Occurrence of Natural Mixed Halogenated Dibenzo-p-Dioxins

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Occurrence of natural mixed halogenated dibenzo-p-dioxins: Specific distribution and profiles in mussels from Seto Inland Sea, Japan Akitoshi Goto, Nguyen Minh Tue, Masayuki Someya, Tomohiko Isobe, Shin Takahashi, Shinsuke Tanabe, and Tatsuya Kunisue Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b03738 • Publication Date (Web): 19 Sep 2017 Downloaded from http://pubs.acs.org on September 20, 2017

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Environmental Science & Technology

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Occurrence of natural mixed halogenated dibenzo-p-

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dioxins: Specific distribution and profiles in mussels

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from Seto Inland Sea, Japan

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Akitoshi Goto †, Nguyen Minh Tue †, Masayuki Someya ‡, Tomohiko Isobe §, Shin Takahashi †, ||,

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Shinsuke Tanabe †, Tatsuya Kunisue †* †

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Matsuyama, Ehime 790-8577, Japan ‡

Tokyo Metropolitan Research Institute for Environmental Protection, 1-7-5 Shinsuna, Koto-ku,

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Tokyo 136-0075, Japan §

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Center for Marine Environmental Studies (CMES), Ehime University, 2-5 Bunkyo-cho,

Center for Health and Environmental Risk Research, National Institute for Environmental Studies (NIES), 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan

||

Center of Advanced Technology for the Environment, Agricultural Faculty, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime 790-8566, Japan

14 15 16

ACS Paragon Plus Environment

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ABSTRACT

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In addition to unintentional formation of polychlorinated (PCDD/Fs), polybrominated

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(PBDD/Fs) and mixed halogenated (PXDD/Fs) dibenzo-p-dioxins/dibenzofurans during

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industrial activities, recent studies have shown that several PBDD and PXDD congeners can be

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produced by marine algal species from the coastal environment. However, multiple exposure

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status of anthropogenic and naturally-derived dioxins in marine organisms remains unclear. The

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present study examined the occurrence, geographical distribution and potential sources of

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PCDD/Fs, PBDD/Fs and PXDD/Fs using mussels and brown algae collected in 2012 from Seto

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Inland Sea, Japan. The results showed the widespread occurrence of not only PCDD/Fs but also

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PBDDs and PXDDs in Seto Inland Sea. The geographical distribution pattern of PBDDs was

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similar to that of PXDDs, which were obviously different from that of PCDDs and PCDFs, and a

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significant positive correlation was observed between the levels of their predominant congeners,

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i.e., 1,3,7-/1,3,8-TrBDDs and DiBMoCDDs. Interestingly, potential precursors of 1,3,7-/1,3,8-

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TrBDDs and DiBMoCDDs, hydroxylated tetrabrominated diphenyl ethers (6-HO-BDE-47 and

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2’-HO-BDE-68) and their mixed halogenated analogue (HO-TrBMoCDE), were also identified

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in the mussel and brown alga samples collected at same site, by comprehensive two-dimensional

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gas chromatography with time-of-flight mass spectrometry (GC×GC–ToFMS) analyses. It is

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noteworthy that residue levels of 1,3,7-/1,3,8-TrBDDs and DiBMoCDDs in the mussel were

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thirty times higher than those in the brown alga, suggesting the bioaccumulation of these natural

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dioxins.

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1. INTRODUCTION


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Polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs) are recognized as unintentional

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contaminants generated during municipal solid waste incineration1,2 and as impurities in specific

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organochlorine formulations such as polychlorinated biphenyl (PCB),3 pentachlorophenol (PCP)

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and chloronitrofen (CNP).4 In many developed countries including Japan, with the

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implementation of highly efficient incinerators and strict regulations on the production and usage

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of hazardous organochlorine formulations, PCDD/F levels in the environment have been

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decreasing during the last few decades. 5–7 However, these contaminants have still been detected

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in various trophic levels of wildlife species due to their persistent and bioaccumulative

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properties.8–10

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Polybrominated dibenzo-p-dioxins/dibenzofurans (PBDD/Fs) and their mixed halogenated

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analogues (PXDD/Fs) have recently been drawing international attention because of similar

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physico-chemical and toxicological properties to those of PCDD/Fs.11 These emerging dioxin-

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related compounds (DRCs) are formed unintentionally during manufacturing, recycling and

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waste incineration processes of electrical and electronic equipment treated with brominated

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flame retardants (BFRs).12,13 PBDD/Fs are also present as impurities in major additive BFRs

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such as technical polybrominated diphenyl ether (PBDE) formulations.14 According to the

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estimation by Persistent Organic Pollutants Review Committee (POPRC), the total global

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production of technical PBDE formulations (Penta-, Octa- and Deca-BDE) during 1970–2005

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was approximately 1.3–1.5 million tonnes.15 Although the production, usage and export-import

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of these PBDE formulations have been banned and/or regulated strictly by the Stockholm

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Convention on POPs, huge amounts of PBDE-containing products and their wastes (e-waste)

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still remain around the world which can be potential sources of PBDD/Fs. Hence, continuing

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release of PBDD/Fs into the environment and their exposure to wildlife are of global concern.


