Article pubs.acs.org/est
Transformation and Removal of Tetrabromobisphenol A from Water in the Presence of Natural Organic Matter via Laccase-Catalyzed Reactions: Reaction Rates, Products, and Pathways Yiping Feng,† Lisa M. Colosi,‡ Shixiang Gao,† Qingguo Huang,§ and Liang Mao†,* †
State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210093, P. R. China ‡ Department of Civil and Environmental Engineering, University of Virginia, Charlottesville, Virginia 22904, United States § College of Agricultural and Environmental Sciences, Department of Crop and Soil Sciences, University of Georgia, Griffin, Georgia 30223, United States S Supporting Information *
ABSTRACT: The widespread occurrence of the brominated flame retardant tetrabromobisphenol A (TBBPA) makes it a possible source of concern. Our experiments suggest that TBBPA can be effectively transformed by the naturally occurring laccase enzyme from Trametes versicolor. These reactions follow second-order kinetics, whereby apparent removal rate is a function of both substrate and enzyme concentrations. For reactions at different initial concentrations and with or without natural organic matter (NOM), reaction products are identified using liquid or gas chromatography with mass spectrometry. Detailed reaction pathways are proposed. It is postulated that two TBBPA radicals resulting from a laccase-mediated reaction are coupled together via interaction of an oxygen atom on one radical and a propyl-substituted aromatic carbon atom on the other. A 2,6-dibromo-4isopropylphenol carbocation is then eliminated from the radical dimer. All but one of the detected products arise from either substitution or proton elimination of the 2,6-dibromo-4-isopropylphenol carbocation. Three additional products are identified for reactions in the presence of NOM, which suggests that reaction occurs between NOM and TBBPA radical. Data from acute immobilization tests with Daphnia confirm that TBBPA toxicity is effectively eliminated by laccase-catalyzed TBBPA removal. These findings are useful for understanding laccase-mediated TBBPA reactions and could eventually lead to development of novel methods to control TBBPA contamination.
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INTRODUCTION Global use of the brominated flame retardant (BFR) tetrabromobisphenol A (TBBPA) ranges from 120 000 to 150 000 tons per year. This makes it the most widely used BFR at present accounting for nearly 60% of the total market share.1−3 It is widely detected in the environment,4−9 biota, and human tissues.10 On the basis of these factors, the European Food Safety Authority (EFSA) was asked by the European Commission to deliver an assessment of TBBA’s likely health impacts from dietary exposure.11 The EFSA concluded that that dietary exposure in the EU does not constitute a health concern; however, water exposure was not evaluated in this study. Measured TBBPA concentrations in municipal wastewater treatment plant effluents have been as high as 85 ng L−1, and industrial landfill leachates may contain TBBPA up to 620 ng L−1.7,9 This is alarming because of the structural similarity of TBBPA to thyroxin and the steroid estrogens enable it to act as both a thyroid hormone agonist and an estrogen agonist.12,13 It is documented that TBBPA also mediates immunotoxicity at © 2012 American Chemical Society
high concentrations and also leads to neurotoxicity effects on mammals.11,14−16 Despite these impacts, TBBPA is currently unregulated worldwide.2 Many BFRs are bioaccumulative and/or persistent, which makes them uniquely challenging from the perspective of water and wastewater treatment.9,17−21 Anaerobic biotransformation and UV-photodegradation have been shown to effectively reduce TBBPA concentrations in water and sediments under certain conditions; however, complete dehalogenation of TBBPA to bisphenol A (BPA) has been observed for these treatments. This is undesirable because BPA exhibits greater estrogenicity than TBBPA, and, in both cases, no further degradation of the BPA was observed.18,19,21 Received: Revised: Accepted: Published: 1001
July 3, 2012 December 12, 2012 December 20, 2012 December 20, 2012 dx.doi.org/10.1021/es302680c | Environ. Sci. Technol. 2013, 47, 1001−1008
Environmental Science & Technology
Article
ultimately be leveraged into development of novel enzymatic treatments for TBBPA and other BFRs.
