2,5,6,9,10-Pentabromocyclododecanols (PBCDOHs): A new class of HBCD transformation products

Chemosphere 88 (2012) 655–662

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2,5,6,9,10-Pentabromocyclododecanols (PBCDOHs): A new class of HBCD transformation products Norbert V. Heeb a,⇑, Daniel Zindel a,b, W. Bernd Schweizer c, Peter Lienemann b a

Swiss Federal Institute for Materials Science and Technology (Empa), Laboratory of Analytical Chemistry, Überlandstrasse 129, CH-8600 Dübendorf, Switzerland Zurich University of Applied Sciences, Institute of Chemistry and Biological Chemistry, Reidbach, 8820 Wädenswil, Switzerland c Swiss Federal Institute of Technology (ETH), Laboratory for Organic Chemistry, Hönggerberg, 8093 Zürich, Switzerland b

a r t i c l e

i n f o

Article history: Received 23 December 2011 Received in revised form 14 March 2012 Accepted 15 March 2012 Available online 20 April 2012 Keywords: Brominated flame retardants HBCD transformation products Crystal structure Stereoisomers HBCDD

a b s t r a c t Pentabromocyclododecanols (PBCDOHs) are potential environmental transformation products of hexa bromocyclododecanes (HBCDs). They are also potential stage one metabolites of biological HBCD transformations. Herein, we present analytical evidence that PBCDOHs are also constituents of technical HBCDs and flame-proofed polystyrenes (FP-PSs). PBCDOHs are possibly formed during the synthesis of technical HBCD, presumably during the bromination of cyclododecatrienes in aqueous isobutanol together with isobutoxypentabromocyclododecanes (iBPBCDs), which have been identified in these materials recently. Of the 64 stereoisomers possible, eight pairs of enantiomers, named a-, b-, c-, d-, e-, f-, g-, and h-PBCDOHs were separated with a combination of normal-, reversed- and chiral-phase LC. Crystal structure analysis revealed the stereochemistry of the a-PBCDOH pair of enantiomers, which was assigned to (1S,2S,5R,6S,9S,10R)-2,5,6,9,10-pentabromocyclododecanol and its enantiomer. Mass spectrometric data are in accordance with the expected isotope patterns. On a C18-RP-column, the polar PBCDOHs eluted before the HBCD and iBPBCD classes of compounds. PBCDOHs were also found in FP-PS materials. The stereoisomer patterns varied considerably in these materials like those of HBCDs and iBPBCDs. Expanded polystyrenes were rich in late-eluting stereoisomers, similar to technical HBCD mixtures. Extruded polystyrenes contained more of the polar, faster-eluting isomers. The presented chromatographic and analytical methods allow a stereoisomer-specific search for PBCDOHs in biota samples, which might have experienced metabolic HBCD transformation reactions. Besides this potential source, it has to be recognized that PBCDOHs are by-products in technical HBCDs and in flame-proofed polystyrenes. Therefore, it is likely that PBCDOHs and iBPBCDs are released to the environment together with HBCD-containing plastic materials. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Polystyrenes (PSs) are high production volume chemicals produced at more than 10 million t y 1 (Wünsch, 2000). They are used in form of expanded, low-density polystyrenes (EPSs) extruded, high-density polystyrenes (XPSs) and some other specialty materials such as styrene–butadiene and acrylonitrile–butadiene–styrene copolymers (ABS). These light-weight polymers are widely applied as insulation materials in the construction sector. For safety reasons, polystyrenes are flame-proofed for these applications. Hexa bromocyclododecanes are currently the most important flame retardants for this purpose (Alaee et al., 2003). HBCDs are mixed with the polymer material but are not covalently bound to it like tetrabromobisphenol, a reactive brominated flame retardant,

