Application Note No. 206/2015 Brominated Flame Retardants in Marine Mammals SpeedExtractor E-916, Syncore

Application Note
No. 206/2015
Brominated Flame Retardants in Marine Mammals
SpeedExtractor E-916, Syncore® Analyst R-12:
Extraction of Alternative Brominated Flame Retardants from Harbour Porpoises (Phocoena
phocoena)
1. Introduction
In recent years, the most widely used brominated flame retardants (BFRs), the polybrominated
diphenyl ethers (PBDEs), have suffered increasing regulation and restrictions on their production
and use. These measures are a consequence of concerns over their persistence, ability to
bioaccumulate and potential for toxicity. Their removal from markets has resulted in a need for
their substitution with other, non-PBDE, BFRs. There are a range of these ‘alternative’ BFRs
(aBFRs) reported to be in use. A recent publication [1] has tried to clarify their abbreviations as
different naming systems are currently in use by different authors. Current knowledge about
aBFRs has been reviewed recently [2] and analytical methodologies used to date to detect them
have been critically reviewed [3]. Several recent studies have confirmed the presence of a range
of aBFRs in the environment [4, 5] and a study has demonstrated endocrine disruption activity for
some aBFRs [6].
In this application note the development and the results of a method for the analysis of >20
aBFRs, plus the chlorinated FR Dechlorane Plus (DP), in biota samples is presented (for details,
see reference [7]). The method utilises pressurized solvent extraction (PSE) and parallel
evaporation followed by clean up using gel permeation chromatography (GPC) and solid phase
extraction (SPE). Sample extracts are analysed by GC-MS/MS. Blubber from harbour porpoises
collected as part of the UK Cetacean Strandings Investigation Programme (CSIP) were analysed
using this method, in order to investigate the occurrence of aBFRs in the UK marine environment
[7].
2. Equipment
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SpeedExtractor E-916, equipped with 40 mL cells
Syncore® Analyst R-12, with R-12 glass tubes, 1 mL residual volume (BUCHI, 046071)
Nitrogen blow-down instrument, TurboVap LV (Zymark, Germany)
Samplicity 0.20 µm Hydrophilic PTFE (Merck KGaA, Darmstadt, Germany).
Agilent Series 1100 HPLC system (Agilent Technologies, Waldbronn, Germany) with two
Envirogel™ columns (Waters Corporation, Milford, MA, USA) (19×150 mm and 19×300
mm) in series protected by an Envirogel™ guard column
Techne Sample Concentrator (Camlab, Cambridge)
Agilent 7890a gas chromatograph coupled to an Agilent 7000B triple quadrupole mass
spectrometer, equipped with a MMI injector and a 7693 autosampler (Agilent
Technologies, Waldbronn, Germany)
3. Chemicals and Materials
Chemicals:
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n-hexane, dichloromethane (DCM), ethyl acetate and cyclohexane, HPLC-grade,
Rathburn (Walkerburn, UK)
n-nonane, Acros Organics (Geel, Belgium)
Florisil® 100-200 mm mesh, Acros Organics (Geel, Belgium)
Anhydrous sodium sulphate, analytical reagent grade, Fisher Scientific (Loughborough,
UK)
Hydromatrix Isolute HM-N, Kinesis (St Neots, UK)
Individual standards or partial mixtures of ATE, DPTE, BATE, OBIND, a-DP, s-DP, HBB,
13C -HBB, PBEB, PBT, EHTBB, pTBX, TBPH, HCDBCO,
-TBCO, -TBCO, BTBPE,
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13C -BTBPE, BDE204,13C -BDE 209 (MBDE-209), and mixes of - and -TBECH, and
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- and -TBECH were from Wellington Laboratories (Guelph, Canada)
BB153, PBBA, PBBB, TBoCT, CB200 (injection standard) and technical mixture FR-651
were from AccuStandard (New Haven, CT, USA)
TBBPA-DBPE, Dr. Ehrenstorfer (Ausburg, Germany)
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13C -2,4,6-TBP
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(MTBP), diMe-TBBPA and 13C12-BDE3 (MBDE3), Cambridge Isotope
Laboratories (Andover, MA, USA)
Neat 2,4,6-TBP (99.5 % purity), Riedel-de-Haen® (Seetze, Germany)
Bottom cellulose filters (BUCHI, 049569)
Cellulose filters for E-916 (BUCHI, 049572)
For a safe handling please refer to all corresponding MSDS.
