Article pubs.acs.org/est
Novel Octabrominated Phenolic Diphenyl Ether Identified in Blue Mussels from the Swedish West Coast Ulrika Winnberg,*,† Andreas Rydén,† Karin Löfstrand,‡ Lillemor Asplund,† Anders Bignert,§ and Göran Marsh† †
Environmental Chemistry Unit, Department of Materials and Environmental Chemistry, Stockholm University, SE-106 91 Stockholm, Sweden. ‡ Department of Applied Environmental Science (ITM), Stockholm University, SE-106 91, Stockholm, Sweden. § Department of Contaminant Research, Swedish Museum of Natural History, Box 50007, SE-104 05, Stockholm, Sweden S Supporting Information *
ABSTRACT: Hydroxylated (OH−) and methoxylated (MeO−) polybrominated diphenyl ethers (PBDEs) are compounds present in the marine environment and OH−PBDEs are of toxicological concern and are therefore of interest to monitor in the environment. A phenolic octaBDE was tentatively identified in the phenolic fraction of previously analyzed mussel samples after methylation of the halogenated phenolic compounds (HPCs). The aim of the present study was to confirm the identity of this compound in blue mussels and investigate whether the analyte is diOH− and/or OH−MeO− octaBDE. Two reference standards, 6,6′-dimethoxy-2,2′,3,3′,4,4′,5,5′octabromodiphenyl ether (6,6′-diMeO−BDE194) and 6-ethoxy-6′methoxy-2,2′,3,3′,4,4′,5,5′-octabromodiphenyl ether (6-EtO-6′-MeO− BDE194) were prepared via O-arylation of 2,3,4,5-tetrabromo-6-methoxyphenol and 2,3,4,5-tetrabromo-6-ethoxyphenol, respectively, with a novel unsymmetrical diaryliodonium salt, 2,3,4,5-tetrabromo-6-methoxydiphenyliodonium triflate. The GC retention time and GC/MS spectrum of the synthesized 6,6′-diMeO−BDE194 correspond well with the analyte in the methylated phenolic fraction of a mussel extract from a previous study. Structural analysis performed in this study indicate that the synthesized 6,6′-diMeO−BDE194 and 6-EtO-6′-MeO−BDE194 correspond well with 6-hydroxy-6′-methoxy2,2′,3,3′,4,4′,5,5′-octabromodiphenyl ether (6-OH−6′-MeO−BDE194) after methylation and ethylation, respectively, of the HPCs in the mussel extracts. The compound 6-OH−6′-MeO−BDE194 was identified and quantified in new mussels, sampled in 2012 from two locations on the Swedish west coast, with geometric mean concentrations of 3700 and 410 ng/g fat, respectively.
■
INTRODUCTION Various hydroxylated (OH−) and methoxylated (MeO−), diOH−, diMeO−, and OH−MeO−polybrominated diphenyl ethers (PBDEs) have been detected in the marine environment in, for example, marine sponges, algae, and mussels.1−8 Some of these compounds are known to be of natural origin9,10 and are presumed to be formed through endogenic production by algae or symbiotic living bacteria.11 Further, biologic O-methylation of halogenated phenols forming the methoxylated analogue has been observed to occur in bacteria12 and fungi.13 MeO−PBDEs can in turn be converted to their hydroxylated analogue through in vivo demethylation, a transformation that has been shown to occur to 6-MeO−BDE47.14 Apart from the natural source of OH− and MeO−PBDEs, flame retardant PBDEs have been shown to form functionalized PBDE analogues, for example, hydroxylated PBDEs (OH−PBDEs)15,16 and methoxylated PBDEs (MeO−PBDEs)17 through metabolism. Naturally occurring OH− and MeO−PBDEs are almost exclusively substituted with the OH− or MeO− group in ortho-position to the ether linkage, while ortho, meta-, and para© 2014 American Chemical Society