⇑ Corresponding author. Tel.: +41 58 765 4257; fax: +41 58 765 4614. E-mail address: [email protected] (N.V. Heeb). URL: http://www.empa.ch (N.V. Heeb). 0045-6535/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemosphere.2012.03.052

which is integrated into the polymer chain. Therefore, it is not unlikely that HBCDs may escape from such materials. HBCDs are now ubiquitous environmental pollutants and their widespread occurrence has been documented on local, regional, and global scales (de Wit, 2002; Remberger et al., 2004; de Wit et al., 2006; Law et al., 2006; Covaci et al., 2006). HBCDs are considered as persistent (P), bioaccumulating (B) and toxic (T) and are therefore included in the PBT-list of the European Chemicals Bureau (ECB, 2010). HBCDs have been identified in humans (Weiss et al., 2004; Thomsen et al., 2005, 2010). They are frequently found in wildlife samples (Law et al., 2003; Sellström et al., 2003; Gerecke et al., 2003; Lindberg et al., 2004; Tomy et al., 2004; Peck et al., 2008) and in environmental compartments such as lake-, river-, and marine-sediments (Sellström et al., 1998; Marvin et al., 2006; Kohler et al., 2008), even in remote areas (Bogdal et al., 2010). Their properties have been reviewed thoroughly by the Persistent Organic Pollutant Review Committee of the Stockholm Convention and a decision is expected by the Conference of Parties in May 2012.

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Transport of HBCDs into the environment is still a matter of debate. They may be released directly from industrial plants, where HBCDs and HBCD-containing polymers are produced and processed. The widespread use of these materials and subsequent degradation and disintegration is another potential HBCD source. The role of plastic debris as a carrier for such compounds in the aquatic environment has also been recognized (Moore et al., 2001; Derraik, 2002; Thompson et al., 2004; Browne et al., 2008). Any time along this transport, transformation of HBCDs may occur. Early occasions are the industrial HBCD synthesis itself and the production and processing of HBCD-containing plastic materials. It was reported that technical HBCD mixtures also contain isobutoxypentabromocyclododecanes, which are possibly formed during the bromination of cyclododecatrienes in aqueous isobutanol (Heeb et al., 2010a). iBPBCDs were also identified in various flameproofed polystyrenes together with HBCDs (Heeb et al., 2010b). Their patterns vary substantially for EPS and XPS. We showed that various isomerization reactions occur during thermal treatment of such materials (Heeb et al., 2008a,b, 2011). These isomerizations are HBCD transformation reactions too. They are fast, in the order of minutes, at temperatures above 110 °C, indicating that HBCDs and structurally-related compounds react at elevated temperatures. It is not clear yet, if such isomerizations are also possible at lower temperatures, e.g., under abiotic environmental conditions. We hypothesized that hydroxylation reactions may also occur during industrial synthesis of HBCDs forming PBCDOHs. Analytical evidence is now presented that PBCDOHs indeed are constituents of technical HBCD mixtures and flame-proofed polystyrenes. But PBCDOHs are also potential environmental transformation products formed by metabolism of HBCDs. Hydroxylated HBCD metabolites have been observed lately (Brandsma et al., 2009; Esslinger et al., 2011). The findings presented here show that PBCDOHs are present in technical HBCD mixtures and may be released from flame-retarded plastic materials.

water. Aliquots (60 ll) of filtered solutions were separated on a permethylated-b-cyclodextrin LC column (PM-b-CP, 200 mm  4 mm, 5 lm, 100 Å, Nucleodex 5, Macherey-Nagel) with acetonitrile/water under isocratic conditions (48% acetonitrile). Fractions containing a-PBCDOH enantiomers were combined and concentrated at 50 °C in a stream of nitrogen. Upon cooling, crystallization occurred. The recovered material was suitable for X-ray diffraction analysis. 2.2.3. Preparation of polystyrene samples Aliquots of the polystyrene samples were dissolved in dichloromethane and concentrated to dryness. Acetonitrile was added to the residues resulting in suspensions of polystyrene materials and clear supernatants. Aliquots of the supernatants were concentrated to dryness and dissolved in methanol/water (80/20) for LCMS analysis. 2.3. Chromatographic characterization Separation of different PBCDOHs was succeeded by a combination of normal- (SiO2, F60, 230–400 mesh), reversed- and chiral-phase liquid chromatography (Spectra System P4000, Thermo Separation Products, San Jose, CA, USA). A C18-reversed-phase column (C18-RP, 125 mm  4 mm, 5 lm, 100 Å, Nucleosil 100–5, Macherey-Nagel, Oensingen, Switzerland) with a methanol–water gradient (80% methanol for 5 min, 80–98% in 15 min, 98% for 4 min) at a flow rate of 1 ml min 1 was used to separate diastereoisomers. Separation of enantiomers was achieved with a permethylated-b-cyclodextrin chiral-phase column (PM-b-CP, 200 mm  4 mm, 5 lm, 100 Å, Nucleodex 5, Macherey-Nagel) at a flow rate of 0.9 ml min 1 with a methanol/water gradient (75–85% methanol in 20 min, 85–98% in 10 min, 98% for 3 min). Samples were dissolved in methanol/water (80:20) and injected at aliquots of 20 ll. Identical chromatographic conditions were used to separate PBCDOHs, iBPBCDs and HBCDs. Therefore, respective data sets are comparable and can be used as reference (Heeb et al., 2010a,b).