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Another emerging issue is the natural formation of PBDDs in the coastal environment. Recent

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studies reported that several PBDD congeners can be produced by marine algal species16,17

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and/or formed photochemically from naturally-derived hydroxylated-PBDEs (HO-PBDEs).18,19

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In the Baltic Sea, these natural PBDD congeners have been detected in not only marine primary

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producers (algae, cyanobacteria, sponge)20–22 but also fish and shellfish.16,21,23 An experimental

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study using zebrafish (Danio rerio) demonstrated that a mixture of natural PBDD congeners (1-

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MoBDD,

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/1,3,6,8-/1,3,7,9-TeBDDs) identified in the Baltic Sea biota induced dioxin-like responses.24

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However, almost all biomonitoring surveys for brominated dioxins have targeted only 2,3,7,8-

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substituted congeners, and hence information on exposure status of natural dioxins are limited in

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biota from the Baltic Sea. In addition, polybrominated monochlorinated dibenzo-p-dioxins

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(PBMoCDDs) were recently found in sponge (Ephydatia fluviatilis) from the Swedish coast,

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which were suspected to be of non-anthropogenic origin,22 but their exposure to the other marine

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organisms and generation sources remain unclear.

2,7-/2,8-DiBDDs,

1,3,7-/1,3,8-/2,3,7-TrBDDs,

1,2,3,7-/1,2,3,8-/1,2,4,7-/1,2,4,8-

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The aim of this study was to elucidate the occurrence, geographical distribution and potential

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sources of anthtopogenic, natural and unknown DRCs (i.e., PCDD/Fs, PBDD/Fs and PXDD/Fs)

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in Japanese coastal waters, by conducting a mussel monitoring survey in Seto Inland Sea, the

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largest semi-enclosed sea in Japan. We also screened potential DRC percursors in mussels and

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marine algae by gas chromatography with high resolution mass spectrometry (GC–HRMS) and

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comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry

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(GC×GC–ToFMS) to determine main DRC sources in the coastal environment.

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2. MATERIALS AND METHODS


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2.1. Sample collection. Seto Inland Sea is a semi-enclosed sea located in the western part of

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Japan (Figure 1), with a surface area of 23,203 km2, an average water depth of 38 m and a

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seawater residence time of 15 months.25,26 This semi-enclosed sea is surrounded by Honshu (the

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main island of Japan), Kyushu and Shikoku Islands, with approximately 30 million inhabitants.

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Many agricultural, industrial and municipal facilities are widely distributed on the shoreline.

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Blue mussels (Mytilus galloprovincialis) and green mussels (Perna viridis) were collected at

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16 locations of Seto Inland Sea during September–November 2012 (Figure 1 and Table S1).

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Mussels were washed with seawater on site and then shucked in the laboratory. The whole soft

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tissues of the mussels from each sampling location were pooled and homogenized using a stain-

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less steel blender (Labo Milser LM-2, Osaka Chemical Co., Ltd., Japan). Brown algae

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(Sargassum sp.) were collected at EH-1 (Figure 1) at the same time as blue mussels. After

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washing with seawater on site, the whole bodies of algae were cut into approximately 1-cm

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segments with scissors in the laboratory. The sliced alga tissues were pooled and homogenized

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using above mentioned blender. All the samples (mussels: n = 16, brown algae: n = 1) were

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stored in glass bottles pre-washed with acetone and kept at –25 °C in the environmental

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Specimen Bank (es-BANK) of Ehime University27 until chemical analysis.

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2.2. Extraction. Freeze-dried tissue (approximately 10–20 g of mussel or alga sample) was

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extracted using a high speed solvent extractor (SE-100, Mitsubishi Chemical Analytech, Japan)

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with acetone/n-hexane (1:1 v/v) at 35 °C for 30 min at a flow rate of 10 mL min-1. The crude

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extract was solvent-exchanged into n-hexane and concentrated to 10 mL using a rotary

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evaporator. An aliquot of crude mussel extract (equivalent to 1–2 g of dried tissue) was used for

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determination of the lipid content according to the gravimetric method reported by Isobe et al.,

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(2011).28


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2.3. Quantitative analysis of PCDD/Fs, PBDD/Fs and PXDD/Fs. PCDD/Fs, PBDD/Fs and

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PXDD/Fs were analyzed based on the method reported by Goto et al., (2017).29 Detailed cleanup

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procedure and instrumental analysis are described in the Supporting Information (SI). Breifly, an

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aliquot (equivalent to 2–4 g of dried mussel and alga samples) was spiked with internal standards

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(13C12-labeled PCDD/Fs: 2,3,7,8-TeCDD/Fs, 1,2,3,7,8-PeCDD/Fs, 2,3,4,7,8-PeCDF, 1,2,3,4,7,8-