substition can be found among OH−PBDEs formed from metabolism of anthropogenic PBDEs through enzymatic CYP450 mediated direct hydroxylation, or via 1,2shift.6,15,16,18 Like other halogenated phenolic compounds (HPCs), OH−PBDEs are of potential toxicological concern.19 Hence, reported adverse effects correlated to OH−PBDE exposure are, for example, disruption of the oxidative phosphorylation,20,21 endocrine disruption through binding competitively to transthyretin (TTR)22 and estrogenic effects.19,23 Blue mussels are a major source of feed for some species of fish and sea ducks in the Baltic Sea. OH−PBDEs exposure may be a possible contributing factor to adverse effects observed in Baltic marine wildlife, such as high fish egg mortality, reduced body fat and possibly also the dramatic decline in sea duck populations observed in the Baltic Sea, as Received: Revised: Accepted: Published: 3319
November 11, 2013 January 24, 2014 February 21, 2014 February 21, 2014 dx.doi.org/10.1021/es404969e | Environ. Sci. Technol. 2014, 48, 3319−3326
Environmental Science & Technology
Article
suggested by Dahlberg et al.20 and references cited therein. Unlike Baltic blue mussels, blue mussels from the west coast of Sweden are consumed by humans. Hence, both wildlife and humans may be exposed to phenolic PBDE compounds present in blue mussels from the Swedish west coast and it is therefore important to gather information on HPCs, such as chemical structures, concentrations, etc. Analytical research is to a large extent limited by the availability of reference standards. Methods for the syntheses of individual OH−PBDE and MeO−PBDE congeners have been summarized elsewhere.24 In addition, a diMeO−PBDE, 6,2′dimethoxy-2,3′,4,5′-tetrabromodiphenyl ether, has been synthesized by Marsh et al.25 using methods described elsewhere.28 A desirable methodology to prepare PBDEs and their derivatives is O-arylation of functionalized phenols with unsymmetrical diaryliodonium triflates. This method has been employed in the present work. Synthesis of 6,6′-diMeO−BDE194 and 6-EtO-6′MeO−BDE194 has not been previously reported in literature. The purpose of this study was to identify an earlier detected but only tentatively identified compound found in the phenolic fraction of blue mussel samples from Fladen and Väderöarna, along the Swedish west coast.6 The unknown compound, a peak referred to as “U5” in the gas chromatography mass spectrometry (GC/MS) chromatogram, was tentatively assigned as diOH− and/or OH−MeO−octaBDE, identified as its methyl derivative based on its GC/MS-electron capture negative ionization (ECNI) fragmentation pattern.6 The peak “U5” was of highest abundance in the GC/MS-ECNI fullscan chromatogram and was therefore considered of highest priority for identification. The analyte in the mussel samples was assumed to be of natural origin, therefore ortho-substituted diOH and/or OH−MeO−octaBDE were hypothesized as the congeners of interest. Thus, the aim was to develop a synthesis method for 6,6′-dimethoxy-2,2′,3,3′,4,4′,5,5′-octabromodiphenyl ether (6,6′-diMeO−BDE194, Figure 1) and 6-ethoxy6′-methoxy-2,2′,3,3′,4,4′,5,5′-octabromodiphenyl ether (6-EtO6′-MeO−BDE194, Figure 1) to use as authentic analytical reference standards. Further, the aim was to identify the previously mentioned “U5” peak in the pooled mussel extract from 2003 to 2005,6 and to identify and quantify the
substance(s) in recently sampled mussels (2012) from Fladen and Väderöarna. Ethyl derivatized analytes (Figure 1) together with the 6-EtO-6′-MeO−BDE194 reference standard were used in order to determine the exact structure of the phenolic analogue of 6,6′-diMeO−BDE194, that is, to determine whether it is substituted with one hydroxyl-group and one methoxy-group, 6-hydroxy-6′-methoxy-2,2′,3,3′,4,4′,5,5′-octabromodiphenyl ether (6-OH-6′-MeO−BDE194, Figure 1), or two hydroxyl-groups, 6,6′-dihydroxy-2,2′,3,3′,4,4′,5,5′-octabromodiphenyl ether (6,6′-diOH−BDE194, Figure 1).