2. Materials and methods

2.4. Mass spectrometry and X-ray diffraction analysis

2.1. Materials

Mass spectrometric analysis of the LC-effluents was performed on a triple stage quadrupole mass spectrometer (TSQ 7000, Thermo Finnigan, San Jose, CA, USA). Sensitivity was higher with atmospheric pressure chemical ionization (APCI) than with electrospray ionization. PBCDOHs were detected in selective ion monitoring mode (SIM), recording prominent anions of the chloride adduct cluster [M + Cl] at m/z 612.7, 614.7 and 616.7 at a corona current of 5 lA, an octapol potential of 0 V and heated capillary and vaporizer temperatures of 150 and 400 °C, respectively. A limit of detection at a signal-to-noise ratio s/n = 3 of 0.2 ng (on column) was determined for a-PBCDOH standards. The crystal structure measurement and data reduction was performed on a Bruker APEX-II Duo single crystal diffractometer with Mo-Ka radiation (k = 0.071073 nm) equipped with a graphite monochromator (Bruker, 2010). Atomic coordinates and crystallographic details have been deposited with the Cambridge Crystallographic Data Center, deposition number CCDC 858571.

Individual PBCDOH diastereoisomers were isolated from a lowmelting, technical grade HBCD mixture (Saytex HP-900Ò, mp = 168–184 °C) by normal phase liquid chromatography (LC) on silica 60 (Merck, Darmstadt, Germany) with mixtures of n-hexane (Merck) and dichloromethane (Merck). HPLC-grade methanol (ROMIL, Cambridge, UK), acetonitrile (ROMIL), and water (Merck) were used for reversed- and chiral-phase LC. The examined plastic materials, a low-density EPS board (q = 19 kg m 3) and a high-density XPS board (q = 48 kg m 3) were obtained from different construction sites. 2.2. Sample purification and crystal growth 2.2.1. Isolation of diastereoisomers A dichloromethane solution of a technical HBCD mixture was adsorbed on activated silica (SiO2, F60, 230–400 mesh). The suspension was homogenized, dried with a stream of nitrogen, and loaded on activated silica and fractionated by normal-phase LC with n-hexane and various n-hexane/dichloromethane mixtures (Supplementary material). Sampled fractions were investigated with LC-MS for the presence of PBCDOHs. 2.2.2. Isolation of enantiomers and crystal growth Fractions containing identical PBCDOH diastereoisomers were combined, concentrated to dryness, and dissolved in acetonitrile/

3. Results and discussion 3.1. Stereochemistry of PBCDOHs and their structural relation to HBCDs PBCDOHs like iBPBCDs can be considered as first-generation transformation products of HBCDs resulting from a substitution of one of the six bromine atoms with a hydroxy- or an isobutoxy-group. Because all three classes of compounds are found in

