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C/12C and 15N/14N Isotope Analysis To Characterize Degradation of Atrazine: Evidence from Parent and Daughter Compound Values Armin H. Meyer† and Martin Elsner*,† Institute of Groundwater Ecology, Helmholtz Zentrum München, Ingolstädter Landstraße 1, 85764 Neuherberg, Germany S Supporting Information *

ABSTRACT: Atrazine (Atz) and its metabolite desethylatrazine (DEA) frequently occur in the environment. Conclusive interpretation of their transformation is often difficult. This study explored evidence from 13 C/12C and 15N/14N isotope trends in parent and daughter compounds when Atz was dealkylated by (i) permanganate and (ii) the bacterium Rhodococcus sp. NI86/21. In both transformations, 13C/12C ratios of atrazine increased strongly (εcarbon/permanganate = −4.6 ± 0.6‰ and εcarbon/Rhodoccoccus = −3.8 ± 0.2‰), whereas nitrogen isotope fractionation was small. 13C/12C ratios of DEA showed the following trends. (i) When DEA was formed as the only product (Atz + permanganate), 13C/12C remained constant, close to the initial value of Atz, because the carbon atoms involved in the reaction step are not present in DEA. (ii) When DEA was formed together with desisopropylatrazine (biodegradation of Atz), 13C/12C increased but only within 2‰. (iii) When DEA was further biodegraded, 13C/12C increased by up to 9‰ giving strong testimony of the metabolite’s breakdown. Two lines of evidence emerge. (a) Enrichment of 13C/12C in DEA, compared to initial Atz, may contain evidence of further DEA degradation. (b) Dual element (15N/14N versus 13C/12C) isotope plots for dealkylation of atrazine agree with indirect photodegradation but differ from direct photolysis and biotic hydrolysis. Trends in multielement isotope data of atrazine may, therefore, decipher different degradation pathways.

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INTRODUCTION Atrazine has been used for broadleaf and grassy weed control in the agricultural production of corn, sugar cane, and other crops,1 and is one of the most commonly detected herbicides worldwide.2−4 Biotic atrazine degradation in the environment occurs either by oxidative dealkylation or by hydrolysis5,6 which can be catalyzed by fungi and bacteria.7−10 Oxidative dealkylation forms the products desethyl- (DEA) and desisopropylatrazine (DIA), which are still herbicidal but more mobile in the aquatic environment due to the loss of the alkyl chain. Biotic hydrolysis of atrazine leads instead to the formation of nontoxic hydroxyatrazine (HAT), which is mainly immobilized by sorption in the upper soil horizons.11 Atrazine degradation is difficult to assess in the environment, (i) because concentrations decrease not only due to degradation but also due to sorption and dilution and (ii) because it is typically not possible to close hydraulic mass balances.12,13 Since dealkylated products were often detected in surface and groundwater systems, the ratio of DEA and DIA to atrazine (DAR) is often used as a proxy for atrazine degradation in the environment.10,14 However, this approach may be biased if metaboliteto-parent compound ratios change (i) as a result of further transformation of the dealkylated products, (ii) as a consequence of selective sorption, or (iii) due to new recharge of atrazine into the system. Moreover, recent studies demonstrated that hydrolysis may be the dominating dissipation process for atrazine in the environment.11,15 This © XXXX American Chemical Society

process is likely underestimated, because its primary product HAT is not considered in the DAR. New approaches are therefore needed to characterize degradation of atrazine and desethylatrazine and to identify the dominant degradation pathway3 in order to support fate models of atrazine in the environment.16−18 Compound specific isotope analysis (CSIA) may detect and quantify degradation of groundwater contaminants such as aromatic19 and chlorinated20 hydrocarbons, RDX21 and MTBE22 in the field, and it may differentiate between different degradation pathways.23−25 CSIA measures the isotopic composition of a compound at natural abundance. Since (bio)chemical reactions are associated with kinetic isotope effects, often an enrichment of the heavier isotope in the remaining parent compound and a depletion of the heavier isotope in the product can be observed. Information on degradation can therefore be obtained from observable isotope fractionation in the parent as well as the daughter compound. Normally, parent compound data are considered, because CSIA is typically employed to discover degradation if metabolites are not detectable. For the parent compound atrazine isotope Special Issue: Rene Schwarzenbach Tribute Received: January 2, 2013 Revised: April 24, 2013 Accepted: April 30, 2013