■
MATERIALS AND METHODS Samples. Blue mussels (Mytilus edulis) were collected in autumn, 2012 from Fladen (57° 18′ N, 11° 54′ E) and Väderöarna (58° 38′ N, 11° 16′ E) in Kattegat and Skagerrak, respectively, along the Swedish west coast. The mussels were stored frozen (−18 °C) until analysis. Also, a pooled extract of blue mussels prepared on mussels collected in the autumns of 2003−2005 from Fladen and reported in a previous study,6 was reanalyzed. Fladen and Väderöarna are reference sites, that is, there are no known point sources in these areas. The selected locations are included in the Swedish Environmental Protection Agency’s Environmental Monitoring Program, in which PBDEs are annually analyzed in blue mussels.27 Chemicals. Solvents and chemicals of highest purity available were used in the analysis procedure. The purity and the suppliers of the solvents and chemicals are described in the Supporting Information (SI). The volumetric standard (VS) 4′chloro-2,2′,3,3′,4,5,5′,6,6′-nonabromodiphenyl ether (ClBDE208) was synthesized in house as described by Christiansson et al.28 and the analytical reference standards 6,6′-diMeO− BDE194 and 6-ethoxy-6′-methoxy-2,2′,3,3′,4,4′,5,5′-octabromodiphenyl ether (6-EtO-6′-MeO−BDE194) were synthesized in this study. Instruments for Analysis. Instrumental analysis information is described in detail in the SI and in brief below and in Table 1. In the qualitative analyses (identification) as well as the quantification analyses of the HPCs after methylation, the samples were analyzed using gas chromatography mass spectrometry (GC/MS) with the MS operating in electron capture negative ionization (ECNI) mode with methane as reagent gas. Identification was performed based on comparison of the GC retention times and the GC/MS-ECNI fragmentation pattern between the analyte in the mussel samples and 6,6′-diMeO−BDE194 (external reference standard). Relative retention times were calculated for the analytes relative to the volumetric standard (VS). For quantification, the MS detector was operating in selected ion monitoring (SIM) mode. The analyte, 6,6′-diMeO−BDE194, was quantified against a seven point calibration curve (R2 = 0.9991) of 6,6′-diMeO−BDE194 (reference standard). The quantification was performed using the phenolate fragment ions for 6,6′-diMeO−BDE194 and the VS (m/z 436.6, 438.6, and 440.6, 442.6, respectively). Structural identification of the ethylated HPCs in a pooled mussel sample was performed using GC/MS-electron ionization (EI) and GC/MS-ECNI. Synthesis. In this study, a synthesis method for 6,6′diMeO−BDE194 was developed and optimized. Also, 6-EtO6′-MeO−BDE194 was synthesized according to the same methodology. A pure analytical reference standard of 6,6′diMeO−BDE194 was successfully prepared through an eight step synthesis route starting from 2-nitrophenol (1). A detailed synthesis description is presented in the SI. First, 1 was
Figure 1. Structures of the two hypothesized analytes: 6-OH-6′MeO−BDE194 and 6,6′-diOH−BDE194, and the possible derivatization products after methylation, 6,6′-diMeO−BDE194, and ethylation, 6-EtO-6′-MeO−BDE194 and 6,6′-diEtO-BDE194. 3320
dx.doi.org/10.1021/es404969e | Environ. Sci. Technol. 2014, 48, 3319−3326
Article
GC/MS-ECNI
GC/MS-EI
full scan (m/z 250−1000) and SIM (m/z for analyte: 421.7, 423.7, 875.7, 877.7, for VS: 753.6, 755.6, 915.7, 917.7) full scan (m/z 33−1000)
brominated using benzyltrimethylammonium tribromide (BTMA-Br3) and calcium carbonate in dichloromethane and methanol according to Kajigaeshi et al.29 to yield 2,4-dibromo6-nitrophenol (2) (Step 1, Scheme 1). The compound 1,5dibromo-2-methoxy-3-nitrobenzene (3) was prepared through O-methylation of 2 in a two-phase system utilizing tetrabutylammonium hydroxide ((Bu)4NOH), sodium hydroxide and iodomethane in dichloromethane and water (Step 2, Scheme 1), in accordance with a general methylation method described by Marsh et al.25 Reduction of 3 to the corresponding aniline, 3,5-dibromo-2-methoxyaniline (4), was carried out using tin chloride dihydrate in ethanol and tetrahydrofurane similar to Chanteau and Tour30 (Step 3, Scheme 1). The compound 2,3,4,5-tetrabromo-6-methoxyaniline (5) was