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HxCDD/Fs, 1,2,3,6,7,8-HxCDD/Fs, 1,2,3,7,8,9-HxCDF, 2,3,4,6,7,8-HxCDF, 1,2,3,4,6,7,8-

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HpCDD/Fs, 1,2,3,4,7,8,9-HpCDF, and OCDD/Fs, Wellington Laboratories, Canada;

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labeled PBDD/Fs: 2-MoBDF, 2,8-DiBDF, 2,4,8-TrBDF, 2,3,7,8-TeBDD/Fs, 1,2,3,7,8-PeBDD,

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2,3,4,7,8-PeBDF,

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OBDD/Fs, Cambridge Isotope Laboratories, USA). The extract was cleaned-up with sulfuric

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acid, multi-layer silica gel and fractionated with activated carbon-impregnated silica gel. The

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cleaned-up solution was spiked with

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1,2,3,7,8-PeBDF (Wellington Laboratories, Canada, and Cambridge Isotope Laboratories, USA)

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and then concentrated under gentle stream of nitrogen gas. The final solution was stored at 4 °C

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until instrumental analysis.

1,2,3,4,7,8-HxBDD/Fs,

13

1,2,3,6,7,8-HxBDD,

1,2,3,4,6,7,8-HpBDF,

13

C12-

and

C12-labeled 1,2,3,4-TeCDD, 1,2,3,7,8,9-HxCDD and

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PCDD/Fs, PBDD/Fs and PXDD/Fs were identified and quantified using a gas chromatograph–

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magnetic sector high resolution mass spectrometer (GC–HRMS, Hewlett-Packard 6890 GC,

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Agilent Technologies, USA; JMS-700D, JEOL, Japan) coupled with a solvent cut large volume

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injection system (SGE Analytical Science, Australia) (Table S2). Procedural blanks (n = 3) were

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analyzed with every batch of 7 samples to check for contamination during clean-up, and no

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interference peaks were observed on each SIM chromatogram. Recoveries of the

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PCDD/Fs and PBDD/Fs ranged from 69 to 128% and from 44 to 98%, respectively.

13

C12-labeled


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2.4. Target screening of potential DRC precursors. The detailed analytical method for

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potential DRC precursors is described in the SI. Breifly, an aliquot of each crude extract

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(equivalent to 10 g of the dried mussel or alga from EH-1 in Figure 1) was spiked with the

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internal standard (13C12-labeled 6-hydroxy-2,2’,4,4’-tetrabromodiphenyl ether (6-HO-BDE-47),

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Cambridge Isotope Laboratories, USA) and subsequently subjected to gel permeation

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chromatography (packed Bio-Bead S-X3, BioRad Laboratories, USA). Target phenolic

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compounds (i.e., HO-PBDEs and HO-PXDEs) in the extract were isolated by liquid-liquid

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partioning with 1 M potassium hydroxide in ethanol/hexane-washed MilliQ water (1:1 v/v), as

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described previously.30 After re-extraction with methyl tert-butyl ether/n-hexane (1:1 v/v), the

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extract was cleaned-up with sulfuric acid and deactivated silica gel. The purified solution was

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spiked with 13C12-labeled 6-MeO-BDE-47 (Cambridge Isotope Laboratories, USA), concentrated

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under

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(trimethylsilyl)trifluoroacetamide/trimethylchlorosilane (BSTFA/TMCS, 99:1 v/v). The final

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solution was stored in a desiccator until instrumental analysis to avoid hydrolysis of the

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trimethylsilylated (TMS) derivatives.

gentle

stream

of

nitrogen

gas

and

then

derivatized

with

N,O-bis-

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Screening of HO-PBDEs and HO-PXDEs (after trimethylsilylation) in the mussel and alga

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samples were performed using a gas chromatograph (Agilent 7890A GC, Agilent Technologies,

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USA) coupled with a KT2006 GC×GC system (Zoex, USA)–high resolution time-of-flight mass

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spectrometer (JMS-T100GCV, JEOL, Japan) (GC×GC–HRToFMS). Detailed instrumental

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conditions are summarized in the Table S4. Briefly, the GC×GC column set was consisted of

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BPX5 (30 m, 0.25 mm i.d., 0.25 µm film thickness), FST (1.5 m, 0.1 mm i.d.) and BPX50 (2 m,

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0.1 mm i.d., 0.1 µm film thickness) (BPX5–FST–BPX50, SGE Analytical Science, Australia).

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The GC oven program was 160 °C (1 min), 20 °C min-1 to 200 °C, and then 4 °C min-1 to 320 °C


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(5 min). The modulation period and hot jet duration were programmed at 5000 ms and 300 ms,

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respectively. The HRToFMS was operated in electron ionization (EI) with full-scan mode (mass

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range: m/z 210–700, sampling rate: 25 Hz) at resolving power of > 5000 (full width at half

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maximum). Acquisition data were processed using GC Image software version 2.1b3 (GC Image

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LLC, USA).

