The application of chiral LC to the study of HBCDs in the environment

Transcription

1 The application of chiral LC to the study of HBCDs in the environment Adrian Covaci 1 Karel Janak 2, Norbert Heeb 3, Stefan Voorspoels 1, Andreas Gerecke 3 1-Toxicological Centre, University of Antwerp, Belgium 2-Norwegian Institute of Public Health, Oslo, Norway 3-Swiss Federal Laboratories for Materials Testing and Research (EMPA), Dübendorf, Switzerland

2 Outline General aspects Stereochemistry Analysis -LC/MS - chiral LC/MS Chiral HBCDs in various species Discussion Acknowledgements

3 General aspects - HBCDs - additive brominated flame retardants (BFRs) - polystyrene foams (up to 2.5% HBCDs) used as thermal insulation in buildings, - in upholstery textiles (6-15% HBCDs) - in electrical equipment housings - In 2001, world market demand t, from which t in the EU - HBCDs - second highest-volume BFR used in the EU, after TBBP-A, but before BDE To date, there are no restrictions on the production or use of HBCDs

4 POP-like properties - resistance to chemical and biological degradation - persistence in environment and biota - accumulation in fatty tissues, resulting in biomagnification in the higher trophic levels of the food chain - long-range transport in the environment (detection in remote areas such as the Arctic)

5 Bioaccumulation α-hbcd β-hbcd γ-hbcd Sediment Soil Sewage sludge Air Aquatic invertebrates Marine fish Freshwater fish Birds Marine mammals % total HBCD - increase in the proportion of α-hbcd 1. from abiotic to biotic matrices 2. going up the food chain

6 Toxicity - Acute toxic effects are low - Oral exposure to HBCDs induces hepatic cytochrome P450 in rats - HBCDs may induce cancer by a non-mutagenic mechanism - HBCDs may disrupt the thyroid hormone system and affect the thyroid hormone receptor-mediated gene expression - HBCDs can also alter the normal uptake of neurotransmitters in rat brain - Further research on the actual levels at which these effects occur is needed

7 Synthesis - Technical grade HBCD mixtures are obtained via bromination of cyclododeca-1,5,9- triene isomers - The commercial mixtures mainly consist of γ-hbcd (75-89%), while α-hbcd and β- HBCD are present in lower amounts (10-13% and 1-12%, respectively) - Two additional stereoisomers, named δ- and ε-hbcd, are present at minor concentrations (Heeb et al., 2005)

8 Stereochemistry (1) - 6 stereogenic centers at positions 1,2,5,6,9,10 - theoretically 16 stereoisomers: 6 pairs of enantiomers 4 meso-forms

9 Stereochemistry (2) α-hbcd - 1R, 2R, 5S, 6R, 9R, 10S - 1S, 2S, 5R, 6R, 9S, 10R β-hbcd - 1R, 2R, 5R, 6S, 9R, 10S - 1S, 2S, 5S, 6R, 9S, 10R γ-hbcd - 1R, 2R, 5R, 6S, 9S, 10R - 1S, 2S, 5S, 6R, 9R, 10S

10 GC - Separation of different HBCD stereoisomers is not possible by GC - A relatively broad, unresolved peak is obtained - HBCDs are subject to thermal rearrangement at temperatures above 160 C, resulting in a specific mixture (78% α-hbcd, 13% β-hbcd and 9% γ-hbcd) - Results reflect total HBCD concentrations BDE47 BDE99 BDE100 BDE153 HBCD Eel

11 LC (1) - In contrast to GC, HBCD diastereoisomers can be easily separated using reversed-phase LC (first report Budakowski and Tomy, 2003). 100 γ γ α Relative Abundance Standards α β Technical mix. α β Thermally equil. technical mix. β γ Time (min) Time (min) Time (min)

12 LC (2) - Furthermore, enantiomeric pairs can be resolved on a chiral, permethylated β- cyclodextrin stationary phase for LC (Heeb et al., 2005; Janák et al., 2005a). 100 Standard (+) β (+) γ (-) γ Relative Abundance (-) β (-) α (+) α Time (min)

13 Chiral LC EF = ( ) ( + ) A A+ ( + ) A - EF = racemic mixture - As a consequence, up to 8 individual HBCD stereoisomers can now be differentiated by LC/MS (Heeb et al., 2005)

14 Analysis (1) Extraction: - liver, muscle, eggs or blubber + Na 2 SO 4 - Soxhlet extraction Clean-up: - acidified silicagel or H 2 SO 4 decomposition - elution with hexane/dichloromethane, concentration - solvent exchange to acetonitrile or methanol Internal standards: - 13 C-HBCDs -d 18 -HBCDs - dibromo-toluene (DBT)

15 Analysis (2) Diastereoisomers: Symmetry C18 (2.1mm x 150mm, 5µm) - Waters Flow: 250 µl/min -H 2 O/MeOH/AcN (60/30/10) to MeOH/AcN (50/50) in 5 min, held 6 min -H 2 O/MeOH (60/40) to MeOH (100) in 5 min, held 6 min. Enantiomers: NUCLEODEX β-pm (4mm x 200mm, 5µm) Macherey-Nagel Flow: 500 µl/min -H 2 O/MeOH/AcN (40/30/30) to MeOH/AcN (30/70) in 8 min, held for 14 min

