq 5 where E was obtained from the slope of the line ln(RE) vs ln(c).

ln(RE) ) E/1000‚ln(c) + ln(RE0/(c0))

(5)

RE ) RE,ref(δEE/1000 + 1)

(6)

with

where RE equals the isotope ratio of element E in the sample

FIGURE 2. Reduction of 2-chloronitrobenzene (2-Cl-NB) to 2-chloroaniline (2-Cl-An) in solutions of 200 µM juglone and 5 mM H2S at pH 7.5: (a) time course of the concentrations; lines represent model fits for sequential pseudo-first-order reduction of the NAC and formation of the corresponding aniline (see Table 1 for rate constants and text for details on modeling), (b) measured δ15N values for 2-Cl-NB and 2-Cl-An and calculated isotope enrichment eqs S11-S16 (SI), and (c) linearized 15N-enrichment of 2-Cl-NB according to eq 2.

TABLE 1. One-Electron Reduction Potential, E1h, Pseudo-First-Order Rate Constants of NAC Reduction, kobs, 15N Isotope Enrichment Factors, EN, and Corresponding Apparent Kinetic Isotope Effects, AKIEN, of Substituted NACs Obtained from Experiments in Suspensions of Fe(II) and Goethite and in Solution Containing Juglone (8-Hydroxy-1,4-naphthoquinone) and H2S at Different pH Fe(II)/goethite NAC

E1ha (mV)

2-CH3-NB 4-CH3-NB 2-Cl-NB 4-Cl-NB

-590 -500 -485 -450

kobs

(h-1)

8.39 × 10-2 8.32 × 10-2 8.04 × 10-1 2.42 × 100

pH 7.1 7.0 7.0 7.0

ENb,c

juglone/H2S AKIENc,d

(‰)

-31.9 ( 1.0 -31.3 ( 1.4 -29.2 ( 0.3 -29.4 ( 0.8

1.0329 ( 0.0010 1.0324 ( 0.0014 1.0301 ( 0.0003 1.0303 ( 0.0008

kobs

(h-1)

nde nde 1.69 × 10-1 3.84 × 10-2

pH

ENc (‰)

AKIEN d

7.5 6.9

-30.2 ( 1.7 -28.0 ( 0.8

1.0312 ( 0.0017 1.0288 ( 0.0008

a Values from ref 27. b Calculated from linear regression using eq 5. c Uncertainties represent (1σ calculated according to procedures proposed in ref 26, samples size n g 6. d Calculated with eq 4. e nd ) not determined, rates of reduction were too slow.

and the reference material (Rref), respectively, E is the isotope enrichment factor, c stands for the concentration, and δE represents the isotope signature of the NAC. Uncertainties of E arise predominantly from linear regression analysis, whereas the contribution of measurement uncertainties are less pronounced ((26) see SI for details). Apparent kinetic isotope effects, AKIEs, were calculated from eq 4. The trends of δ15N- and δ13C-values of NACs, reaction intermediates, and anilines were modeled on the basis of a kinetic model including light and heavy isotopomers as independent species and isotopic rate constants (see equations discussed in the SI).

Results and Discussion Extent and Variability of N- and AKIEN-Values. The reduction of 4-chloronitrobenzene (4-Cl-NB) in suspensions of Fe(II)/goethite (Figure 1, panel a) and of 2-chloronitrobenzene (2-Cl-NB) in solutions of juglone/H2S (Figure 2, panel a) followed pseudo-first-order kinetics. The reactions were accompanied by a continuous and substantial 15N-isotope enrichment of the parent compound (Figures 1b and 2b) but only small 13C enrichment (Figure 1d). This behavior is consistent with a primary kinetic isotope effect for a reaction at the NO2-group and a secondary effect of the nonreactive aromatic C atoms, which is reduced due to isotopic dilution. In all cases, the δ15N data could be fit well with eq 2 (R2values >0.98, see examples in Figures 1c and 2c). The derived N-values are summarized in Table 1 together with the oneelectron reduction potentials, E1h, and the observed pseudofirst-order rate constants, kobs, of the NACs investigated. Table 1 also shows the AKIEN-values of the NACs calculated from N using eq 4, which can be applied since there is only one nitrogen atom present per molecule. There are several striking features in the data presented in Figures 1 and 2 and in Table 1. First, when considering