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dx.doi.org/10.1021/es305242q | Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Scheme 1. Different Scenarios for Expected Carbon Isotope Effects in Atrazine and DEA and DIA Associated with Dealkylation of Atrazinea

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(A) Formation of desethylatrazine (DEA) by loss of reactive ethyl group. (B) Competitive dealkylation at isopropyl- or ethyl group of atrazine leads to 13C/12C enrichment in both alkyl chains. One of both is transferred to the product. (C) Carbon isotope enrichment in DEA due to the kinetic isotope effect of its degradation. Reactive groups in atrazine and its metabolites are indicated by gray boxes.

isotope signature over the whole degradation process, because its atoms were simply not involved in the transformation and can, therefore, also not show any isotope effects. In the case of the dealkylation of atrazine leading to DEA, an analogous scenario for the carbon isotope ratio can be expected, since the reacting carbon atom(s) (gray box in Scheme 1A) are split off in the leaving ethyl group. Therefore, in such a case the 13 C/12C isotope ratio of DEA would be expected to be constant throughout the transformation. Depending on the intramolecular isotope distribution in atrazine, however (i.e., if the ethyl group contains less or more 13C/12C than the molecular average) this constant isotope ratio of DEA may contain more, less, or an equal proportion of 13C/12C compared to the initial parent compound. The situation is different for nitrogen, of which all atoms are transferred to the daughter compound. If a small nitrogen isotope effect occurs, it should, therefore, be observable in both atrazine and DEA. In comparison, Scheme 1B illustrates the scenario typically observed in biotic dealkylation of atrazine: DEA and DIA are formed simultaneously. Since both the N-ethyl and the Nisopropyl groups may react (leading to DIA and DEA, respectively) 13C/12C isotope ratios increase in both molecular positions of atrazine during transformation (gray boxes in parent compound) when the average of many atrazine

fractionation has been investigated in great detail in recent studies, demonstrating that degradation processes in nature biotic hydrolysis, oxidative dealkylation, and photooxidation are associated with significant pathway-dependent 13C/12C and (smaller) 15N/14N isotope shifts.26−28 In groundwater monitoring, however, atrazine is often detected together with its daughter compounds DEA and DIA. Most recent data from Schreglmann et al.29 report for the first time δ13C isotope values of both atrazine and DEA in natural groundwater samples. This raises the question how isotope values of parent and daughter compounds can be interpreted relative to each other, and what information is contained about their natural degradation. Scheme 1 illustrates three different scenarios which lead to different carbon isotope trends in the metabolites DEA and DIA: Scheme 1A illustrates a scenario which transforms atrazine selectively to DEA. Normally, an enrichment of 13C over 12C in the parent compound leads to a corresponding depletion of 13C relative to 12C on the side of the products. Such depletion, however, can only be observed if the atoms located in the reacting position (gray box in Scheme 1A) are still present in the transformation product. Earlier studies on dealkylation of MTBE, ETBE, and TAME have shown30 that the transformation product (there: TBA) otherwise has a constant B