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HO-PBDEs and HO-PXDEs were identified based on their retention time, accurate mass,

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isotope ratio of halogen cluster and fragment pattern, which were verified by comparing to those

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of authentic standards and/or literature data.31–34 All the HO-PBDE and HO-PXDE congeners

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detected in this study were quantified using the isotope dilution method to 13C12-6-HO-BDE-47,

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as 1.0 of relative response factor (RRF). A procedural blank was analyzed with 2 samples, and

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there was no substantial interference on each two-dimensional total ion chromatogram (2D TIC),

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except for some column bleed. Recoveries of the 13C12-labeled 6-HO-BDE-47 ranged from 87 to

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113%.

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2.5. Statistical analysis. The relationships among the concentrations of PCDD/Fs, PBDD/Fs

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and PXDD/Fs in the mussel samples were assessed using Spearman’s rank correlation

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coefficients with R statistical software package (R for Windows, version 3.3.2). Statistical

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significance was assumed at p < 0.05. Concentrations below the detection limits were excluded

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from the statistical analysis.

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3. RESULTS AND DISCUSSION

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3.1. Residue levels, congener profiles and potential sources of DRCs. Concentrations of

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PCDD/Fs, PBDD/Fs and PXDD/Fs detected in mussel samples from Seto Inland Sea are shown

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in Table 1 and their congener data are summarized in Table S5–7.


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3.1.1. PCDD/Fs. PCDD/Fs were detected in all the mussel samples analyzed (1200–32,000,

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median 4200 pg g-1 lw), suggesting their widespread contamination in Seto Inland Sea. The

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geographical distribution of PCDD/F levels in mussels showed an increasing trend in the eastern

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part, compared to the western part, of Seto Inland Sea (Figure 2), and PCDDs were accounted for

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60–91% (median: 80%) of the total PCDD/Fs (Table S5). Thus, it is likely that mussels from the

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estern Seto Inland Sea are exposed to relatively high levels of PCDDs even in recent years.

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The highest level of PCDD/Fs was found at HY-1 located in the northeastern Seto Inland Sea

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(Figure 2), and 1,3,6,8-/1,3,7,9-TeCDDs and OCDD were the predominant congeners accounting

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for 67% (44% and 23%, respectively: see Table S5) of the total PCDD/Fs. Interestingly, these

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PCDD congeners were also detected at higher proportions in all the other locations (37–72% of

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the total PCDD/Fs). Previous studies reported that 1,3,6,8-/1,3,7,9-TeCDDs and OCDD are the

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most abundant impurities of CNP and PCP formulations, respectively.6 In Japan, these

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organochlorine formulations had been mainly used as paddy herbicides during 1958–1990 for

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PCP and 1965–1996 for CNP.35 The massive usage of PCP and CNP herbicides has also been

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confirmed in the catchment areas of Seto Inland Sea, notably in the northeastern region,36 which

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was consistent with the geographical distribution pattern of PCDD levels in mussels observed in

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this study (Figure 2). These results indicate that widespread PCDD contamination in Seto Inland

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Sea was mainly caused by the past usage of PCP and CNP herbicides.

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Although a previous study reported that OCDD was the most abundant PCDD/F congener in

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sediment from Seto Inland Sea,37 higher concentrations of 1,3,6,8-/1,3,7,9-TeCDDs than OCDD

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were detected in mussels analyzed in this study. Similar phenomena have also been observed

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between marine shellfish and sediments from Tokyo Bay, Japan, and the authors suggested that it

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is attributed to a higher biota–sediment accumulation factor (BSAF) of TeCDDs than OCDD.38,39


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Thus, mussels from Seto Inland Sea could preferentially accumulate CNP-derived PCDD

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congeners, 1,3,6,8-/1,3,7,9-TeCDDs.

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3.1.2. PBDD/Fs. PBDD/Fs were also detected in all the mussel samples (640–46,000, median

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11,000 pg g-1 lw) with the predominance of PBDDs (> 90% of the total PBDD/F levels) except

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for HY-2 (Table S6). The geographical distribution pattern of PBDDs was clearly different from

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that of PCDDs (Figure 2), and no correlation was observed between the levels of PBDDs and

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PCDDs in the mussels (Spearman's ρ = –0.50, p = 0.05). These results indicate that PBDDs and

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PCDDs are originated from different sources.

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The highest level of PBDD/Fs was found at HR-4 located in the northwestern Seto Inland Sea

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(Figure 2), and 1,3,7-/1,3,8-TrBDDs were the predominant congeners accounting for 89% of the

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total PBDD/Fs (Table S6). The remaining 11% was composed of only Di–TeBDDs: Pe–OBDDs

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and Mo–OBDFs were below the detection limits. 1,3,7-/1,3,8-TrBDDs was also detected at

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higher porportions in mussels from all the other locations (75–93% of thr total PBDD/Fs), except

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for HY-2. These results suggest the ubiquity of 1,3,7-/1,3,8-TrBDDs in Seto Inland Sea.