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technical-grade mixtures, a common formation mechanism is assumed and it is worthwhile to discuss the structural relations among HBCDs, PBCDOHs, and iBPBCDs in more detail. Figure S1 (Supplementary material) displays a schema of all 2,5,6,9,10-pentabromocyclododecanols possible. These PBCDOHs as well as the related isobutyl-ethers are all substituted at positions 1,2,5,6,9,10 and therefore derivatives of 1,2,5,6,9,10-HBCDs. The numbering scheme introduced previously for iBPBCDs (Heeb et al., 2010a), is also applicable for PBCDOHs. With six stereogenic centers, 64 stereoisomers (2n, n = 6), or in other words, 32 diastereomeric pairs of enantiomers (1a/b–32a/b) have to be expected. Individual stereoisomers are distinguished according to the R/S nomenclature. Substituents can either be cis or trans with respect to the cyclododecane ring. The relative configurations of substituents in 1,2- (C/T) and 1,4-distance (c/t) are also distinguished and mirror planes (dashed lines) separate both enantiomers (Fig. S1). Fig. 1 shows the relation of three generations of cyclododecane derivatives. The technical synthesis of HBCDs is achieved via bromination of 1,5,9-cyclododecatrienes, which are produced from cyclotrimerization of butadiene. Technical-grade cyclododecatriene mixtures consist of the CTT and some TTT-isomers. As a consequence, technical-grade HBCDs mainly contain stereoisomers with configurations VI to X (Fig. 1. red, black and blue branches). It was claimed that the HBCD synthesis in aqueous isobutanol is favorable with respect to product yield (Ransford, 1991). However, the presence of various isobutoxy- and hydroxy-derivatives in technical-grade HBCDs also proves that by-products are formed during this process. All crystal structures obtained so far, show that the respective compounds indeed are 1,2,5,6,9,10-substituted cyclododecanes (Heeb et al., 2005, 2007a,b, 2010a, 2011). Even though 10 different relative configurations (I–X) are possible, only stereoisomers with

13a/b 12a/b 11a/b 10a/b 9a/b 8a/b 7a/b 6a/b

14a/b

15a/b

16a/b

configurations VI–X were found (Fig. 1). Stereoisomers 17a/b–19a/ b (Fig. S1) with a CtCcTc-configuration (VI) are related to a-HBCDs, compounds 20a/b–25a/b with CcCcTt- (VII) and 26a/b–28a/b with CtCtTt-configurations (VIII) are related to b- and c-HBCDs, respectively. Stereoisomers 29a/b–31a/b with CcCtCt- (IX) and 32a/b with CcCcCc-configurations (X) are related to d- and e-HBCDs. So far, 8 pairs of PBCDOH enantiomers were separated by normal-, reversed- and chiral-phase LC. We hypothesize that they have configurations similar to the hitherto identified HBCDs and iBPBCDs. In other words, the PBCDOHs found in such materials possibly belong to families VI–X rather than to I–V (Fig. 1, gray branches). The CtCtTt configuration (Fig. 1, VIII, blue branch) is most prominent in technical HBCD mixtures. Both c-enantiomers dominate the HBCD fraction (Heeb et al., 2005) and the d- and estereoisomers (26a/b and 28a/b, Fig. S1) are most prominent in iBPBCD class of compounds (Heeb et al., 2010a, 2011). Assuming a common formation mechanism, it is likely that the most prominent PBCDOHs also belong to CtCtTt-family (VIII). In conclusion, because HBCDs, PBCDOHs and iBPBCDs are formed under the same conditions, it is plausible that similar stereoisomers are obtained. This might facilitate the structure elucidation of individual stereoisomers to some degree. However, the PBCDOH and iBPBCD classes of compounds are more diverse (n 6 64) than the HBCDs (n 6 16) and the structure elucidation of all stereoisomers remains a considerable challenge. 3.2. Chromatographic patterns of PBCDOHs A combined approach using normal-, reversed- and chiralphase LC was necessary to separate the various PBCDOH stereoisomers. Fig. 2 displays chromatographic pattern of PBCDOHs as

