Reproductive changes in American kestrels (Falco sparverius) in relation to exposure to technical hexabromocyclododecane flame retardant
Kim J. Fernie * , Sarah C. Marteinson
†
, David M. Bird
†
, Ian J. Ritchie
†
, Robert J. Letcher
‡
*† Wildlife and Landscape Science Directorate, Science and Technology Branch, Environment Canada, 867
Lakeshore Rd., Burlington, Ontario, Canada, L7R 4A6; kim.fernie@ec.gc.ca
†
Avian Science and Conservation Centre, McGill University, 21111 Lakeshore Road, Ste Anne de
Bellevue, Quebec, Canada, H9X 3V9; david.bird@mcgill.ca; ian.ritchie@mcgill.ca; sarah.marteinson@mcgill.ca
‡
Wildlife and Landscape Science Directorate, Science and Technology Branch, Environment Canada,
National Wildlife Research Centre, 1125 Colonel By Drive, Ottawa, Ontario, Canada K1S 5B6; robert.letcher@ec.gc.ca
*Corresponding author: kim.fernie@ec.gc.ca.; Tel. 1-905-336-4843; Fax: 1-905-336-6430.

Hexabromocyclododecane isomer analysis
The first egg produced by each kestrel pair was also used for determining the

-, β- and γ-
HBCD isomer concentrations and burdens. The contaminant burdens in the first egg of each kestrel clutch may not reflect concentrations in other eggs within the same clutch, although contaminant burdens, including PBDEs, did not vary with laying order as reported for other species [22].
All HBCD analysis was performed in the Organic Contaminants Research Laboratory
(OCRL) at NWRC in Ottawa. The analytical method for the determination of the

-, β- and γ-
HBCD isomers in the kestrel eggs and plasma was the same as reported elsewhere [12,15]. The original solid and safflower oil solution were also analyzed for HBCD isomers to determine the original isomer compositions and if any isomerization had occurred during the preparation process. In addition, contaminant levels in the cockerel diet were reflected by the control eggs.
All samples were spiked with internal standards
13
C
12
-labeled-

-,

-,

-HBCD. The original dosing solutions and all plasma or egg samples were analyzed for isomer-specific
HBCDs using high performance liquid chromatography-electrospray ionization (negative mode)tandem quadrupole mass spectrometry (HPLC-ESI(-)-MS/MS) as described elsewhere [12].
Briefly, tissue and injection stock solution samples were weighed, transferred to a glass mortar, and ground with ~5 g of diatomaceous earth; 10 ng of each internal standard (
13
C
12
labeled surrogates of α-, 
- and

-HBCD; Wellington Laboratories; Guelph, ON, Canada) were spiked into the sample prior to extraction using accelerated solvent extraction (ASE200; Dionex
Company, Sunnyvale, CA) with dichloromethane/hexane (50/50, vol/vol). The operation

parameters were three cycles of: heat for 5 min, static for 5 min, flush 100%, and purge for 30 s
(pressure, 1500 psi; temperature, 100

C). The extract was concentrated and cleaned up by sulfuric acid silica (50%). The eluant, containing target compounds, was collected, concentrated, and solvent exchanged to methanol for analysis by HPLC-ESI(-)-MS/MS.
The fraction analysis was performed using a Waters 2695 HPLC equipped with an ACE 3
C
18
analytical column (2.1 3 50 mm, 3 lm particle size; Advanced Chromatography
Technologies, Aberdeen, UK). The temperature of the HPLC column was kept at 40

C. The mobile phase consisted of water, acetonitrile, and methanol, and the LC flow rate was 0.25 ml/min. The following gradient was employed: water/acetonitrile/methanol (40/40/20) linear gradient changed to acetonitrile/methanol (60/40) after 3 min and held for 8 min, followed by a return to the initial gradient conditions for 30 s and equilibrated for 9.5 min. A 10 μL aliquot of the sample was injected into the HPLC system.
The mass spectrometer was a Waters QuattroUltima tandem-quadrupole mass spectrometer (Waters, Milford, MA) with an ESI(-) source. Nitrogen was used as a nebulizing and dissolvent gas, and argon was used as the collision gas. Multiple reaction monitoring (MRM) mode was used and the monitored transitions were m/z 640.5 > 81 for the three non-labeled analytes (

-, β- and γ-HBCD) and m/z 652.5 > 81 three isotopically-labeled surrogates used as internal standards (
13
C
12
-labeled

-, β- and γ-HBCD). The data analysis was performed using
QuanLynx software, Version 4.0. Quantification was performed using an internal standard method with a five-point calibration curve spanning the range of anticipated analyte concentrations in the samples.
Quality control for HBCD analysis

For isomer-specific HBCD analysis in eggs, an in-house (NWRC) reference material
(RM) was used, and was a pool of double-crested cormorant ( Phalacrocorax auritus ) (DCCO) egg homogenate (generated from eggs collected in 2003 from the Great Lakes). As there were no readily available reference materials for HBCD isomers, the DCCO-RMwas the chosen option. .
A sample of polar bear plasma was used as a reference material for the plasma samples taken after the uptake period, to use a similar matrix.
A blank and DCCO-RM were analyzed with approximately every 8 samples for HBCDs.
Based on n=29 replicate analysis in various batches, the blanks contained mean concentrations of
0.2 ± 0.3, 0.1 ± 0.3 and 0.5 ± 0.4 ng/g wet weight of α-,

- and

-HBCD, respectively. Based on n=23 replicate analysis in various batches, the reference samples contained mean concentrations of DCCO reference sample contained 10.0 ± 0.8, 0.1 ± 0.2 and 0.3 ± 0.4 ng/g wet weight of α-,

- and

-HBCD, respectively.
The method detection limit (MDL) value was measured by performing replicate analyses
(n = 8) of chicken egg samples, which were spiked with analytes. The MDLs were determined by calculating the standard deviation (SD) of the 8 replicate analyses spiked with 3-5 times the estimated limits of detection (LODs) and multiplying by 3, i.e., MDL = SD x 3. The LOD was calculated based on a ratio, peak to peak, of 3 between the signal of the analyte and the baseline noise. The MDL and LOD of the replicate analyses and the MDL and LOD calculated for the tissue and plasma samples from the kestrel dosing study were provided in table. The LODs for eggs were 0.3 ng/g ww and 0.1 ng/g ww for plasma for all HBCD isomers. The MDLs were 0.01 ng/g ww (1.0 ng/g lipid weight (lw)) for eggs and 0.01 ng/g ww (or 0.1 ng/g lw) for plasma samples for the three

-,

- and

-HBCD isomers.

References
12. Gauthier LT, Hebert CE, Weseloh DVC, Letcher RJ. 2007. Current-use flame retardants in the eggs of herring gulls ( Larus argentatus ) from the Laurentian Great Lakes Environ Sci Technol 41:4561-
4567.
15. Crump D, Egloff C, Chiu S, Letcher RJ, Chu SG, Kennedy SW. 2010. Pipping success, isomerspecific accumulation, and hepatic mRNA expression in chicken embryos exposed to HBCD.
Toxicol Sci 115:492-500.
22. van den Steen E, Dauwe T, Covaci A, Jaspers VLB, Pinxten R, Eens M. 2006. Within- and amongclutch variation of organohalogenated contaminants in eggs of great tits ( Parus major ). Environ
Pollut 144:355-359.