Samples:
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First experiments to test extraction performance were done using standards without a
matrix (Spiked blank samples). These samples were extracted with SpeedExtractor E916 and concentrated with Syncore® Analyst and then analysed directly with GC-MS/MS
(without clean-up procedure).
Method validation was carried out by spiking cod liver at 3 different levels. These samples
are used as in-house laboratory reference materials.
Twenty-one porpoise blubber samples were obtained from selected dead animals which
were found stranded, or were bycaught around the UK coast. Samples were taken for
post-mortem study in order to establish cause of death within the UK Cetacean
Strandings Investigation programme funded by the UK government.
Tissue samples were stored frozen at −20 °C prior to analysis. Skin was removed and
samples were homogenised before extraction.
4. Procedure
The determination of aBFRs in biota samples includes the following steps:
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Preparation of the samples and the cells
Pressurized solvent extraction using SpeedExtractor E-916
Concentration of the extracts using Syncore ® Analyst R-12
Filtration using Samplicity 0.2 µm Hydrophilic PTFE
Clean-up using gel permeation chromatography
Clean-up using solid phase extraction (SPE) with Florisil® cartriges
Concentration of the extracts using Syncore® Analyst R-12 and nitrogen blow-down
instrument and Techne Sample Concentrator.
Analysis and quantification using gas chromatography - tandem mass spectrometry (GCMS/MS) using electron ionization mode (EI).
4.1 Preparation of the samples and the cells
1. Mix approx. 5 g of porpoise blubber (or 0.4 g cod liver) with approx. 20 g of sodium
sulphate, spike with 13C-labelled flame retardants analogues and leave overnight in the
fridge.
2. Mix the samples, once at room temperature, with Hydromatrix 1:1 vol:vol.
3. Fill the cell with approx. 2 cm of Hydromatrix followed by the mixture of sample +
sodium sulphate + Hydromatrix. Fill up the volume left empty in the cell with
Hydromatrix.
4. Place cellulose filters at each end.
For the experiments with the spiked blank samples, the same materials were used and the
cell was filled the same way, but without sample.
4.2 Pressurized Solvent Extraction
1. Preheat the instrument to the set temperature.
2. Insert the 220 mL collection vessels into the collection unit.
3. Insert the cells and extract the samples using the method shown in Table 1.
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Table 1: Extraction method of SpeedExtractor E-916.
Parameter
Value
Temperature
100 °C
Pressure
120 bar
Solvent
Hexane 50 % : Acetone 50 %
Cells
40 mL
Vials
220 mL
Cycles
3
Heat-up
Hold
Discharge
1 min
5 min
3 min
Flush with solvent
2 min
Flush with gas
3 min
Total extraction time
49 min
4.3 Concentration with Syncore
Concentrate the collected samples in the Syncore using the parameters shown in Table 2.
Table 2: Parameters for evaporation of the solvent using Syncore® Analyst R-12, using glasses with working volume up
to 120 mL.
Parameter
Value
Rack temperature
40 °C
Cover temperature
60 °C
Speed
250 rpm
Residual volume
1 mL
4.4 Clean-up with Gel Permeation Chromatography and SPE
1. Clean-up the concentrated extracts using gel permeation chromatography (GPC) using
the parameters shown in Table 3.
Table 3: Parameters for clean-up with GPC .
Type of GPC
Agilent Series 1100 HPLC system
GPC column
Envirogel™ columns (19×150 mm and 19×300 mm) in series
protected by an Envirogel™ guard column.
Mobile phase
Ethyl acetate : Cyclohexane [1:1]
Flow
5 mL/min
2. Concentrate the cleaned-up extracts using Syncore followed by nitrogen blow-down
(TurboVap)
3. Do a second clean-up step using SPE under the conditions shown in Table 4.
Table 4: Parameters for clean-up with SPE.