16 Analysis (3) Analysis by LC/MS-MS, electrospray negative ion mode SRM for [M-H] - (m/z=640.6) Br - (m/z=79.0 and 81.0) This transition cannot be monitored with ion-trap MS!! Matrix effects: - suppression or enhancement of signals Detection of diastereomers: triple quadrupole, single quadrupole, ion-trap Detection of enantiomers: triple quadrupole

17 Scheldt Estuary (1) Sampling - based on 1. food web positioning 2. sample availability 3. HBCD plant in Terneuzen Janák, Covaci, Voorspoels, Becher, Environ Sci Technol, 2005a

18 Scheldt Estuary (2) Common sole (Solea solea) Plaice (Pleuronectus platessa) Bib (Trisopterus luscus) Eel (Anguilla anguilla) Whiting (Merlangius merlangus)

19 Relative Abundance Sediment γ Diastereoisomers Eel muscle α 20 0 α β Time (min) β Time (min) γ Relative Abundance Bib liver (-) α (-) β (+) α (+) β (+) γ (-) γ Enantiomers (-) α (+) α Whiting liver (+) β (+) γ Time (min) Time (min)

20 Scheldt Estuary (4) EFs for α-hbcd n EF SD Range Bib L 3P Sole L 3P Sole M 2P Whiting L 1P + 2I Eel M 1I EF species specific - high differences in between species - no difference in between liver and muscle of the same species (sole)

21 Swedish samples (1) Herring (Clupea harengus) Guillemot (Uria algae) Peregrine falcon (Falco peregrinus) White-tailed sea eagle (Haliaeetus albicilla) anák, Sellström, Johansson, Becher, de Wit, Lindberg, Helander, Orghalog. Compounds, 2005b

22 Swedish samples (2) EFs for α-hbcd n EF SD Range Falcon E 6I Sea Eagle E 3I Guillemot E 2P + 2I Herring M 3P + 1I EFs in guillemot differs from EFs in their prey, herring 2. Very small differences within a colony 3. Minor differences between colonies 4. High differences between birds species

23 Swedish samples (3) Peregrine falcon (-)α (+)α Sea eagle (-)α (+)α Common tern (-)α Relative abundance Guillemot (-)α (+)α (+)α?? Herring (-)α (+)α

24 Dolphin samples from NIST EFs of α-hbcd n EF Range EFs blubber liver Atlantic white-sided dolphin (Lagenorhynchus acutus) EF liver y = 0.444x r = 0.58, p= Most of EFs in liver were higher than the corresponding EFs in blubber 2. No correlation between EF and α-hbcd levels EF blubber Peck, Tuerk, Keller, Kucklick, Schantz, Orghalog. Compounds, 2005

25 Overview α-hbcds L liver M muscle E egg B blubber Error bars: SD Herring M Sole M Sole L Eel M Bib L Whiting L Salmon M Mean EFs Falcon E Guillemot E Sea Eagle E Dolphin B Dolphin L 1. Clear differences in EFs of α-hbcds between the investigated species 2. EFs > 0.5 and EFs < Enantioselective absorption and/or metabolism of α-hbcds vary between species

26 Overview γ-hbcds Mean EFs L liver M muscle Error bars: SD Bib L Whiting L Salmon M 1. γ-hbcd is not measurable in many species (detected only in fish) 2. Apparently, EF > 0.5

27 Concluding remarks Diastereomers Increase of proportion of α-hbcd with the trophic level - Bioaccumulation from fish to birds or marine mammals Enantiomers - EFs insufficient data - Indication for species specific accumulation of enantiomers - The predator does not necessarily follow the pattern of the prey

28 References chiral HBCDs 1. Janák K, Thomsen C, Becher G. BFR 2004, 2004, Heeb NV, Schweizer WB, Kohler M, Gerecke AC. Chemosphere 2005, 61, Janák K, Covaci A, Voorspoels S, Becher G. Environ. Sci. Technol. 2005a, 39, Janák K, Sellström U, Johansson AK, Becher G, de Wit C, Lindberg P, Helander B. Organohalogen Compounds 2005b, 67, Law RJ, Kohler M, Heeb NV, Gerecke AC, Schmid P, Voorspoels S, Covaci A, Becher G, Janák K, Thomsen C. Environ. Sci. Technol. 2005, 39, 281A-287A. 6. Peck AM, Tuerk KJS, Keller J, Kucklick JR, Schantz MM. Organohalogen Compounds 2005, 67,

29 Acknowledgements - Flanders Institute for the Sea (VLIZ) for providing logistic support during sampling in the Scheldt Estuary - Swedish samples kindly provided by the Contaminant Research Group, and the Environmental Monitoring Group at Stockholm University, and the Swedish Museum of Natural History, Stockholm - Dr. Aaron Peck (NIST) for dolphin data

30 THANK YOU