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(Budapest, Hungary). Degradation experiments and quantification of atrazine and its metabolites were done in a similar way as in our previous study28,32 (see also the SI), with the exception that the current experiments were investigated over a longer period (700 h) in order to follow not only atrazine degradation but also further degradation of DEA and DIA. Product distribution was analogous to our recent study28,32 meaning that besides DEA and DIA, also hydroxylated products in α- and β-position of the side chains were detected (see also Figure S3). Since all of them form by oxidation of either the ethyl or the isopropyl group, they correspond to the scenario of Scheme 1B. Selective Formation of DEA by Permanganate Oxidation. The detailed setup of this experiment is described in Meyer et al.28,32 Shortly, triplicate experiments were conducted to oxidize atrazine (80 μM) to DEA with potassium permanganate (0.1 M) in an aqueous phosphate-buffer (pH 7.1) at room temperature (21 °C). Carbon and Nitrogen Isotope Analysis. Samples for compound-specific isotope analysis of atrazine, DEA, and DIA (15 mL−200 mL) were extracted with 5−10 mL of dichloromethane, which was subsequently evaporated at room temperature under the hood. Extracts of the permanganate experiment were in addition filtered through glass wool. Tests with standards showed no significant isotope fractionation during the preparation steps. Each sample was analyzed in duplicate. A 4 μL portion of the extracts was injected with a GC Pal autosampler (CTC, Zwingen, Switzerland) onto a GC-CIRMS system consisting of a TRACE GC Ultra gas chromatograph (Thermo Fisher Scientific, Milan, Italy), a GC-III combustion interface, and a Finnigan MAT253 IRMS (both Thermo Fisher Scientific, Bremen, Germany). The injector contained a split/splitless liner (Thermo Electron S.p.A.; Milan, Italy) and was operated for 1 min in splitless and then in split mode (1:10), at 250 °C with a column flow of 1.4 mL min‑1. A DB-5 column (60 m × 0.25 mm; 1 μm film; J&W Scientific, Folsom; CA, USA) was used with a GC oven program of 140 °C (hold: 1 min), ramp 18 °C/min to 155 °C, ramp 2 °C/min to 240 °C, ramp 30 °C/min to 260 °C (hold: 5 min). For carbon isotope analysis target compounds were combusted to CO2 in a GC IsoLink oven (Thermo Fisher Scientific, Bremen, Germany) at 1050 °C.33 The analytical uncertainty for atrazine was ±0.7‰33 and for DEA and DIA ± 1‰. For N isotope analysis, target compounds were converted to N2 using the setup described in Hartenbach et al.26 with an analytical uncertainty of ±1‰. The δ15N-and δ13C-values are reported relative to Vienna PeeDee Belemnite (V-PDB) and air, respectively:

molecules in an environmental sample is analyzed. Of the two groups, however, only one is cleaved off at a time. The enrichment of 13C over 12C from the remaining group is, therefore, transferred to the respective daughter compound (gray box). Unlike in scenario 1A, 13C/12C isotope ratios of DEA and DIA are therefore expected to slightly increase over time relative to the initial isotope value of atrazine. Scheme 1C illustrates a third, more obvious reason why 13 C/12C values in DEA may increase during biodegradation over time. If the daughter compound itself is subject to further transformation, it can be hypothesized that 13C/12C isotope ratios increase in DEA over time, due to the isotope effect associated with DEA degradation and in analogy to the isotope fractionation observed in atrazine.27,28 In summary, three reasons may explain why 13C/12C values in DEA can be higher than in the initial atrazine. Two of them result from factors other than DEA degradation: an uneven intramolecular isotope distribution of 13C in the initial atrazine (discussed in case A) and an enrichment of 13C in atrazine which is passed on to the daughter compounds (discussed in case B). In contrast, enrichment of 13C/12C values in DEA may also be truly indicative of its further degradation (discussed in case C). Such a line of evidence is warranted to detect elimination of the problematic metabolite from the environment, similarly as recently outlined for the herbicide metabolite 2,6-dichlorobenzamide.31 Most recently, the first data on DEA and atrazine in natural groundwater samples have been reported for 6 sampling locations in Austria and Bavaria (Germany).29 Remarkably, DEA in all samples contained more 13C/12C than atrazine from the same location. Consequently, the question needs to be addressed whether this enrichment of 13C in DEA can be taken as evidence of further DEA degradation, or whether it may alternatively be caused by the concurrent formation of DEA and DIA as discussed above. The aim of our study was, therefore, to explore C and N isotope trends of the degradation products DEA and DIA during oxidative dealkylation of atrazine. Scenario A was investigated in oxidative dealkylation of atrazine by permanganate, which forms selectively only DEA, whereas scenarios B and C were investigated during biodegradation of atrazine by Rhodococcus sp. NI86/21.