Enzymatic reactions are highly efficient and specific offering biochemical transformation of trace contaminants without unreasonably long reaction times or overly intensive energy consumption. 22 The peroxidase enzymes, including horseradish peroxidase (HRP) and lignin peroxidase (LiP), are known to mediate efficacious removal of various emerging contaminants such as pharmaceuticals and perfluorooctanoic acid.22,23 Laccases are another class of enzymes capable of mediating oxidative coupling reactions for trace contaminants. It has been shown that laccase treatment can effectively remove both pharmaceutical compounds (such as acetaminophen and hormones) and their associated toxicity from synthetic water under simulated environmental conditions.24−26 Laccase is capable of polymerizing phenols, and the phenolic polymer products are themselves good substrates for further laccasecatalyzed coupling reactions. This process continues until the polymers become sufficiently large to precipitate from solution.27−29 Because the polymerized products formed from such coupling reactions can readily settle from water and/or become immobilized in soil/sediment systems, laccase-mediated oxidative coupling reactions have potential applications for water treatment. Laccases also possess one very important operational advantage compared to peroxidases, which is that they can use dissolved molecular oxygen rather than hydrogen peroxide to initiate catalysis.24,25 Like other blue multicopper oxidases, laccases catalyze oneelectron oxidation of four substrate molecules with concomitant four-electron reduction of molecular oxygen to water. Laccases generally contain one type-1 (T1) copper, together with one type-2 (T2) and two type-3 (T3) copper ions, arranged in a trinuclear cluster. The substrates are oxidized by the T1 copper, and the electrons are transferred through a strongly conserved His-Cys-His tripeptide motif, to the T2/T3 site, where oxygen reduction occurs.29 The laccase catalytic cycle is shown in Figure S1 of the Supporting Information. Natural organic matter (NOM) is ubiquitously present in environmental systems, and it has been previously demonstrated that phenolic/anilinic functionalities within the NOM structure enable it to participate as a substrate in reactions mediated by HRP, LiP, and laccase.30−34 In these studies, it has been widely assumed that the ability of NOM to act as enzyme cosubstrate results in radical-mediated cross-coupling between the NOM structure and micropollutants. Additionally, because NOM is generally present at concentrations that are orders of magnitude higher than that of the contaminants we desire to remove, it is necessary to understand how NOM could impact laccase performance during micropollutant transformation. In this study, we explored the potential of laccase-mediated oxidative coupling processes to achieve efficient removal of TBBPA from water. Reaction kinetics and mechanisms were systematically investigated based on identification of reaction products by a combination of liquid chromatography−mass spectrometry (LC/MS) and gas chromatography−mass spectrometry (GC/MS) techniques. The influence of NOM on TBBPA transformation was also investigated for systems containing extremely low concentrations (nM levels) of the target contaminant. Mass spectrometric analyses of TBBPA’s two stable bromine isotopes (79Br and 81Br) were used to generate conclusive proof of direct reactions between TBBPA radical and NOM and improve understanding of how NOM impacts laccase-mediated TBBPA reactions in natural systems. Taken together, these results provide useful information for understanding laccase-mediated TBBPA reactions, which could
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EXPERIMENTAL SECTION Materials. All reagents were of ACS grade or higher grade. TBBPA (97%) was purchased from Sigma-Aldrich (St. Louis, MO). A stock solution of 1 mM TBBPA was prepared in HPLC-grade methanol and stored at 4 °C. Working solutions were prepared by dilution in phosphate buffer (PBS) and contained less than 0.5% (v/v) methanol, which has no impact on the enzyme.22 Laccase from Trametes versicolor was obtained from Sigma-Aldrich, and dosing solutions were prepared in PBS. Aquatic NOM (Suwannee River NOM) was purchased from International Humic Substances Society. Enzyme Activity Assay. Laccase activity was quantified using a colorimetric assay in which ABTS (2,2′-azinobis-(3ethyl benzthiazoline-6-sulfonic acid) is oxidized by the enzyme in a 10 mM sodium acetate buffer (pH 5), with continuous monitoring of absorbance at 420 nm over time. One unit of laccase activity is defined as the amount of enzyme required to mediate oxidation of 1 μmol of ATBS per min.25 Assessment of TBBPA Removal at Varying Reaction Conditions. Experiments were conducted in glass test tube batch reactors, which were incubated on a rotary shaker at 150 rpm. Reaction solutions (2 mL) were prepared in PBS (0.01 M, pH 7.0) containing 10 μM TBBPA and one of six enzyme dosages ranging from 0.025 to 0.2 U mL−1. Seven sets of reactors were prepared in triplicate for each enzyme dosage. Identical reactors without laccase were used as controls. At prespecified intervals (1, 3, 5, 10, 20, 30, and 60 min) reactors were dosed with 2 mL of acetonitrile (to terminate reaction) and sacrificed to measure TBBPA concentration. Details of the HPLC (high pressure liquid chromatography) method for TBBPA analysis are given in section II of the Supporting Information. Reaction Efficiencies at Different EnvironmentallyRelevant TBBPA Concentrations. Experiments were conducted in 500 mL reactors and reaction solutions (200 mL) were prepared in PBS (0.01 M, pH 7.0) with predetermined doses of laccase and one of six TBBPA concentrations ranging from 5 to 100 nM. Four sets of reactors were prepared in triplicate for each TBBPA concentration. Identical reactors without laccase were used as controls. Experimental and blank reactors were sacrificed at 1, 2, 3, and 4 h. Samples were then concentrated via solid phase extraction (SPE) on C18 cartridges (6 mL, 500 mg) and analyzed for TBPPA concentration using HPLC. The minimum detection limit (MDL) of SPE-assisted HPLC analysis was 0.18 nM corresponding to three times the standard deviation of the background noise signals. Laccase-mediated TBBPA removal for the range of initial concentrations noted above was also evaluated in a separate experiment in which all reactors also contained 5 mg L−1 NOM on a TOC (total organic carbon) basis. Detailed information about SPE extraction and recovery, HPLC analysis, and MDL determination for reactions with and without NOM are given in sections II and III of the Supporting Information. Products Characterization. Samples for product characterization were prepared in a 250 mL flask reactor containing 100 mL of reaction solution comprising 10 μM TBBPA, 0.3 U mL−1 of laccase, with or without the presence of 5 mg L−1 NOM (as TOC). These reactors were continuously mixed via magnetic stirring for 60, 120, or 240 min. At sampling time, 1002
dx.doi.org/10.1021/es302680c | Environ. Sci. Technol. 2013, 47, 1001−1008
Environmental Science & Technology
Article
which contained neonates and laccase in MPSB buffer medium. Toxicity was also evaluated for a series of TBBPA standards. Molecular Modeling. HyperChem 8.0 was used to perform ab initio calculations with a 3-21 G basis set for determination of the charge and spin densities of the TBBPA radical.