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Previous studies have reported that these PBDD congeners can be produced by marine algal

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species16,17 and/or formed photochemically from natural HO-PBDE congeners.18,19 In fact, 1,3,7-

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/1,3,8-TrBDDs were detected as predominant congeners in the brown alga sample analyzed in

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this study (84% of the total PBDD/F levels), which was consistent with the result (88%)

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observed in the mussel sample from the same point (Table S8). Similar compositions have been

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reported also in marine organisms from the Baltic Sea (e.g., algae, cyanobacteria, sponge, fish

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and shellfish).40 In addition, our recent study on Japanese marine sediment cores, 1,3,7-/1,3,8-

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TrBDDs have been detected not only in the surface layer but also in the deeper layers

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corresponding to the pre-industrial era.29 Furthermore, it has been demonstrated that 1,3,7-/1,3,8-


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TrBDDs were below the detection limits in DecaBDE-containing plastics and were unformed

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duirng their thermal processes.41 These observations strongly support that 1,3,7-/1,3,8-TrBDDs

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detected in the mussels from Seto Inland Sea are marine natural products.

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On the other hand, 1,2,3,4,6,7,8-HpBDF was below the detection limits in all the mussel

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samples analyzed, despite being the most abundant congener in Japanese marine sediments.29,42

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Similar phenomena have been reported also in Swedish marine fish and shellfish.16 In fact, it is

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estimated that bioavailability of higher brominated dioxin congeners are low due to their large

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molecular size and strong affinity for organic particles.43

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3.1.3. PXDD/Fs. PXDDs were detected in all the mussel samples analyzed (230–28,000,

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median 5500 pg g-1 lw), whereas PXDFs were below the detection limits. Intriguingly, the

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geographical distirbution pattern of PXDDs was similar to that of PBDDs (Figure 2) and a

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significant positive correlation was observed between the levels of PXDDs and PBDDs in the

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mussel samples (Spearman's ρ = 0.85, p < 0.01) (Figure S1 (a)), implying that PXDDs are

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derived from similar sources to PBDDs.

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The highest level of PXDDs was found at EH-4 located in the southwestern Seto Inland Sea

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(Figure 2), and 7-MoB-2,3-DiCDD was the dominant identified congener accounting for 23% of

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the total PXDDs. The remaining 77% was composed of unknown PXDD homologues (Table S7),

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which could not be completely identified due to the lack of authentic standards, and they were

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semi-identified as MoBDiCDDs, MoBTrCDDs, DiBMoCDDs and DiBDiCDD by integrated

240

analyses of using GC–HRMS and GC×GC–HRToFMS (Figures S2 and S3, and Table S9).

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Among PXDD homologues, higher proportions of DiBMoCDDs, the full-scan mass spectra of

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which are illustrated in Figure S4, were found in all the mussels (46–100% of the total PXDDs),

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except the sample from EH-4 (14%). Similar geographical distribution patterns (data not shown)


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and a significant positive correlation (Spearman's ρ = 0.91, p < 0.01, Figure S1 (b)) were

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observed between the levels of DiBMoCDDs and 1,3,7-/1,3,8-TrBDDs in the mussel samples.

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Furthermore, it is noteworthy that DiBMoCDDs were detected in the brown alga sample

247

analyzed (Table S8). These results clearly suggest that DiBMoCDDs are naturally formed in

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Seto Inland Sea, as well as 1,3,7-/1,3,8-TrBDDs.

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A previous study reported the occurrence of DiBMoCDDs in sponge from the Baltic Sea,22 but

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their significant source and formation process remain unclear so far. Steen et al. (2009)18

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demonstrated in a laboratory experiment that ortho-hydroxylated PBDE or PXDE that are

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halogenated in at least one ortho position on the adjacent phenyl ring, such as 6-HO-BDE-47 and

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3Cl- or 5Cl-6-HO-BDE-47, were photochemically transformed to 1,3,7-TrBDD and their

254

potential TrBMoCDDs via intramolecular cyclization under natural sunlight (at 45°N latitude).

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In addition, a previous study verified the formation of TrBDDs and TeBDDs from naturally

256

abundant 2,4,6-tribromophenol via bromoperoxidase(BPO)-mediated coupling and secondary

257

debromination.17 In the proposed pathway, it has been shown that pentabrominated

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phenoxyphenol with a hydroxyl group on one ortho position is formed as an intermediate,17

259

indicating that TrBDD might be formed from natural ortho-hydroxylated PBDEs also by BPO-

260

mediated catalysis. In fact, a few research groups investigating the Baltic Sea area have

261

suggested that naturally abundant 6-HO-BDE-47 and 2’-HO-BDE-68, which are ortho-

262

hydroxylated PBDEs that are halogenated in one ortho position on the adjacent phenyl ring,

263

synthesized by marine organisms can act as precursors to the formation of 1,3,7- and 1,3,8-

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TrBDDs in surface marine waters or organisms.16,17,19,40 Considering these observations, to

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comprehend the sources and generating mechanisms of DiBMoCDDs present in Seto Island Sea,


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further analyses on occurrence and chemical structure of their potential precursor(s), HO-

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TrBMoCDE, are required.