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MATERIALS AND METHODS Chemicals. Atrazine (1-chloro-3-ethylamino-5-isopropylamino-2,4,6-triazine, CAS: 1912-24-9) was purchased from Tropitzsch (97.7%, Marktredwitz, Germany). Ethyl acetate (99.8% Riedel-de Haën, supplied by Sigma Aldrich, Taufkirchen, Germany) was used as a solvent for standard solutions (GC-IRMS). Aqueous HPLC standards contained atrazine, hydroxyatrazine (CAS: 2163-68-0), desethylatrazine (CAS: 6190-65-4), and desisopropylatrazine (CAS: 1007-28-9) (96.0%, 99.9%, and 96.3%, respectively; all Riedel de Haën, supplied by Sigma-Aldrich, Seelze, Germany) and had concentrations of 4.7, 11.6, 23.3, 46.6, and 93.2 μM of the analytes. Potassium permanganate (99%) as oxidative agent was from Sigma Aldrich (Seelze, Germany). Acetonitrile used as HPLC eluent was from Roth (Karlsruhe, Germany), and Na3PO4·12H2O and Na2HPO4·2H2O (for aqueous puffer solution) and dichloromethane for extraction were purchased from Merck (Darmstadt, Germany). Biotic Degradation of Atrazine by Rhodococcus sp. Strain NI86/21. The strain was purchased from the National Collection of Agricultural and Industrial Microorganisms

δ13C = [(13C/12CSample − 13C/12CStandard )/13C/12CStandard ] (1)

δ15 N = [(15 N/14 NSample − 15 N/14 NStandard)/15 N /14 NStandard]

(2)

CO2 and N2 monitoring gases were calibrated against VPDB and air using the international reference materials RM 8562, RM 8563, RM 8564 (for CO2) and NSVEC (for N2).34 Carbon and nitrogen isotope enrichment factors ε associated with atrazine degradation were derived according to the linearized Rayleigh equation C


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⎛ 1 + δ hE ⎞ Rt t ⎟⎟ = ε ·ln f = ln⎜⎜ h R0 ⎝ 1 + δ E0 ⎠

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(3)

in which Rt and R0 describe the compound-specific isotope ratios of heavy versus light isotopes at a given time and at the beginning of the reaction. δhEt and δhE0 are the isotope values of the compound in the delta notation (eqs 1 and 2) determined for the element E at times t and zero, respectively, while ct/c0 (c: concentration of atrazine) is the fraction f of the remaining atrazine.

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RESULTS AND DISCUSSION Isotope Values of DEA during Its Selective Formation. In our previous study,28 we observed that oxidation of atrazine by permanganate forms selectively DEA. This setup was therefore chosen to investigate scenario A: what isotope value DEA would have if it forms exclusively from the nonreacting positions in atrazine (atoms outside of the gray box in Scheme 1A). Figure 1 illustrates two trends. Due to the kinetic isotope

Figure 2. Carbon isotope signatures of atrazine during degradation of atrazine by Rhodococcus sp. NI86/21. The dashed black line indicates the Rayleight fit yielding an enrichment factor εcarbon of −3.8 ± 0.2‰.