product mixtures were concentrated using SPE (section II of the Supporting Information) prior to GC/MS or LC/MS characterization. Additional HPLC analysis with a UV−vis detector (section II of the Supporting Information) was used to quantify the relative yields of each product. Derivatization was required for samples analyzed via GC/ MS. One milliliter of SPE-concentrated sample was transferred to a 1.5 mL vial, which was then evaporated to dryness via gentle nitrogen flow. Vial contents were reconstituted in 100 μL n-hexane plus 100 μL of silylation reagent N,O-bis (trimethylsilyl) trifluoroacetamide. This mixture was incubated at 60 °C for 60 min to ensure completion of the silylation reaction. Afterward, the derivatization solution was evaporated to dryness (again using nitrogen), and vial contents were reconstituted into 1 mL n-hexane. This solution was then used for GC/MS analysis. GC/MS characterization was performed on a Thermo Finnigan Trace gas chromatograph equipped with a Polaris Q ion trap tandem mass spectrometer (GC-ITMS/MS, Thermo, Finnigan, USA). A DB-5 fused-silica capillary column (30 m × 0.32 mm i.d., 0.25 μm film thickness) was used for GC separation, and helium was used as the carrier gas at 1 mL min−1. The oven temperature was initially 100 °C for 1 min, increased to 290 °C at 20 °C min−1, then increased to 320 °C at 10 °C min−1, and finally held at 320 °C for 20 min. The injector, interface, and source temperatures were set at 280, 280, and 230 °C, respectively. LC/MS analysis was carried out on a Thermo liquid chromatograph connected to a Thermo LCQ Advantages (Quest LCQ Duo, USA) mass spectrometer through an ESI interface. Separation was performed on a C18 reverse-phase column (150 × 2.1 mm, 5 μm particle, Agilent). Injection volume was 10 μL. The mobile phase was made up of methanol (85%) and water (15%), and eluted at 0.2 mL min−1. The mass spectrometer was operated in negative ionization mode over the range m/z = 50−1200. Capillary voltage and cone voltage were 4.5 kV and 25 V, respectively. Desolvation and source temperatures were 300 and 120 °C, respectively. Nitrogen was used as sheath gas at a flow rate of 35 arb units and as auxiliary gas at a flow rate of 5 arb units. Acute Toxicity Testing. Postreaction TBBPA solutions with or without 5 mg L−1 NOM was assessed for toxicity to Daphnia as a function of laccase reaction time. Reactions were performed in 250 mL glass flasks initially containing 100 mL of 3(N-morpholino) propane-sulfonic buffer (MPSB, 3.6 mM, pH 7), 2 μM TBBPA, 0.2 U mL−1 of laccase. Flasks were prepared in triplicate and reacted for 1, 3, 5, 10, 20, 30, or 60 min. Reactors were then dosed with NaOH (6 mM) to achieve pH 10.0, effectively terminating laccase activity. Sample pH was then readjusted to 7.0 using HCl and reactor contents were apportioned into two samples: one for acute toxicity testing (below), and the other for SPE-assisted HPLC determination of residual TBBPA concentration. In this way, it was possible to determine what TBBPA concentrations correspond to various toxicity levels. Daphnia were used for acute toxicity testing, as performed using a standard method, OECD 202 (OECD/OCDE, 2004). There were four replicates per treatment. Each replicate consisted of five Daphnia neonates (