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3.2. Screening of potential PXDD precursors. We conducted target screening and structural

269

analysis of HO-PXDEs in brown alga and blue mussel collected at the same sampling point (EH-

270

1 in Figure 1), using GC×GC–HRToFMS. As shown in Figure 3 and Table 2, peaks of six HO-

271

TrBDEs, two HO-TeBDEs, and one HO-TrBMoCDE were identified from the analyses of total

272

ion chromatogram of GC×GC (2D TIC), which are based on retention time, accurate mass,

273

isotope ratio of halogen cluster and fragment pattern. 6-HO-BDE-47 (Blob No. 8 in Figure 3),

274

2’-HO-BDE-68 (Blob No. 9), and HO-TrBMoCDE (Blob No. 6) that can be precursors of 1,3,7-

275

TrBDD, 1,3,8-TrBDD, and DiBMoCDD, respectively, were found in both of brown alga and

276

blue mussel. Intriguingly, the mass spectrum of HO-TrBMoCDE (after trimehtylsilylation;

277

TMSO-TrBMoCDE) detected in this study showed a quite similar pattern to that of 6-TMSO-

278

BDE-47 (trimehtylsilylation of authentic 6-HO-BDE-47 standard) (Figure 4). Previous studies

279

showed that the substitution position (ortho-, meta-, or para-position) of MeO- or TMSO-group

280

on the HO-PBDE derivative after methylation or trimehtylsilylation of a hydroxyl group is the

281

most important key factor to determine the intensity and profile of fragment ions.33,34 Thus the

282

similarity of fragment patterns between TMSO-TrBMoCDE and 6-TMSO-BDE-47 illustrated in

283

Figure 4 strongly suggests that the detected HO-TrBMoCDE substitutes a hydroxyl group on its

284

ortho position. Furthermore, it is noteworthy that a fragment ion of [M–CH3–C6H3Br]+ was

285

observed in the mass spectrum of TMSO-TrBMoCDE. This distinctive fragment ion represents

286

the cleavage of C–O bond in the diphenyl ether skeleton and conclusively proves that a chlorine

287

atom is substituted on the phenyl ring with a TMSO-group (Figure 4). Given the above


13


Page 14 of 32

288

observations, it is highly likely that HO-TrBMoCDE found in brown alga and blue mussel from

289

Seto Island Sea is 6-HO-TrBMoCDE-47 with the similar chemical structure to 6-HO-BDE-47.

290

3.3. Predicted formation pathway of DiBMoCDDs. As described earlier, it has been

291

suggested that natural PBDDs can be formed by two major pathways, biosynthesis in marine

292

organisms and photochemical transformation in surface seawater, via intramolecular cyclization

293

of their possible precursor HO-PBDEs.16,17,19,40 Considering that detection of not only HO-

294

PBDEs but also possible HO-PXDE peaks in red algae and blue mussels from the Baltic Sea by

295

GC-MS analyses have been previously reported,32 natural PXDDs might be inevitably generated

296

via intramolecular cyclization of HO-PXDEs in the marine environment. Assuming that 6-HO-

297

TrBMoCDE-47 semi-identified in this study using GC×GC–HRToFMS analyses is

298

intramolecularly cyclized by enzyme-mediated catalysis in marine organisms and/or

299

photochemical reaction in surface seawater, 1,7-DiB-3MoCDD and 3,7-DiB-1-MoCDD can be

300

theoretically formed in the marine environment (Figure 5). In fact, two potential peaks (pp1 and

301

pp2) of DiBMoCDDs were detected in brown alga and blue mussel from Seto Island Sea

302

analyzed in this study and their compositions (percentages of pp1 and pp2: 22% and 78% for

303

brown alga, 16% and 84% for blue mussel) were almost similar (Table S8), supporting the

304

possibility of natural DiBMoCDD formation. Intriguingly, concentrations of TrBDDs and

305

DiBMoCDDs in the blue mussel were one–two orders of magnitude higher than those in the

306

brown alga, whereas their possible HO-TeBDE and HO-TrBMoCDE precursors showed lower or

307

comparable levels in both of the samples (Table S8). This result clearly suggests the

308

bioaccumulation of TrBDDs and DiBMoCDDs.

309

3.4. Toxic implications. Toxic equivalents (TEQs) of 2,3,7,8-substituted PCDD/F congeners

310

in mussels from Seto Island Sea calculated based on the World Health Organization toxic


14

Page 15 of 32


311

equivalency factors (WHO-TEFs)44 were in the range of 6.6–310 pg TEQs g-1 lw (median: 31 pg

312

TEQs g-1 lw). TEQs of PBDD/Fs were also calculated using the WHO-TEFs corresponding to

313

their PCDD/F analogues,44 for the mussel samples from HR-4 (87 pg TeBDD-TEQ g-1 lw) and

314

HR-5 (35 pg TeBDF-TEQ g-1 lw), the only two samples with detectable concentrations of

315

2,3,7,8-substituted PBDD/Fs (Table S10). It is noteworthy that these TeBDD- and TeBDF-TEQ

316

values were higher than or comparable to PCDD/F-TEQs at each site. Although the sources of

317

TeBDD/Fs detected at HR-4 and -5 are unclear, it has been recently shown that relatively high

318

levels of 2,3,7,8-TeBDD/Fs were formed by thermal process of PBDE-containing plastics.41

319

Elucidation of local emission sources of these toxic PBDD/F congeners are required because

320

huge amounts of PBDE-containing products and their waste still remain in the terrestrial

321

environment.

