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

The application of The application of chiralchiral LC to the study of LC to the study of HBCDsHBCDs in the environmentin the environment

Adrian Covaci1

Karel Janak2, Norbert Heeb3, Stefan Voorspoels1, Andreas Gerecke3

1-Toxicological Centre, University of Antwerp, Belgium2-Norwegian Institute of Public Health, Oslo, Norway

3-Swiss Federal Laboratories for Materials Testing and Research (EMPA), Dübendorf, Switzerland

Outline

General aspects

Stereochemistry

Analysis

- LC/MS

- chiral LC/MS

Chiral HBCDs in various species

Discussion

Acknowledgements

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 - 16 700 t, from which 9 500 t in the EU

- HBCDs - second highest-volume BFR used in the EU, after TBBP-A, but before BDE 209

- To date, there are no restrictions on the production or use of HBCDs

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)

Bioaccumulation

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Sedi

men

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Soi

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Sew

age

slud

ge Air

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inve

rtebr

ates

Mar

ine

fish

Fres

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h Bird

s

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mam

mal

s

% to

tal H

BCD

α-HBCDβ-HBCDγ-HBCD

- increase in the proportion of α-HBCD 1. from abiotic to biotic matrices

2. going up the food chain

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

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)

Stereochemistry (1)

- 6 stereogenic centers

at positions 1,2,5,6,9,10

- theoretically 16 stereoisomers:

6 pairs of enantiomers

4 meso-forms

Stereochemistry (2)

β-HBCD - 1R, 2R, 5R, 6S, 9R, 10S

- 1S, 2S, 5S, 6R, 9S, 10R

α-HBCD - 1R, 2R, 5S, 6R, 9R, 10S

- 1S, 2S, 5R, 6R, 9S, 10R

γ-HBCD - 1R, 2R, 5R, 6S, 9S, 10R

- 1S, 2S, 5S, 6R, 9R, 10S

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

BDE4

7

HBC

DBD

E153

BDE9

9BD

E100

Eel

- Separation of different HBCD stereoisomers is not possible by GC

LC (1)

- In contrast to GC, HBCD diastereoisomers can be easily separated using

reversed-phase LC (first report Budakowski and Tomy, 2003).

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Standards

α β

γ

6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0Time (min)

α β

γ

Technical mix.

Time (min)6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0

α

βγ

Thermally equil .technical mix.

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Standards

α β

γ

6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0Time (min)

α β

γ

Technical mix.

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6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.06.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0Time (min)

α β

γ

Technical mix.

Time (min)6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0

Time (min)6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.06.0 7.0 8.0 9.0 10.0 11.0 12.0 13.06.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0

α

βγ

Thermally equil .technical mix.

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).

(-) α

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Time (min)

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Standard

(-) β

(+) α

(+) β(-) γ(+) γ

(-) α

8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.08.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0

Time (min)

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Standard

(-) β

(+) α

(+) β(-) γ(+) γ

Chiral LC

- EF = 0.5 - racemic mixture

AAAEF

)()(

)(

++−

+=

- As a consequence, up to 8

individual HBCD stereoisomers can

now be differentiated by LC/MS

(Heeb et al., 2005)

Analysis (1)

Internal standards: - 13C-HBCDs- d18-HBCDs- dibromo-toluene (DBT)

Extraction: - liver, muscle, eggs or blubber + Na2SO4

- Soxhlet extraction

Clean-up: - acidified silicagel or H2SO4 decomposition- elution with hexane/dichloromethane, concentration- solvent exchange to acetonitrile or methanol

Analysis (2)

Diastereoisomers: Symmetry C18 (2.1mm x 150mm, 5µm) - Waters

Flow: 250 µL/min- H2O/MeOH/AcN (60/30/10) to MeOH/AcN (50/50) in 5 min, held 6 min- H2O/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- H2O/MeOH/AcN (40/30/30) to MeOH/AcN (30/70) in 8 min, held for 14 min

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)

Matrix effects: - suppression or enhancement of signals

Detection of diastereomers: triple quadrupole, single quadrupole, ion-trap

Detection of enantiomers: triple quadrupole

This transition cannot be monitored with ion-trap MS !!

