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Dual carbon-bromine stable isotope analysis allows distinguishing transformation pathways of ethylene dibromide Kevin Kuntze, Anna Kozell, Hans Hermann Richnow, Ludwik Halicz, Ivonne Nijenhuis, and Faina Gelman Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b01692 • Publication Date (Web): 16 Aug 2016 Downloaded from http://pubs.acs.org on August 16, 2016
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Dual carbon-bromine stable isotope analysis allows distinguishing transformation pathways of ethylene dibromide
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Kevin Kuntze1, Anna Kozell2, Hans H. Richnow1, Ludwik Halicz2,3, Ivonne Nijenhuis1*, and Faina Gelman2 1 Department of Isotope Biogeochemistry, Helmholtz-Centre for Environmental Research – UFZ, Permoserstrasse 15, 04318 Leipzig, Germany 2 Geological Survey of Israel, 30 Malkhei Israel St., Jerusalem, 95501, Israel 3 Faculty of Chemistry, Biological and Chemical Research Centre, University of Warsaw, 02089Warsaw, Poland *corresponding author: phone: +49 341 235 1356, fax: +49 341 235 450822, e-mail:
[email protected] 20 21
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Abstract
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The present study investigated dual carbon-bromine isotope fractionation of the
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common groundwater contaminant ethylene dibromide (EDB) during chemical and
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biological transformations, including aerobic and anaerobic biodegradation, alkaline
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hydrolysis, Fenton-like degradation, debromination by Zn(0) and reduced corrinoids.
29
Significantly different correlation of carbon and bromine isotope fractionation (ΛC/Br)
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was observed not only for the processes following different transformation pathways,
31
but also for abiotic and biotic processes with, the presumed, same formal chemical
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degradation mechanism. The studied processes resulted in a wide range of ΛC/Br
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values: ΛC/Br = 30.1 was observed for hydrolysis of EDB in alkaline solution; ΛC/Br
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between 4.2 to 5.3 were determined for dibromoelimination pathway with reduced
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corrinoids and Zn(0) particles; EDB biodegradation by A. aquaticus and S.
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multivorans resulted in ΛC/Br = 10.7 and 2.4, respectively; Fenton-like degradation
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resulted in carbon isotope fractionation only, leading to ΛC/Br ∞. Calculated carbon
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apparent kinetic isotope effects (13C-AKIE) fell with 1.005 to 1.035 within expected
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ranges according to the theoretical KIE, however, biotic transformations resulted in
40
weaker carbon isotope effects than respective abiotic transformations. Relatively
41
large bromine isotope effects with
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observed for nucleophilic substitution and dibromoelimination, respectively, and
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reveal so far underestimated strong bromine isotope effects.
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Br-AKIE of 1.0012-1.002 and 1.0021-1.004 were
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Introduction
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Brominated organic compounds (BOCs) are widely used and play an important role
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in the production of e.g. agrochemicals, pharmaceuticals or dyes. However, many of
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these compounds are considered to be toxic, carcinogenic or even mutagenic 1. One
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of these brominated contaminants is ethylene dibromide (EDB, 1,2-dibromoethane),
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extensively used in the past as a lead scavenger in gasoline as well as an agriculture
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fumigant
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oxic and anoxic conditions
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environment can follow several different mechanistic pathways: nucleophilic
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substitution (e.g. hydrolysis), dehydrobromination, dibromoelimination or radical
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oxidation (via proton abstraction). Under certain conditions, EDB degradation may
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occur through multiple reaction types. Thus, for example, in alkaline solutions both
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hydrolysis and dehydrobromination may compete
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nucleophiles
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(e.g.dibromoelimination) may occur during reaction with FeS or sulfur species 11.
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During the last decades it has been demonstrated that compound-specific stable
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isotope analysis (CSIA) can be used to characterize transformation reactions of
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organic contaminants
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methods for carbon, hydrogen and nitrogen was extended by the development of
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highly sensitive bromine isotope analyses as a tool for investigating the
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transformation of BOCs
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formed by heavy isotopes (e.g. 2H,
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therefore, cleaved slower than bonds between lighter isotopes. As a result, the
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residual (not yet degraded) fraction of the substrate becomes enriched in the heavier
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isotopes as a reaction proceeds. So far, it has been demonstrated that single
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element isotope fractionation can serve as an indicator for a specific reaction
2-4
. EDB is susceptible to abiotic reactions and can be biodegraded under
combined
with
5-13
. In general, all EDB transformations in the
two-electron
10,
transfer
14
; SN2 substitution by
reductive
debromination
15, 16
. Recently, the set of the well-established analytical
17-20
. The approach of CSIA is based on the fact that bonds 13
C,
37
Cl,
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Br) are slightly more stable and,
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pathway without the need to trace reaction products, which can be challenging in
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complex environmental systems15. However, other steps preceding the isotope-
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sensitive bond cleavage, e.g. uptake, transport and binding of substrate to the
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enzyme, may significantly affect isotope enrichment factors, even for similar
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reactions
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than intrinsic kinetic isotope effects (KIEs). Additionally, a possible lack of variability
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in isotope fractionation patterns among different reaction pathways may limit its
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diagnostic value for reaction identification
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analysis allows the correlation of isotope ratios of several elements to each other and
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reveals pathway-specific information. When the changes in isotope fractionation of
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two elements are correlated, a correlation factor specific for a given reaction pathway
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and corresponding bond cleavage is expected
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already extensively applied
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up recently for evaluation of reaction mechanisms
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large potential for environmental studies, the implementation of the dual carbon-
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bromine isotope analysis is still in its infancy. To our knowledge, thus far, only two
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studies applied dual carbon-bromine isotope analysis for getting insight into the
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mechanism during the abiotic transformation of bromophenols and tribromoneopentyl
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alcohol, respectively 20, 34.