322

Concentrations of 1,3,7-/1,3,8-TrBDDs and DiBMoCDDs detected in the mussel from HR-4

323

were 470 and 140 times higher than 2,3,7,8-TeBDD level, respectively (Table S6). High

324

accumulation of 1,3,7-/1,3,8-TrBDDs has been reported also in shellfish from the Baltic Sea.16,23

325

An in vivo study using zebrafish (Danio rerio) demonstrated that a PBDD mixture containing 1-

326

MoBDD,

327

/1,3,6,8-/1,3,7,9-TeBDDs, which have been detected in marine organisms from the Baltic Sea,

328

interacted with aryl hydrocarbon receptor, leading to dioxin-like responses.24 Given these

329

observations, risk assessment by non-2,3,7,8-substituted PBDD congeners that WHO-TEFs are

330

not established should be addressed for marine organisms.

2,7-/2,8-DiBDDs,

1,3,7-/1,3,8-/2,3,7-TrBDDs,

1,2,3,7-/1,2,3,8-/1,2,4,7-/1,2,4,8-

331

A recent study has suggested that seasonal variations in 1,3,7-/1,3,8-TrBDD levels observed in

332

blue mussels from the Baltic Sea might be attributed to the change in biomass of marine algal

333

species.45 Thus, human-induced climate change and eutrophication of coastal waters may lead to


15


Page 16 of 32

334

local overgrowth and activation of algal species and cyanobacteria which can synthesize PBDDs

335

and PXDDs. Because these naturally-produced dioxins may act as environmental stressor to

336

marine organisms, comprehensive studies on their generation process, bioaccumulation property

337

and ecotoxicological risk are required in the marine environment.

338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356


16

Page 17 of 32


357 358

Figure 1. Sampling locations of bivalves analyzed in this study. Closed circles: blue mussels

359

(Mytilus galloprovincialis), open cricles: green mussels (Perna viridis). Brown algae (Sargassum

360

sp.) were collected at EH-1 together with blue mussels.

361 362 363 364 365 366 367


17

PCDD/Fs (pg g -1 lw)


40000 30000

Page 18 of 32

PCDDs PCDFs

20000 10000 0

PBDD/Fs (pg g -1 lw)

50000 40000

PBDDs PBDFs

30000 20000 10000 0

PXDD/Fs (pg g-1 lw)

30000 20000

PXDDs PXDFs

10000 0

368

YM-1 HR-1 HR-2 HR-3 HR-4 HR-5 EH-1 EH-2 EH-3 EH-4 EH-5 EH-6 OK-1 OK-2 HY-1 HY-2

369

Figure 2. Concentrations of PCDD/Fs, PBDD/Fs and PXDD/Fs (pg g-1 lw) in mussels collected

370

at 16 locations of Seto Inland Sea.

371 372 373 374 375 376 377


18

Page 19 of 32


378 379

Figure 3. Two-dimensional total ion chromatogram (2D TIC) in phenolic fractions (after

380

trimethylsilylation) of brown alga and blue mussel samples collected at EH-1 from Seto Inland

381

Sea, Japan. Detailed information on each blob (assigned peak) is shown in Table 2.

382 383 384 385 386 387 388 389 390 391


19


Page 20 of 32

392 393

Figure 4. Full-scan mass spectra of HO-TrBMoCDE and 6-HO-BDE-47 analyzed using

394

GC×GC–HRToFMS. (a) brown alga, (b) blue mussel, (c) authentic standard trimetylsilylated by

395

BSTFA/TCMS (99: 1 v/v).

396 397 398


20

Page 21 of 32


Br

OH

Br O

O Br

OH

Cl

Br

Br

Br

Cl

Intramolecular cyclization by BPO16,17 and/or hν18,19

Cl

Br

O

399

Br

O

O Br

Br

O

Cl

400 401

Figure 5. Predicted structures and formation pathways of DiBMoCDDs detected in the brown

402

alga and blue mussel based on the previous studies on photochemical (hν)18,19

403

bromoperoxidase(BPO)-mediated16,17 formation of PBDD/PXDDs.

and

404 405 406 407 408 409 410 411 412 413 414 415 416 417


21


Page 22 of 32

418

Table 1. Concentrations of PCDD/Fs, PBDD/Fs and PXDD/Fs in mussels from Seto Inland Sea,