the Fe(II)/goethite system, independent of the type and position of substitution, and thus of their corresponding properties (E1h, tendency to adsorb to mineral surfaces, etc.), all NACs exhibited virtually identical N- or AKIEN-values. Similarly, no correlation was observed between N and the reduction rate constants of the NACs. This implies that the reaction steps determining the relative rates of NAC reduction by mineral-bound Fe(II) species, that is, the tendency of the NACs to adsorb to reactive surface sites (precursor complex formation (17-19)), had either the same or no influence on 15N isotope fractionation. Second, the same AKIE -values as N found for NAC reduction by mineral-bound Fe(II) were also obtained for the reaction in the juglone/H2S system. In contrast to reactions in Fe(II)/goethite suspensions, rates of homogeneous NAC reduction are very sensitive to the NACs tendency to acquire an electron from the reductant as quantified by the compounds’ E1h. Obviously, different E1h of the NACs did not cause the AKIEN-values found in the juglone/H2S system to deviate from each other. Moreover, AKIEN-values are identical to the ones observed in heterogeneous suspensions, which led us to the conclusion that the first electron transfer from the reductant to the NAC did not contribute significantly to the overall isotope fractionation. This also means that regardless of the involved reductant an apparently very similar fractionation step was responsible for the AKIEN-values and that this step can be preceded by different nonfractionating reactions. Third, the AKIEN-values for reduction of the aromatic NO2 group are of the same magnitude as the ones obtained for the reduction of NO2- to NH2OH and of NH2OH to NH3 in an Fe(II)/NaOH system (see Introduction). From all these findings, it is rather unlikely that in the three very different systems considered, nonfractionating steps preceding the isotopic reaction step masked 15N isotope fractionation in exactly the same way. Hence, it seems reasonable to assume that an AKIEN-value VOL. 40, NO. 24, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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SCHEME 2

of about 1.03 is representative for the intrinsic kinetic nitrogen isotope effect, KIEN, associated with the breaking of one N-O bond. Additional features of the isotope fractionation during NAC reduction are reflected in the 15N-signatures of the final reduction products (substituted anilines). From Figures 1b and 2b it can be seen that in both cases the δ15NAn-values of the anilines could be described very well (solid line) based on the N-values derived from δ15NNAC-values of the NACs and the initial NAC isotope signatures (δ15NNAC,0). The evolution of δ15NAn-values was calculated successfully using eq 7, which is similar to eq 2 used for the derivation of Nvalues (see ref 5 for details):

δ15NAn )

δ15NNAC,0 + 1000 ‚(1 - f N/1000 + 1) - 1000 1-f

(7)

As alternative to eq 2, N-values of NAC reduction can be obtained from the extrapolation of the δ15N-values for anilines to the time zero of the experiment (f f 1, arrows in Figures 1b and 2b). Using data points at low degree of NAC conversion, this procedure described by eq 8 (see ref 5 for details on its derivation) yields N-values of approximately -30 ( 2‰, which are consistent with the N-values derived from the linearized Rayleigh-plots and eq 2 (Figures 1c and 2c, Table 1).

δ15NAn + 1000 N≈1000‚ 15 - 1000 ff1 δ NNAC,0 + 1000

(8)

The fact that δ15N signatures of NACs and anilines could be described with a single N-value and the good agreement of N calculated from eqs 2 and 8 point out that in both systems investigated, only the breaking of the first N-O bond yielding the nitroso compound (Scheme 1) caused the observed N isotope fractionation. Even though a second N-O bond is broken during reduction of the hydroxylamine to aniline, this step apparently did not contribute to the δ15N signatures of substituted anilines. This conclusion also holds for NAC reduction in solutions of juglone/H2S, where the amount of aniline recovered was not stoichiometric (Figure 2a). Here, reduction of the NACs led to a more pronounced formation of reaction intermediates compared to reactions in suspensions of Fe(II)/goethite, since in the presence of juglone/H2S intermediates are converted to the corresponding anilines at much slower rates. As illustrated in Figure 2, after complete disappearance of 2-Cl-NB (time >30 h, f f 0), the δ15NAn of 2-Cl-An was almost identical to the initial δ15NNAC,0 of 2-Cl-NB, although only 16% of 2-Cl-NB had been reduced to 2-Cl-An. Since δ15NAn of 2-Cl-An at this stage of the reaction already corresponded to δ15NNAC,0 of 2-Cl-NB, the reduction of the remaining reaction intermediates to 2-Cl-An was either not isotope-fractionating or its isotope effect was masked by a preceding, slower, and nonisotopic reaction. This observation indicates that after complete NAC reduction, the reaction intermediates had δ15N signatures, which were also virtually identical to the initial NAC-value (δ15NNAC,0, Figure S2). If, in our experimental systems, the 7714