Columns
Florisil
Filling
8g
1. Elution
Hexane, 55 mL
2. Elution
Dichloromethane, 35 mL
4. Combine both eluted fractions.
5. Concentrate the fractions to 1 mL using Syncore and TurboVap.
6. Evaporate fo 25 µL using a gentle current of nitrogen and add CB200 (2,2 ,3,3 ,4,5,6,6 octachlorobiphenyl) dissolved in n-nonane as internal quantification standard to the
concentrated extracts.
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4.5 Analysis with GC-MS/MS
The configuration of the GC-MS/MS system is specified in Table 5.
Table 5: Configuration of the GC-MS/MS system for the determination of the aBRF.
GC-MS/MS
Agilent 7890a GC coupled to an Agilent 7000B triple quadrupole MS
Mode
Electron ionization at 70eV
Column
DB-5 MS 15 m x 0.25 mm x 0.1 µm
Injection volume
1 µL
Injector
Multimode injector, pulsed splitless injection mode
Inlet conditions
30 psi, 1.5 min, 300 °C
Temperature program
90 °C (1 min), 10 °C/min to 310 °C, 13 min
Temperatures: Transfer line
300 °C
Source
300 °C
Quadrupole
150 °C
Details of the precursor and products ions studied in the MS/MS transitions and instrumental limits
of detection for all 30 alternative flame retardant compounds investigated in this study are given
in [7].
The BDEs were also recovered using this method and so their analysis was included as well. A
suite of 17 BDE congeners (BDE17, BDE28, BDE47, BDE66, BDE85, BDE99, BDE100, BDE126,
BDE138, BDE153 BDE154, BDE183, BDE204, BDE206, BDE207, BDE208 and BDE209) was
also determined using the same method.
Quality control was achieved through the analysis of an in-house laboratory reference material
(cod liver tissue spiked with alternative BFR compounds prepared and characterised in-house)
and procedural blanks for each sample batch.
5. Results and Discussion
5.1 Separation of the standard mixtures
An example of the separation and retention orders observed for the selected compounds is shown
in Figure 1.
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Figure 1: MS/MS chromatograms of two standard mixtures of aBFRs and PBDEs (details in [7]).
5.2 Recovery of the spiked blank samples
The recoveries of the extraction of the spiked blank samples are presented in Table 6
Table 6: Mean values of recoveries of aBFR in the spiked blank sample (n= 3).
Recovery in spiked blank samples [%]
ATE
95
HBB
94
-TBECH
109
PBBB
97
-TBECH
104
PBBA
92
BATE
116
HCDBCO
98
pTBX
88
EHTBB
100
-TBCO
159
BB153
96
-TBECH
115
diMeTBBPA
74
-TBECH
126
BTBPE
106
TBoCT
89
TBPH
102
PBCC
40
s-DP
93
PBT
52
a-DP
77
PBEB
891
OBIND
97
DPTE
255
DBDPE
84
The recoveries in the spiked blank samples are between 70-120 % for most of the analytes. A
few analytes have recoveries outside the acceptable range. The method was developed for
screening of a wide set of flame retardants in biota, but does not suit all compounds perfectly. For
these compounds, the method is not absolute, but orientative.
5.3 Recovery of the spiked cod-liver samples
The recoveries for cod liver spiked at a medium level using the developed extraction and cleanup procedure are presented in Table 6.
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Table 7: Mean values of recoveries of aBFR in the spiked cod liver (n= 6), rsds are in brackets.