Sampling - based on 1. food web positioning

2. sample availability3. HBCD plant in Terneuzen

Scheldt Estuary (1)

Janák, Covaci, Voorspoels, Becher, Environ Sci Technol, 2005a

Common sole (Solea solea)Plaice (Pleuronectus platessa)

Whiting (Merlangius merlangus)

Bib (Trisopterus luscus)

Eel (Anguilla anguilla)

Scheldt Estuary (2)

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Time (min)6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0

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βγ

Eel muscle

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(-) α

(-) γ(+) β

(+) γ

Bib liver

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Time (min)

(-) α

(+) α

(+) β (+) γ

Whiting liver

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(-) β

(+) α

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(+) γ

Bib liver

Time (min)8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.08.0 9.0 10.0 11.0 12.0 13.0 14.0 15.08.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0

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(-) β(-) β

(+) α(+) α

(-) α(-) α

(-) γ(-) γ(+) β(+) β

(+) γ(+) γ

Bib liver

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(-) α

(+) α

(+) β (+) γ

Whiting liver

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Time (min)

(-) α(-) α

(+) α(+) α

(+) β(+) β (+) γ(+) γ

Whiting liver

Enantiomers

Diastereoisomers

Scheldt Estuary (4)

- EF species specific

- high differences in between species

- no difference in between liver and muscle of the same species (sole)

EFs for α-HBCD

n EF SD RangeBib L 3P 0.58 0.02 0.56 - 0.59Sole L 3P 0.43 0.05 0.38 - 0.46Sole M 2P 0.42 0.02 0.40 - 0.43

Whiting L 1P + 2I 0.70 0.06 0.65 - 0.76Eel M 1I 0.54

Swedish samples (1)

Peregrine falcon

(Falco peregrinus)

Guillemot

(Uria algae)

White-tailed sea eagle

(Haliaeetus albicilla)

Herring

(Clupea harengus)

Janák, Sellström, Johansson, Becher, de Wit, Lindberg, Helander, Orghalog. Compounds, 2005b

Swedish samples (2)

1. 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

EFs for α-HBCD

n EF SD RangeFalcon E 6I 0.21 0.12 0.11 - 0.35

Sea Eagle E 3I 0.77 0.07 0.72 - 0.83Guillemot E 2P + 2I 0.53 0.05 0.50 - 0.60Herring M 3P + 1I 0.25 0.07 0.18 - 0.31

Rel

ativ

e ab

unda

nce

(-)αPeregrine falcon

(+)α

(+)αSea eagle(-)α

(+)α(-)αGuillemot

Herring(+)α

(-)α

(+)αCommon tern (-)α

? ?

Swedish samples (3)

Atlantic white-sided

dolphin (Lagenorhynchus acutus)

Dolphin samples from NIST

EFs of α-HBCD

n EF Range EFsblubber 89 0.41 0.26 - 0.59

liver 16 0.44 0.37 - 0.58

1. Most of EFs in liver were higher than the corresponding EFs in blubber

2. No correlation between EF and α-HBCDlevels

Peck, Tuerk, Keller, Kucklick, Schantz, Orghalog. Compounds, 2005

y = 0.444x + 0.2731r = 0.58, p=0.02

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EF

liver

Overview α-HBCDs

L – liver M – muscle E – eggB – blubber

Error bars: SD

1. Clear differences in EFs of α-HBCDs between the investigated species

2. EFs > 0.5 and EFs < 0.5

3. Enantioselective absorption and/or metabolism of α-HBCDs vary between species

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Eag

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Dol

phin

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Dol

phin

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Mea

n EF

s

Overview – γ-HBCDs

L – liver M – muscle

Error bars: SD

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1. γ-HBCD is not measurable in many species (detected only in fish)

2. Apparently, EF > 0.5

Concluding remarks

Enantiomers- EFs – insufficient data

- Indication for species specific accumulation of enantiomers

- The predator does not necessarily follow the pattern of the prey

Diastereomers – Increase of proportion of α-HBCD with the trophic level

- Bioaccumulation from fish to birds or marine mammals

References – chiral HBCDs

1. Janák K, Thomsen C, Becher G. BFR 2004, 2004, 313-316.

2. Heeb NV, Schweizer WB, Kohler M, Gerecke AC. Chemosphere 2005, 61, 65-73.

3. Janák K, Covaci A, Voorspoels S, Becher G. Environ. Sci. Technol. 2005a, 39, 1987-1994.

4. Janák K, Sellström U, Johansson AK, Becher G, de Wit C, Lindberg P, Helander B. Organohalogen Compounds 2005b, 67, 204-207.

5. 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 Compounds2005, 67, 1259-1262.

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

THANK YOU