17a/b

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5a/b

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30a/b

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Fig. 1. Structural relationship of HBCDs and their first-generation transformation products PBCDOHs and iBPBCDs. The 64 stereoisomers, consisting of 32 diastereomeric pairs of enantiomers, can be classified in 10 families (I–X) with different configurations. All compounds are related to the four 1,5,9-cyclododecatriene isomers and are derivatives of cyclododecane. Kinetically- (VIII, CtCtTt, blue branches) and thermodynamically-favored (VI, CtCcTc, red branches) stereoisomers are distinguished. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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found in technical HBCDs and some fractions their-of. Fig. S2 (supplementary material) presents other fractions containing all assigned diastereoisomers and the respective chromatographic conditions are indicated. Stereoisomers were partially separated on C18 reversed- (RP, left) and permethylated-b-cyclodextrin chiral-phase LC columns (CP, right). From these samples, eight diastereoisomers named a-, b-, c-, d-, e-, f-, g-, and h-PBCDOHs were distinguished so far (Fig. S2). Table S1 reports absolute and relative retention times of these stereoisomers and their proportions in technical-grade HBCD. e-PBCDOHs were most abundant with a proportion of 50%, whereas a-, f-, and g-PBCDOHs accounted for 13%, 10%, and 14%, respectively, and b-, c-, d-, and h-PBCDOHs were minor constituents of 4%, 1%, 4%, and 3%. Proportions were estimated assuming identical mass spectrometric response of all stereoisomers. The chromatographic patterns obtained on the CP column (Fig. 2, right) are even more complex with several co-eluting compounds. PBCDOHs could be isolated from HBCDs by fractionating using normal phase chromatography. Furthermore, normal-phase LC allowed the separation of c-, d-, e-, f-, and g-PBCDOHs which elute in about 1 min from the RP-column (Table S1, Fig. S2). Chromatograms of some of these PBCDOH-containing fractions are shown in Figs. 2 and S2. Fraction A predominantly contains a-PBCDOHs, which elute as two enantiomers a1 and a2 from the CP column (Fig. 2, right). Retention times of 0.35 and 0.44 relative to (-)cHBCD were obtained (Table S1). Smaller proportions of both aPBCDOHs were also found in other fractions. Fractions B and C mainly contain e-PBCDOHs, which elute as two enantiomers e1 and e2 from the CP column (r.r.t = 0.60 and 0.93). d-PBCDOHs are also found in fraction B, eluting as two enantiomers d1 and d2 from the CP column (r.r.t = 0.63 and 0.74). Two additional pairs of enantiomers are found in fraction D. They were assigned to f- and gPBCDOHs, with the f1-enantiomer eluting first (r.r.t = 0.38) from the CP column and the g2-enantiomer last (r.r.t = 0.63), whereas the f2- and g1-enantiomers eluted in between, at r.r.t of 0.50 and 0.42. Table S1 (Supplementary material) reports absolute and relative retention times of these PBCDOHs and relates them to (-)c-HBCD, the last-eluting isomer. Relative retention times of other HBCDs and iBPBCDs have been reported before (Heeb et al., 2005, 2010a,b). This chromatographic data set can now be used as reference to compare PBCDOH patterns of other plastic materials and of environmental and biota samples. The 16 stereoisomers identified so far represent only 1/4 of all possible PBCDOHs. Therefore, it is likely that other co-eluting stereoisomers are present in these materials and chromatographic separation has to be improved to distinguish among all 64 stereoisomers. 3.3. Spectroscopic properties of PBCDOHs Polybrominated alicyclic compounds such as HBCDs have been identified by mass spectrometry based on their characteristic isotope clusters. The formation of halide-adduct anions such as chloride[M + Cl] and bromide-adducts [M + Br] are frequently observed for these compounds under APCI conditions. As discussed above, the various PBCDOHs can be separated from HBCDs and iBPBCDs under the given RP conditions. Therefore, interferences can be excluded. Fig. S3 displays mass-to-charge traces of the most abundant chlorideadduct ions [M + Cl] at m/z 612.7 (C12H1979Br381Br235ClO, 89.7%), 614.7 (C12H1979Br281Br335ClO, 100%), and 616.7 (C12H1979Br181Br435ClO, 61.2%) for PBCDOHs in technical-grade HBCD. The observed relative intensities of 93.5%, 100% and 65.8% are in accordance with the proposed chemical composition. Several attempts were made to obtain pure materials suitable for single crystal analysis by X-ray diffraction. Finally, colorless, cube-like crystals were obtained from acetonitrile/water solutions. From a suitable crystal of 0.14 mm  0.04 mm  0.008 mm size