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cleavage of the second N-O bond, that is, the reduction of the hydroxylamino compound to the substituted aniline contributed to the isotope signature of the latter, δ15NAnvalues of anilines at the initial stage of the NAC reduction (i.e., f f 1) would reflect two consecutive isotope-fractionating reactions. Consequently, these δ15NAn-values would be more than 30‰ more negative than δ15NNAC,0. As is shown in Figures 1b and 2b, this was obviously not the case here. An example for the trends of δ15N-values of NACs, intermediates, and substituted anilines in reactions involving two observable, consecutive isotopic N-O cleavages is illustrated in Figure S2. Mechanistic Considerations. For an explanation of the findings that all N-values of the NACs were similar, it is necessary to take a closer look at the underlying reaction mechanism. As is shown in Scheme 2, the reduction of an aromatic NO2 group consists of a series of sequential electron and proton transfers before a first N-O bond is irreversibly broken (28). At the neutral pH-values in our experiments (pH 6.9-7.5), N-O cleavage can occur either in the intermediate Ar-NO(OH) (3) as elimination of OH- or in ArN(OH)(OH2) (5) as dehydration (H2O elimination). Since we postulated above that the observed large N isotope fractionation is representative for an intrinsic KIEN of a N-O bond cleavage, either of these elimination steps is ratelimiting for the overall NAC reduction. This interpretation is in agreement with studies on the electrochemical reduction of substituted NACs, which suggest that elimination of H2O from 5 limits the rates of NAC conversion to aromatic nitroso (7) or hydroxylamino compounds over a pH range of -6 to 10 (29). However, according to this kinetic scheme, the overall NAC reduction rate depends not only on the first N-O cleavage but also on the preceding e-- and H+-transfers. Thus, observed rate constants are the product of the isotopesensitive rate constant for bond breaking and all equilibrium constants for each preceding e-- and H+-transfer. Even though isotopic contributions from reversible e--and H+transfers cannot a priori be ruled out (equilibrium isotope effects), these effects are expected to be small since only small mass changes at the N-O bond occur, thus not affecting N-O vibrational frequencies in such a way to cause the large observed AKIEN. As was shown in our previous studies with different NAC reductants (see Introduction), pre-equilibria such as the reversible first-electron transfer can be strongly influenced by substituents at the aromatic ring. This is particularly true for homogeneous reductants such as reduced hydroquinone species where reduction rate constants can vary over several orders of magnitude (18, 20, 30). Whereas these pre-equilibria determine the population of molecules up to the rate-limiting elimination step and thus the different observed reaction rate constants of NACs, these reactions do not significantly change the population’s isotopic composition prior to N-O bond cleavage. As a result, AKIEN of NACs are independent of the reductants and not influenced by aromatic substituents and thus varying reaction rates. The similar AKIEN also suggest that a very similar transition state is encountered during N-O cleavage, which raises the question whether substituent effects on the AKIEN were

similar or inexistent. From the AKIENs shown in Table 1, it becomes obvious that ortho vs para substitution at the NACs did not affect the extent of isotope fractionation. Ortho substitution prevents complete coplanarity of the molecule and thus optimal resonance of the NO2-group with the aromatic ring (31). The lack of ortho-effect suggests that electronic interactions of the aromatic π-electrons with the N-O bonds were apparently not relevant for the KIEN of the N-O bond cleavage. The similar AKIENs between Cl-NBs and CH3-NBs suggest that inductive effects of Cl- and CH3substituents did not significantly affected N-O bonding during the isotopic elimination step. This hypothesis is supported by very similar 14C-KIEC (12k/14k) reported for the carbonyl center (C*) during ester hydrolysis of p-CH3C6H4C*OOEt (1.078) and p-Cl-C6H4C*OOEt (1.081) with hydroxide anion in ethanol ((32) note that 14C-KIEC are 1.9 times larger than 13C-KIEC (5)). Environmental Significance and Outlook. The results of this study demonstrate that nitrogen isotope signatures of individual organic compounds can be used successfully to investigate environmentally relevant transformations. The virtually identical N-values obtained for the abiotic reduction of substituted NACs in homogeneous and heterogeneous systems mimicking important environmental reductants suggest that these values could be used to quantify the extent of NAC reduction F in complex field situations (eq 9) substituting an average N-value of -30.0‰ into eq 3.

Fabiotic NAC reduction ) 1 -

(

1000 + δ15NNAC

1000 + δ15NNAC,0

)

-33.3

(9)

However, since the data presented in this paper cover a rather narrow range of contaminants, reductants, and experimental conditions, more work is needed to ascertain this N-value and/or to identify the origins of its variability. In cases, in which the cleavage of the N-O bond is catalyzed by complexation of the oxygen to a metal center, as was shown for the reduction of nitrate (NO3-) by Fe(II) in alkaline solution in the presence of silver ions (Ag(I)), much higher N-values (approximately -70‰) were observed (14). A similar interpretation could be valid for the observed Nvalue of -40‰ (33) for the enzymatic reduction of NO3- by a nitrate reductase where reduction takes place at a molybdopterin, a Mo-containing cofactor known to catalyze oxygentransfer reactions (34-36). For enzyme catalyzed N-O cleavages such as denitrification, a great variability with regard to N-values is reported (37), which is presumably due to the occurrence of enzyme- and system-specific masking effects and/or commitments to catalysis (see above). Consequently, future work should include studies on N isotope fractionation occurring during biologically mediated NAC reduction. Furthermore, the set of compounds investigated in abiotic and biologically mediated reductions should be extended to more reactive and more environmentally relevant compounds including dinitrobenzenes and -toluenes as well as trinitrotoluene (TNT). Finally, more mechanistic work is also needed to evaluate the effect of environmental conditions such as pH on the observed fractionation.

Acknowledgments Silvio Canonica, Kathrin Fenner, Anke Neumann, Michael Sander, and Nicole Tobler are kindly acknowledged for valuable comments and for reviewing the manuscript.

Supporting Information Available Description of the SPME-GC-IRMS method used for CSIA, uncertainty consideration of isotopic enrichment factors, and procedures for modeling of isotope signatures. This material

is available free of charge via the Internet at http:// pubs.acs.org.

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Received for review May 5, 2006. Revised manuscript received September 1, 2006. Accepted September 8, 2006. ES061074Z