prepared through bromination of 4 using N-bromosuccinimide (NBS) and triflic acid (TfOH) in acetonitrile (Step 4, Scheme 1) similar to Oberhauser,31 who brominated phenols with NBS and strong acids. Diazotisation and subsequent halogenation of 5 was carried out using tert-butyl nitrite (t-BuONO) and iodine in a Sandmeyer type reaction to form 1,2,3,4-tetrabromo-5iodo-6-methoxybenzene 6 (Step 5, Scheme 1) similar to Ek et al.32 Novel 2,3,4,5-tetrabromo-6-methyoxydiphenyliodonium triflate (7) was synthesized by reacting 6, 3-chloroperoxybenzoic acid (m-CPBA) and TfOH with benzene in dichloromethane at 70 °C (Step 6, Scheme 1), similar to previously published methods.24,33,34 Bromination of 2-methoxyphenol (8) gave 3,4,5,6-tetrabromo-2-methoxyphenol (9), performed though a solvent-free reaction by addition of bromine to the phenol, 9, and calcium carbonate, using an ultrasonic bath as a means of mixing (Step 7a, Scheme 1).25 The novel 6,6′diMeO−BDE194, was finally synthesized by O-arylation of 9 with 7 (Step 8a, Scheme 2) using potassium tert-butoxide (tBuOK) as base to protonate the phenol, similar to a previously published method.35 6-EtO-6′-MeO−BDE194 was prepared in a similar manner as 6,6′-di-MeO−BDE194. Hence, 2ethoxyphenol (10) was perbrominated using the same method as for perbromination of 2-methoxyphenol described above, yielding 3,4,5,6-tetrabromo-2-ethoxyphenol (11) (Step 7b, Scheme 1). Subsequent O-arylation of 11 with 7 (Step 8b, Scheme 2) yielded 6-EtO-6′-MeO−BDE194. Extraction, Clean-Up and Analysis. Extraction and cleanup were, with some adjustments, performed according to Jensen et al.36 Individual mussels (5−12 g fresh weight) were homogenized and extracted with 2-propanol, cyclohexane, and diethyl ether.36 The organic phase was extracted with hydrochloric acid (0.2 M) in aqueous sodium chloride (0.9% w/v) and the aqueous phase was washed with cyclohexane/ methyl tert-butyl ether (9:1). The combined organic phases were pooled, the solvent was evaporated at room temperature and the fat weight was determined gravimetrically. The organic phase was partitioned with potassium hydroxide (0.5 M) in ethanol/water (1:1) and cyclohexane to separate the phenolic compounds from neutral compounds. The cyclohexane phase containing the neutral compounds was cleaned up as described below. The aqueous phase was re-extracted with cyclohexane/ diethyl ether (3:1) after acidification to pH 1 with hydrochloric acid (2 M). The phenolic compounds were derivatized using either diazomethane (methylation) or ethyl iodide (ethylation). Derivatization with an excess of diazomethane was carried out as previously described.37 CAUTION: Diazomethane must be used with great care and can only be prepared and handled on basis of a national permission, which in our case is No. IMS 2012/39924 (Swedish Work Authority, Sweden). A phase-
mussels from 2012: •Fladen (pooled extract of 5 individual mussels) •Väderöarna (1 sample) structural identification (6-EtO-6′-MeO−BDE194)
DB5-HT
SIM (m/z 79, 81, 436.6, 438.6, 440.6, 442.6) GC/MS-ECNI mussels from 2012: •Fladen (8 samples) •Väderöarna (7 samples) quantification (6,6′-diMeO−BDE194)
CP-SIL 5CB
full scan (m/z 33−1000) GC/MS-ECNI TR5-MS mussels from 2012: •Fladen (1 sample) •Väderöarna (2 samples) Pooled mussel extract from 2003 to 20056 identification (6,6′-diMeO−BDE194)
scan range/ions method GC column samples goal of analysis (analyte)
Table 1. Instrumental Analysis Methods Used for Identification, Quantification and Structural Identification Analyses of HPCs in the Mussel Samples
Environmental Science & Technology
3321
dx.doi.org/10.1021/es404969e | Environ. Sci. Technol. 2014, 48, 3319−3326
Environmental Science & Technology
Article
Scheme 1. Synthetic Pathway for the Preparation of an Unsymmetrical Diaryliodonium Triflate (7) and Two Functionalized Phenols (9, 11). a Yield According to Marsh et al.25