23 450 (7589 unique) reflexions were measured in the orthorhombic space group P212121, with cell parameters a = 1.24197(9) nm, b = 1.3623(1) nm, c = 1.9754(2) nm, V = 3.3422(5) nm3, z = 8. The structure was solved by direct methods (Sheldrick, 2008) and refined by full matrix least squares. All non-H atoms were refined anisotropically and constrained H-positions were included in the structure factor calculation (Sheldrick, 2008; Dolomanov et al., 2009). The crystal shows some twinning with separation of the axis by about 1.5°. Final R-values R = 0.059 for 5616 reflexions with I > 2r(I) and R = 0.100 for all 7589 reflexions and an absolute structure parameter of 0.02(3) were determined (Flack, 1983). Fig. 3 displays the solid-state structure of the early eluting a1PBCDOH enantiomer, which was determined as (1S,2S,5R,6S,9S,10R)2,5,6,9,10-pentabromocyclododecanol. This stereoisomer corresponds to structure 17b, its enantiomer is assigned to structure 17a (Fig. S1). The six substituents of a-PBCDOHs (17a/b) have the CtCcTc configuration (VI), like a-HBCDs (Fig. 1 red branch). The solid state conformation of a1-PBCDOH is very similar to the one of racemic a-HBCDs (Heeb et al., 2005). In both crystals, square-like cyclododecane ring conformations are adopted with four equally-oriented turns (Fig. 3). This conformation was also found in cyclododecane at lower temperatures (Dunitz and Shearer, 1960). Additional elements of symmetry are present in these conformations. In case of cyclododecane, one fourfold- and four twofold-rotation axes are found. In case of a-HBCDs, one twofoldrotation axis is present, resulting in a C2-symmetry. With respect to the cyclododecane ring conformation, these symmetry elements can also be found in the a1-PBCDOH structure. 3.4. Patterns in flame-proofed polystyrenes Fig. 4 compares diastereoisomer patterns of PBCDOHs, HBCDs and iBPBCDs as found in technical-grade HBCD, flame-proofed expanded (EPS) and extruded polystyrenes (XPSs). The given chromatographic conditions allow a separation of the three classes of compounds, but not a baseline separation of all individual diastereoisomers. In accordance to their polarity, PBCDOHs elute first (500–750 s), followed by HBCDs (870–1050 s) and iBPBCDs (1050–1250 s) from a C18-RP column. This elution order can be expected due to the extra hydroxy- or isobutoxy-group. However, on the CP column, these classes of compounds are only partially separated resulting in several co-eluting compounds. The diastereoisomer patterns of technical-grade HBCD (Fig. 4, top) and FP-EPS (middle) are similar for the three classes of compounds, but differ considerably from those of the XPS sample (bottom). In all cases, more polar and faster-eluting isomers were enriched in the XPS sample. Current data indicate that those isomers with a CtCtTt configuration (Fig. 1, VIII, blue branches) are kinetically-favored and dominate in technical HBCD mixtures and FP-EPS. On the other hand, the thermodynamically-favored stereoisomers with a CtCcTc configuration (Fig. 1, VI, red branches) elute earlier and dominate in FP-XPS samples. A preferential migration of vicinal bromine atoms in like configurations (RR and SS) explains the observed isomerizations from CtCtTt- to CtCcTc-configurations (Heeb et al., 2008a,b, 2010b). With two C-Br bonds breaking and reforming, such isomerizations have to be considered as HBCD transformation reactions too. They can occur during the thermal treatment of flame-proofed materials, e.g., in an extruder, during the industrial XPS production. Albeit the accurate quantification of PBCDOH levels is currently not possible, one can notice substantial variations of the stereoisomer patterns in different materials. e-PBCDOHs account for about 50% and 42% in technical HBCD and FP-EPS, whereas a-PBCDOHs dominate with 40% in FP-XPS. Similarly, c-HBCD proportions of 60% and 70% were found in technical HBCD and FP-EPS, but aHBCDs dominate with 40% in FP-XPS. Accordingly, the later-eluting d-, g-, and h-iBPBCDs were most abundant in technical HBCD and