419

Japan Location ID YM-1 HR-1 HR-2 HR-3 HR-4 HR-5 EH-1 EH-2 EH-3 EH-4 EH-5 EH-6 OK-1 OK-2 HY-1 HY-2

Pool n 50 50 30 30 50 125 19 30 27 19 2 24 38 150 65 160

Shell length (mm) 50–70 50–70 50–70 50–70 50–70 30–50 50–70 50–70 50–70 50–70 70–90 50–70 50–70 30–50 30–50 30–50

Moisture (%) 77 74 72 74 75 77 83 77 79 77 75 80 81 81 88 87

Lipid (%) 0.99 1.3 1.2 1.3 1.0 0.78 0.86 1.0 0.57 0.99 1.7 0.82 0.41 0.28 0.35 0.43

PCDDs 1500 860 1400 2800 2700 3000 3400 2500 3000 6200 800 21000 8900 16000 28000 24000

PCDFs 950 400 270 730 1500 1300 560 1700 340 1000 370 4400 1900 1700 3900 6800

Concentrations (pg g-1 lw) PBDDs PBDFs 10000 ND 16000 ND 19000 ND 25000 ND 46000 ND 3300 35 16000 ND 39000 29 29000 ND 11000 ND 2400 ND 11000 30 4700 ND 1400 ND 640 50 750 880

PXDDs 3300 5900 6000 6700 13000 1500 5100 20000 13000 28000 2400 8300 1400 980 230 230

PXDFs ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND

420 421

ND: not detected.

422 423 424 425 426 427 428 429


22

Page 23 of 32


430

Table 2. Identified OH-PBDE and OH-PXDE peaks on 2D TIC of brown alga and mussel

431

samples analyzed using GC×GC–HRToFMS

Specimen Brown alga

Blue mussel

1

Blob No.

tR (min)

2

tR (sec)

Accurate mass

Exact mass

Mass error

Possible

Compound name

(m/z)

(m/z)

(ppm)

formula

(underivatized)

1

19.52

2.30

495.83347

495.83519

-3.5

C15H15Br3O2Si

HO-TrBDE

2

20.10

2.34

495.83951

495.83519

8.7

C15H15Br3O2Si

HO-TrBDE

3

20.27

2.18

495.84002

495.83519

9.7

C15H15Br3O2Si

HO-TrBDE

4

20.93

2.30

495.83331

495.83519

-3.8

C15H15Br3O2Si

HO-TrBDE

5

20.93

2.46

495.84018

495.83519

10

C15H15Br3O2Si

HO-TrBDE

6

22.43

2.38

529.79607

529.79598

0.17

C15H15Br2ClO2Si

HO-TrBMoCDE

7

23.35

2.50

495.83822

495.83519

6.1

C15H15Br3O2Si

HO-TrBDE

573.74878

573.74566

5.4

C15H14Br4O2Si

6-HO-BDE-47

585.78901

585.78582

5.4

C15H14Br4O2Si

8

24.02

2.66

9

25.02

2.70

573.74082

573.74566

-8.4

C15H14Br4O2Si

10

25.27

3.73

527.76514

527.76196

6.0

C15H14Br4O2Si

6

22.43

2.38

529.79878

529.79598

5.3

C15H15Br2ClO2Si

7

23.35

2.46

495.83657

495.83519

2.8

C15H15Br3O2Si

HO-TrBDE

573.74301

573.74566

-4.6

C15H14Br4O2Si

6-HO-BDE-47

585.77977

585.78582

-10

C15H14Br4O2Si

8

24.02

2.62

9

25.02

2.70

573.74859

573.74566

5.1

C15H14Br4O2Si

10

25.27

3.73

527.75852

527.76196

-6.5

C15H14Br4O2Si

13

C12-6-HO-BDE-47 2'-HO-BDE-68

13

C12-6-MeO-BDE-47 HO-TrBMoCDE

13

C12-6-HO-BDE-47 2'-HO-BDE-68

13

C12-6-MeO-BDE-47

432 433 434 435 436 437 438 439 440 441


23


Page 24 of 32

442

ASSOCIATED CONTENT

443

Supporting Information

444

The Supporting data contains 20 pages, with 10 Tables and 4 Figures, and is available free of

445

charge via the Internet at http://pubs.acs.org/.

446 447

AUTHOR INFORMATION

448

Corresponding Author

449

*Phone: +81-89-927-8162; e-mail: [email protected]

450

Notes

451

The authors declare no competing financial interest.

452 453

ACKNOWLEDGMENTS

454

We thank H. Onishi and M. Muto for their cooperation to sample collection. This study was

455

supported by Grants-in-Aid for Scientific Research (A) (No. 16H01784) and (B) (No.

456

16H02963) from Japan Society for the Promotion of Science (JSPS) and by the Ministry of

457

Education, Culture, Sports, Science and Technology, Japan (MEXT) to a project on Joint

458

Usage/Research Center – Leading Academia in Marine and Environment Pollution Research

459

(LaMer).

460 461

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Occurrence of Natural Mixed Halogenated Dibenzo-p-Dioxins

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