Cleanup of the analytes in both the phenolic (derivatized) and neutral fractions were performed through repeated concentrated sulfuric acid treatments until colorless (two to five times) followed by a packed activated silica gel column eluting the analytes with dichloromethane, as described by Löfstrand et al.6 The solvent was changed to n-hexane and the samples were spiked with a volumetric standard (Cl-BDE208) prior to instrumental analysis. Due to the lack of a suitable surrogate standard, quantification was performed using an external standard of 6,6′-diMeO−BDE194 and a volumetric standard. Consequently, compensation could not be made for any losses of analyte during the workup of the mussel samples and the quantified concentrations represent the least amount of the phenolic analogue of 6,6′-diMeO−BDE194 present in the blue mussels. A total of nine and eight mussels from Fladen and Väderöarna, respectively, were extracted as individual samples, and eight and seven mussels, respectively, of these were quantified. The phenolic fraction of the sample extracts were derivatized in order to enable analysis by GC/MS. Out of the mussel extracts from Fladen (nine samples) and Väderöarna
Scheme 2. Synthetic Pathway for the Preparation of 6,6diMeO−BDE194 and 6-EtO-6′-MeO−BDE194
transfer reaction was employed for ethylation using aqueous tetrabutylammonium hydroxide, in a slightly modified method developed by Lindqvist et al.,38 where ethyl iodide was used in this study instead of methyl iodide. 3322
dx.doi.org/10.1021/es404969e | Environ. Sci. Technol. 2014, 48, 3319−3326
Environmental Science & Technology
Article
(eight samples), HPCs present in whole extracts from Fladen (four samples) and Väderö arna (seven samples) were methylated. HPCs in one whole extract of mussels from each location were ethylated. Four extracts of mussels from Fladen were divided; any HPCs in 20% aliquots were methylated while the remaining 80% were ethylated. The purpose of dividing the mussel samples was to collect enough material for the structural identification analyses and to be able to quantify 6,6′-diMeO− BDE194 in the methylated HPC-fraction of each individual mussel. The ethylated HPCs in the Fladen samples were pooled before analysis. The sample distribution in the analysis procedure is schematically presented in the SI. The methylated HPCs in the phenolic fraction of the mussel extracts from Fladen and Väderöarna were used for identification and quantification, while the ethylated HPCs were used for structural identification. The analyte 6,6′-diMeO−BDE194 was also searched for in the neutral fraction of the mussel samples from Fladen and Väderöarna, from 2012. In addition to the mussel samples from 2012, identification was also performed on the phenolic fraction of a pooled mussel extract of mussels sampled in 2003−2005 from a previous study,6 which was reanalyzed in this study. Statistics. An F-test and a two-tailed student t-test were performed on the individual logged mussel specimen concentrations and the geometric mean concentrations of the phenolic analogue of 6,6′-diMeO−BDE194 in the mussel samples from Fladen and Väderöarna. A significance level of 5% (α = 0.05) was chosen.
Figure 2. GC/MS-ECNI chromatograms of methylated HPCs in the phenolic fraction of a blue mussel sample from Fladen (2012), and the 6,6′-diMeO−BDE194 reference standard. The upper chromatogram depicts a sample run in full scan mode, and the lower chromatogram represents the same full scan chromatogram after extraction of the phenolate ions. The peak appearing at a retention time of 20 min is identified as 6,6′-diMeO−BDE194, after methylation of the HPCs.
■
RESULTS AND DISCUSSION Synthesis. This study presents synthetic pathways for 6,6′diMeO−BDE194 and 6-EtO-6′-MeO−BDE194, which to our knowledge have not been previously published. The method involves the use of a novel unsymmetrical brominated diaryliodonium triflate, which in turn is a useful reagent in the synthesis of other OH- and MeO−PBDEs. Overall, high yields were achieved in most of the reactions in the synthetic route for 6,6′-diMeO−BDE194, ranging from 65 to 99% (Scheme 1), giving the overall yield of 8%. The lowest yields were obtained for the preparation of 6,6′-diMeO− BDE194 (34%, Step 8a, Scheme 2) and 6-EtO-6′-MeO− BDE194 (29%, Step 8b, Scheme 2). The low yields of the Oarylation reactions could be a result of sterically hindered starting materials. Higher yields have been reported for monoMeO−PBDEs, substituted with