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PM-β-CP (chiral)

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Fig. 2. Chromatograms of PBCDOH stereoisomers as found in a technical grade HBCD mixture and some fractions there-of. So far, eight pairs of enantiomers were separable with a combination of normal-, reversed- and chiral-phase chromatography. A C18-RP column (left, 125 mm  4 mm, 5 lm, 100 Å, Nucleosil 100–5, 80% methanol, 5 min, 80– 98%, 15 min, 98%, 4 min) and a PM-b-cyclodextrine-CP column (right, 200 mm  4 mm, 5 lm, 100 Å, Nucleodex 5, 75–85% methanol, 20 min, 85–98%, 10 min, 98%, 3 min) were used. Different diastereoisomers were named according to their retention time on the C18-RP column. Additional chromatographic data is also given in Fig. S2 and Table S1 (Supplementary material).

EPS accounting for about 70%, but proportions of the early-eluting a-, b-, e-, and f-iBPBCDs increased to about 80% in XPS (Fig. 4). In summary, patterns and levels of individual stereoisomers vary to a large extent for different flame-proofed materials. A thermal treatment may further alter these patterns. But stereoisomer patterns may also change during environmental transport. Depending on the physicochemical properties of individual stereoisomers such as water solubilities and octanol/water partioning coefficients and their chemical reactivities, patterns in different environmental compartments are expected to further change. The interaction with biota may also affect ratios of diastereoisomers and enantiomers in these classes of compounds. Such pattern

variations have been observed for HBCDs at various occasions (Gerecke et al., 2003; Tomy et al., 2004; Janak et al., 2005; Law et al., 2005; Covaci et al., 2006). Nevertheless, close to point sources, pattern changes are also related to the amounts of individual stereoisomers released. In other words, if the amounts of released EPS and XPS change over time, respective stereoisomer patterns have to follow accordingly. 4. Conclusions Flame-proofed polystyrenes contain variable amounts of different HBCD stereoisomers. They also include small amounts

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Fig. 3. Crystal structure of the early-eluting a1-PBCDOH stereoisomer 17b (left), which was assigned as (1S,2S,5R,6S,9S,10R)-2,5,6,9,10-pentabromocyclododecanol. Its structure is related to (+)a-HBCD, which has the same configuration and a similar square-like conformation (right).

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Time (s)

Fig. 4. Chromatographic patterns of PBCDOHs (left), HBCDs (middle), and iBPBCDs (right) as found in a technical-grade HBCD mixture and in flame-proofed expanded and extruded polystyrenes from a C18-RP LC column (125 mm  4 mm, 5 lm, 100 Å, Nucleosil 100–5, 80% methanol, 5 min, 80–98%, 15 min, 98%, 4 min).

of hydroxy- and isobutoxy-pentabromocyclododecanes. With a combination of normal-, reversed- and chiral-phase LC, we could distinguish 16 PBCDOH and 16 iBPBCD stereoisomers. All identified compounds are substituted at positions 1,2,5,6,9,10 and adopt similar configurations and conformations. They are comparably reactive at temperatures above 110 °C. Despite these similarities, some of their physicochemical properties such as polarity, solubility and partitioning between water, air and hydrophobic phases may be different. Therefore, also the environmental distribution, persistence and bioaccumulation potential of PBCDOHs and iBPBCDs may differ from their parent compounds.