compound (ε = −3.8 ± 0.2‰, see also Figure S2A). In contrast to the results from permanganate, atrazine degradation by Rhodococcus sp. NI86/21 (a) led to formation of DEA and DIA and (b) δ3C values in these products showed an enrichment of 13C/12C compared to the initial atrazine. Results from triplicates in Figure 3 show that this enrichment of 13 C/12C can be attributed to two different phases of the experiment. (i) Daughter compound formation. In replicate A and in the initial phase of replicates B and C (0−ca. 80 h) (Figure 3A, B, C) concentrations of DEA and DIA steadily increased indicating that the metabolites were formed rather than further degraded. This is also indicated by the fact, that the mass balance was nearly closed if all metabolites (also intermediates that were hydroxylated in α- and β-position) are considered28 (Figure S3). In this initial phase the value δ3C = −26.8‰ of DEA was already moderately “heavier” than the initial value of atrazine. As expected from Scheme 1B, enrichment of 13C/12C therefore occurred in both side chains of atrazine (gray boxes), but only one of them was cleaved off during reaction. The enrichment from the remaining group was, therefore, transferred to the respective daughter compound which consequently achieved slightly higher 13 C/12C values compared to the original atrazine. (ii) Daughter compound degradation. In the second phase of degradation (after ca. 80 h in replicates B and C) concentrations of DEA and DIA decreased markedly indicating that the metabolites were further degraded (Figure 3). At the same time their 13 C/12C ratios increased much more strongly than in replicate A resulting in isotope signatures of δ13C = −19.2‰ for DEA and −19.9‰ for DIA after about 700 h (Figure 3B and C). This more pronounced isotope enrichment (changes of δ13CDEA/DIA became significantly greater than twice the total uncertainty of the analytical method, 2‰) can be attributed to the isotope effect of DEA and DIA degradation rather than of their formation. However, while metabolite isotope ratios exceeded the initial value of atrazine, even under these circumstances they never exceeded the value of atrazine present at the same time - not even in this second stage of the experiments. During oxidation of atrazine by Rhodococcus sp. NI86/21 small nitrogen isotope changes (smaller than twice the analytical uncertainty of N isotope analyses, 2‰) were observed, as indicated by slightly increasing δ15N values of atrazine (εnitrogen = −1.5 ± 0.3‰, Figure 4, Figure S2B).

Figure 1. Carbon isotope signature of DEA and atrazine during selective dealkylation of atrazine. Isotope values of atrazine are taken from Meyer et al.28 Straight blue line represent the average of all δ13C measurements of DEA with the standard deviation of ±0.8‰ (blue dotted lines). Black dashed line indicates the Rayleigh fit of atrazine according to eq 3 with a carbon enrichment factor ε of −4.6 ± 0.6‰.

effect in the ethyl group, 13C/12C isotope values of atrazine increased during the reaction, as reflected by increasingly less negative δ3C values and an enrichment factor of ε = −4.6 ± 0.6‰ according to eq 3 (Figure 1). In contrast, δ3C values of DEA were, as expected, constant throughout the whole reaction, because the reactive positions of the ethyl group were cleaved off during reaction. With a value of −28.7 ± 0.8‰ (1σ standard deviation) DEA was of the same 13C/12C isotope composition as the initial δ3C value of atrazine (−28.3 ± 0.7‰) (Figure 1) indicating the absence of large intramolecular isotope variations in the original atrazine. Consistent with previous results on permanganate,28 no nitrogen isotope fractionation was observed, neither in the parent nor the daughter compound of the reaction (Figure SI1). This indicates that the N atom was little involved in the initial step of oxidative dealkylation: the oxidative cleavage of an adjacent C− H bond. Isotope Values of the DEA and DIA during Their Concurrent Formation and Degradation. Figure 2 shows that biodegredation of atrazine by Rhodococcus sp. NI86/21 was associated with increasing 13C/12C ratios in the parent D



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Figure 3. Concentrations (upper graphs) as well as carbon isotope signatures (lower graphs) of atrazine, DEA, and DIP during biotic oxidation by Rhodococcus sp. NI86/21 of atrazine for three replicates over time. Straight blue lines indicate the average δ13C value (−28.7‰) of selective DEA formation by permanganate. Light gray areas indicate the first phase of the experiments (DEA and DIA were formed), dark gray areas the second phase of the experiments (DEA and DIA were further degraded).