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While carbon and chlorine isotope effects in the chlorinated analogue 1,2-
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dichloroethane (1,2-DCA) have been studied in many biological and chemical
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transformations
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investigated. To the best of our knowledge, only two studies regarding carbon
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isotope effect during biological and chemical degradation of EDB are available so far
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8, 11
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still unclear how different factors affect isotope effects during transformations of
21, 22
, and lead to apparent kinetic isotope effects (AKIEs) that are smaller
31, 35-37
25-29
23
. Potentially, multi-elemental isotope
24
. Dual δ2H/δ13C isotope analysis is
and studies applying dual δ13C/δ37Cl analysis came 30-33
. In contrast, probably having
, isotope effects during transformations of EDB are still poorly
. Due to the limited data on isotope effects in brominated organic compounds, it is
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brominated organic compounds. In the present study we aimed to investigate carbon
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and bromine isotope fractionation of EDB during several environment-relevant EDB
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degradation processes: i) chemical hydrolysis in alkaline solution; ii) Zn(0) reduction;
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iii) reduction by reduced corrinoids;
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biotransformation by Sulfurospirillum multivorans crude extract; and vi) aerobic
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biotransformation by Ancylobacter aquaticus crude extract.
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The chosen set of the chemical and biotic transformations of EDB enabled us to
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compare carbon and bromine isotope fractionation and to evaluate the potential of
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dual carbon-bromine isotope slopes for differentiating processes following the same
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mechanistic pathway. The obtained results were compared to isotope effects
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observed for chlorinated analogues
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model, a difference in isotope effects was expected for C-Cl (KIEC=1.0572;
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KIECl=1.013)23 vs. C-Br (KIEC=1.0420; KIEBr=1.002) bond cleavage (see calculations
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in Supplementary Information). Thus the C-KIE is expected to be ~1.1-1.2 times
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larger for chlorinated compounds and Cl-KIE is ~ 5-6 times higher than Br-KIE34, 38.
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Comparison between the isotope effects observed for EDB in the present study to
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the isotope effects reported for chlorinated analogues enables to evaluate similarities
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and differences in behavior of these two important classes of halogenated organic
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pollutants.
34, 38
iv) Fenton oxidation; v) anaerobic
. Based on Streitwieser semi-classical limits
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Materials and Methods
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Cultivation of bacterial cultures. Sulfurospirillum multivorans (DSMZ 12446) was
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cultivated at 28°C and 120 rpm in an anoxic mineral medium as previously described
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39
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mmol L-1) and tetrachloroethene (10 mmol L-1 dissolved in hexadecane) as electron
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acceptor. Ancylobacter aquaticus AD20 (DSMZ 9000) was cultivated under oxic
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condition in mineral medium as described previously
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with 1,2-DCA (1 mmol L-1) and incubated at 28°C and 120 rpm. EDB was not added
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in the pre-cultivations to ensure that the same enzymes were expressed as in
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previous studies. Furthermore, preliminary tests showed that S. multivorans could not
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grow with EDB, likely due to toxicity effects.
with pyruvate (40 mmol L-1) as carbon source and electron donor and fumarate (40
40
. The culture was amended
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Bacterial crude extract preparation. Bacterial cells were harvested at the end of
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the logarithmic growth-phase. Crude extracts were prepared in 0.1 mol L-1 Tris-HCl
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buffer adjusted to pH 7.5 under anoxic conditions (S. multivorans)
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L-1 Tris-HCl buffer with 1 mmol L-1 EDTA adjusted to pH 7.5 under oxic conditions (A.
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aquaticus)
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mixture was incubated for 10-15 min at room temperature. Subsequently, the cells
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were disrupted via French press (Thermo Scientific, Waltham, USA) at 20.000 psi.
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The produced crude extract was stored (under anoxic conditions for S. multivorans)
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at -20°C until further use.
40
21
and in 50 mmol
. For cell lysis 10 mg lysozyme and 1 mg DNase I was added and the
138 139
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Transformation experiments.
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Biotic transformation with crude extracts. In both experiments crude extracts were
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used to avoid rate-limitation, such as substrate uptake, affecting isotope fraction prior
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to bond cleavage typical for biodegradation experiments and to characterize the
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enzyme-related transformation of EDB. The enzymatic dehalogenation reaction with
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crude extract of S. multivorans was done as described previously
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be found in the Supporting Information (SI). The enzymatic reaction with crude
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extract of A. aquaticus was performed in sealed 10 ml vials filled with 5 ml 50 mmol
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L-1 Tris-HCl buffer (pH 7.5) and 1 mmol L-1 EDTA. EDB was added at a final
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concentration of 1 mmol L-1. The reactions were started by adding bacterial crude
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extract to the reaction vials and were incubated at room temperature on a rotary
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shaker (160 rpm). Abiotic controls without adding cell extracts showed no significant
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decrease of EDB (data not shown). The reactions were stopped by acidification to
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pH