It is assumed that PBCDOHs and iBPBCDs are formed during the industrial HBCD synthesis, most probably during the bromination of 1,5,9-cyclododecatrienes in aqueous isobutanol, a process which is claimed to be favorable with respect to HBCD yield (Ransford, 1991). Interestingly, the stereoisomer patterns of these classes of compounds are related. Higher proportions of less polar stereoisomers are found in technical HBCDs and expanded polystyrenes. These kinetically-favored stereoisomers can rearrange to more polar stereoisomers under thermodynamic control. PBCDOHs are also potential HBCD metabolites, which may form in vivo via enzyme-mediated reactions. Brandsma et al., 2009

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presented mass spectrometry data indicating that several pentabromocyclododecenols and hexabromocyclododecanols are present in liver tissue of HBCD-exposed wistar rats. Esslinger et al., 2011 confirmed that cytochrome P450-dependent liver enzymes oxidize HBCDs to hydroxylated HBCDs, but no indications were found for a formation of PBCDOHs. Furthermore, PBCDOHs are also potential environmental HBCD transformation products, possibly formed via hydrolysis in aquatic environments. We have shown here that PBCDOHs are also impurities in technical HBCD mixtures and flame-proofed plastic materials and it is very likely that they are released together with HBCD-containing materials. It will be an analytical challenge to distinguish PBCDOHs that are either formed from abiotic processes during environmental transport of HBCDs or in biota from metabolic transformations or originate from primary sources. The presented analytical methods allow a more specific search for PBCDOHs in various matrices but more research is needed to elucidate the stereochemistry of other prominent stereoisomers. Currently, a combined approach with normal-, reversed- and chiral-phase LC allows the separation of various stereoisomers but improved chromatography may simplify this procedure. Furthermore, reliable analytical standards are needed to quantify PBCDOHs in plastic materials, environmental samples and in biota. In addition, the persistence, bioaccumulation potential and the toxicity of PBCDOHs have to be addressed too. The impact of these first-generation transformation products on human health and the environment should also be assessed to complete the risk assessment of HBCDs and their by-products. Acknowledgments This work was part of a bachelor’s thesis performed at Empa and was supported by the Zurich University of Applied Sciences, Wädenswil. We also acknowledge helpful discussions with J. Tremp, BAFU and the financial support. Furthermore, we thank V. Azara for her help during the PBCDOH fractionation. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemosphere. 2012.03.052. References Alaee, M., Arias, P., Sjödin, A., Bergman, A., 2003. An overview of commercially used brominated flame retardants, their applications, their use patterns in different countries/regions and possible modes of release. Environ. Int. 29, 683–689. Bogdal, C., Schmid, P., Zennegg, M., Blüthgen, N., Anselmetti, F.S., Kohler, M., Heeb, N.V., Scheringer, M., Hungerbühler, K., 2010. Temporal trends of hexabromocyclododecanes in urban, rural and remote lakes in Switzerland. Organohalogen Compd. 72, 338–341. Brandsma, S.H., van der Ven, L.T.M., de Boer, J., Leonards, P.E.G., 2009. Identification of hydroxylated metabolites of hexabromocyclododecanes in wildlife and 28days exposed wistar rats. Environ. Sci. Technol. 43, 6058–6063. Browne, M.A., Dissanayake, A., Galloway, T.S., Lowe, D.M., Thompson, R.C., 2008. Ingested microscopic plastic translocates to the circulatory system of the mussel, Mytilus edulis (L.). Environ. Sci. Technol. 42, 5026–5031. Bruker, 2010. SAINT Release 7.68A. Integration Software for Single Crystal Data. Bruker AXS Inc., Madison, USA. Covaci, A., Gerecke, A.C., Law, R.J., Voorspoels, S., Kohler, M., Heeb, N.V., Leslie, H., Allchin, C.R., De Boer, J., 2006. Hexabromocyclododecanes (HBCDs) in the environment and humans: a review. Environ. Sci. Technol. 40, 3679–3688. de Wit, C.A., 2002. An overview of brominated flame retardants in the environment. Chemosphere 46, 583–624. de Wit, C.A., Alaee, M., Muir, D.C.C., 2006. Levels and trends of brominated flame retardants in the Arctic. Chemosphere 64, 209–233. Derraik, J.G.B., 2002. The pollution of the marine environment by plastic debris: a review. Mar. Pollut. Bull. 44, 842–852. Dolomanov, O.V., Bourhis, L.J., Gildea, R.J., Howard, J.A.K., Puschmann, H., 2009. OLEX2: a complete structure solution, refinement and analysis program. J. Appl. Cryst. 42, 339–341.

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