Patterns of Carbon Isotope Values in Parent and Daughter Compounds. With regard to the processes of Scheme 1, we could therefore confirm that DEA maintains a constant isotope ratio if it is formed as the only product of atrazine (Scheme 1, Scenario A). Through selective transformation, we could also establish that intramolecular isotope variations between the ethyl group and the rest of the molecule were negligible in the atrazine of our experiments. As discussed above, theoretical considerations predict a slight enrichment of 13 C/12C in DEA and DIA when they are formed as parallel products during biodegradation (Scheme 1, Scenario B). Although we could confirm this trend, we also found that DEA and DIA isotope ratios still matched the value of the initial atrazine to within 2‰ as long as they were not further transformed (Figure 3, first phase). In contrast, as soon as DEA and DIA were further biodegraded (Scheme 1, Scenario C), their δ13C values showed a pronounced enrichment of 13C/12C by up to 9‰, which strongly reflects their further breakdown (Figure 3, second phase). Finally, even though 13C/12C ratios of DEA increased compared to the initial value of atrazine, in our experiments they never exceeded atrazine isotope ratios of the same sample, as it was observed by Schreglmann et al.29 Interpretations of isotope ratios in such a consecutive degradation cascade (atrazine → DEA →) depend on several factors. Besides isotope effects and transformation rates, parent-to-daughter concentrations are key factors which determine whether DEA isotope ratios are more strongly influenced by influx of freshly formed DEA or by the effect of further DEA degradation. However, even without detailed knowledge of these parameters it can already be concluded that - to explain these observations in natural groundwater samples - even more extensive DEA

Figure 4. Nitrogen isotope signatures of DEA, DIA, and atrazine during dealkylation of atrazine by Rhodococcus sp. NI86/21. Dotted dashed black lines indicate isotope signatures for DEA and DIA according to the Rayleigh-fit for products36,37 using the nitrogen enrichment factor εnitrogen of −1.5 ± 0.3‰ obtained from the atrazine data (dashed black line). The horizontal gray line represents the initial δ15N value of atrazine of −2‰, together with the associated analytical uncertainty of ±1‰.38

Consequently, δ15N values of DEA and DIA were initially slightly more negative but reached the value of the original atrazine at the end of the reaction. (Note that in contrast to carbon, the reactive N atom of the alkyl chain remains within the metabolites.) In addition, it seems that further degradation of DEA and DIA did not have a pronounced impact on their isotope signature: values did not exceed the initial value of atrazine, despite the fact that some of them stemmed from the second phase of the experiment. E



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Figure 5. (A) Isotope fractionation of atrazine and DEA, expressed by dual C and N isotope plots, during dealkylation of atrazine. (B) Dual C and N isotope plots of atrazine reflect different natural degradation pathways, as reported in recent studies by Meyer et al.,27,28 Hartenbach et al., and Hofstetter et al.26,35 (dashed arrows). Δ indicate the slopes of the different regression lines associated with the dual isotope plots. Dotted lines represent 95% confidence intervals of regression.

identified in environmental samples, trends may therefore detect these atrazine transformation routes also in natural systems. (ii). Combined Consideration of Atrazine and Deethylatrazine Values. Compared to previous investigations26−28 35 this study brings forward an additional line of evidence to assess the degradation state of atrazine and its dealkylated products DEA and DIA: the comparison of parent and daughter compound isotope values. While nitrogen isotope ratios of DEA displayed little variation (Figure 5A), unique information was contained in associated carbon isotope values. In our study we observed that carbon isotope ratios of DEA matched the initial atrazine value to within 2‰ as long as DEA was not further degraded (Scheme 1, scenarios A and B). If this is a general trend, δ13CDEA could even serve as a proxy for the initial isotope ratio of atrazine δ13Catrazine,0 meaning that values of δ13Catrazine and δ13Catrazine,0 could potentially be obtained from the same groundwater sample. However, since the trend depends also on the intramolecular isotope distribution of atrazine (i.e., between the ethyl group and the rest of the molecule) this influence needs to be investigated in further studies. In contrast, when DEA itself was further degraded (Scheme 1, scenario C) 13C/12C ratios of DEA increased strongly compared to the initial value of atrazine. Results from natural groundwater samples showed an even stronger enrichment of 13 C/12C in DEA compared to atrazine suggesting further DEA degradation in the sampled groundwater.29 Presently, this interpretation is not yet conclusive, because an uneven intramolecular isotope distribution with an unusually “light” ethyl group cannot be ruled out as alternative explanation. However, our work delineates an expedient approach to eliminate this possibility in future investigations. Atrazine from groundwater extracts may be isolated by preparative HPLC 29 and selectively be degraded to DEA by permanganate as

degradation must have occurred compared to our study. Alternatively, the possibility remains that the herbicide in these samples had a much stronger intramolecular isotope variation than the atrazine of our experiments. Considering that 2 out of 8 carbon atoms are in the ethyl group of atrazine, a difference of δ13CDEA − δ13CAtz, initial = 2‰ would require the eliminated ethyl group to have 8/2·2‰ = 8‰ more 13C/12C than the rest of the unreacted atrazine molecule. The intramolecular discrepancy would actually even need to be greater, since atrazine in the groundwater samples was partially degraded and since in our experiments we observed an opposite trend during degradation (δ13CAtz, t became increasingly greater than δ13CDEA, see Figure 3).

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ENVIRONMENTAL SIGNIFICANCE Since atrazine and its metabolite DEA are frequently detected in groundwater, it is important to understand and assess their fate in the environment. When results of this study are combined with insight from previous work, two lines of evidence emerge to assess their natural degradation. (i). Dual Element Isotope Fractionation in the Parent Compound Atrazine. When carbon and nitrogen isotope values of atrazine observed in this study (Figure 5A) are combined with trends reported in previous experiments, characteristic carbon and nitrogen isotope patterns emerge for biotic hydrolysis, biooxidation, and photolytic degradation of atrazine, respectively (Figure 5B).26,32 Specifically, with Δ = 0.36 ± 0.06 (Δ: slope of the regression line in a dual element (δ13C vs δ15N) isotope plot) the trend for biotic dealkylation is significantly different from the slope obtained for biotic hydrolysis (Δ = −0.62 ± 0.06) and direct photolysis (Δ = 1.05 ± 0.14). In contrast, the trend is similar to slopes for transformation pathways which produce the same products: photoxidation with OH• radicals or mediated by the excited triplet state of 4-carboxybenzophenone (34-CBBP*).26 When F



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described in this study. The DEA from the decomposed atrazine may subsequently be analyzed and compared to the DEA from the same groundwater sample. If it contains less 13 C/12C, this provides conclusive evidence for enrichment of 13 C/12C in the natural groundwater sample and, therefore, for natural DEA degradation. Complementing the well-established use of DEA/atrazine ratios (DAR), this study therefore brings forward two additional lines of evidence to assess the degradation state of atrazine and desethylatrazine in natural samples: (i) dual element isotope measurements to distinguish different degradation pathways of atrazine and (ii) carbon isotope ratios of DEA and atrazine to detect further degradation of DEA. The importance of the second approach is already emphasized by recent first isotope measurements of δ13C in DEA and atrazine in groundwater samples.29

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ASSOCIATED CONTENT

S Supporting Information *

Further experimental description and N isotope data of atrazine and DEA associated with atrazine oxidation by permanganate. C and N enrichment factors of atrazine calculated according to the Rayleigh equation for biotic oxidation of atrazine. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +49(0)89 3187 2565. Fax: +49(0)89 3187 2565. Email: [email protected]. Author Contributions

† The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. These authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was conducted in a Helmholtz Young Investigator Group supported by funding of the Helmholtz Initiative and Networking Fund.

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ABBREVIATIONS atrazine 2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine DEA 2-amino-4-chloro-6-isopropylamino-1,3,5-triazine DIA 2-amino-4-chloro-6-ethylamino-1,3,5-triazine HAT hydroxyatrazine

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REFERENCES

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