Recovery in spiked cod liver [%]
ATE
52.3 (18.3 %)
HBB
78.1 (10.6 %)
-TBECH
75.3 (8.9 %)
PBBB
65.1 (12.9 %)
-TBECH
79.1 (9.0 %)
PBBA
42.8 (34.2 %)
BATE
79.4 (7.5 %)
HCDBCO
85.4 (10.6 %)
pTBX
78.3 (8.2 %)
EHTBB
46.0 (22.6 %)
-TBCO
92.1 (5.4 %)
BB153
95.9 (9.2 %)
-TBECH
99.0 (5.1 %)
diMeTBBPA
85.7 (35.4 %)
-TBECH
103 (6.8 %)
BTBPE
44.4 (48.7 %)
TBoCT
76.8 (5.5 %)
TBPH
41.7 (50.0 %)
PBCC
65.0 (6.8 %)
s-DP
80.7 (14.9 %)
PBT
56.8 (11.2 %)
a-DP
89.4 (8.6 %)
PBEB
224 (45.6 %)
OBIND
122 (15.3 %)
DPTE
74.9 (10.4 %)
DBDPE
133 (13.1 %)
Recoveries for most alternative BFR compounds in the cod liver reference materials using the
full method were in the range 70–120 %. For -TBCO and DPTE the recoveries after the cleanup were on average of 92.1 % and 74.9 % respectively, suggesting interferences during its
quantification in the experiments with spiked blank samples (see Table 6). Similarly, for PBEB
the recoveries were 224 %, suggesting also interferences and an additional problem with the
blanks. The reason for these interferences remains to be investigated.
5.4 Monitoring of aBRF porpoise blubber samples
Figure 2 displays the chromatogram obtained for one of the samples showing some of the
compounds detected.
Figure 2: Extracted MRM chromatograms showing some aBFRs and PBDEs present in a harbour porpoise that was
stranded in the UK in 2008.
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Of the 30 individual compounds determined within the alternative (non-PBDE) flame retardants
group, 19 were not present. Of the remaining 11 compounds, some were detected under the limit
of quantification. Concentrations were in general low, the highest value observed being 35 g/kg
wet weight for PBEB (pentabromoethylbenzene), see Table 8.
Table 8: Concentration of aBFRs detected in harbor porpoise blubber (mg/kg wet weight)
Sample
TBTCC
pTBX TBoCT PBT PBEB DPTE EH-TBB HCDBCO BB 153
SW2008/26
nd
<0.12
<0.12
<0.12
2.39
<1.23
1.64
SW2008/56
<0.16
<0.12
<0.12
<0.12
2.18
<1.21
SW2008/73
nd
<0.12
<0.12
1.8
1.8
nd
SW2008/64
nd
<0.12
<0.12
<0.12
2.24
SW2008
nd
<0.12
nd
nd
2.71
SW2008/98a
nd
<0.16
nd
nd
1.32
SW2008/98b
nd
<0.15
nd
<0.15
35
SW2008/104f
nd
<0.15
nd
<0.15
3.52
nd
SW2008/113c
nd
<0.14
nd
<0.14
<0.34
nd
SW2008/117b
nd
<0.15
nd
<0.15
1.93
nd
SW2008/120
nd
<0.13
<0.13
<0.13
1.36
SW2008/120c
nd
<0.13
nd
nd
SW2008/123b
nd
<0.13
<0.13
1.43
SW2008/134
<0.18
<0.13
<0.13
SW2008/149a
<0.17
<0.13
nd
SW2008/158a
nd
<0.13
SW2008/161
0.21
SW2008/176
a-DP
s-DP
0.2
<0.12
2.38
0.35
1.22
nd
0.24
0.18
0.15
0.8
1.21
0.62
<0.12
<0.12
nd
0.9
1.26
0.39
0.21
0.12
<1.23
<0.61
1.48
0.19
0.23
<0.12
nd
1.57
1.07
1.26
0.21
0.17
nd
0.84
0.88
0.37
0.3
<0.15
1.12
0.7
0.63
0.31
<0.15
<0.68
2.48
0.65
<0.14
<0.14
<0.74
nd
4.57
0.25
<0.15
nd
1.02
2.66
0.7
0.16
<0.13
2.64
nd
3.12
0.48
0.22
0.26
0.15
1.43
nd
1.02
1.76
0.32
<0.13
<0.13
<0.13
1.1
nd
0.95
2.48
0.76
0.15
<0.13
<0.13
13.2
nd
1.78
1.15
2.09
0.17
<0.13
nd
<0.13
2.3
nd
<0.63
3.33
0.63
0.16
<0.13
<0.13
nd
<0.13
2.37
nd
3.44
nd
0.93
0.36
<0.13
nd
<0.12
nd
<0.12
4.54
<1.17
1.68
1.35
0.33
0.14
0.13
SW2008/179a
nd
<0.12
nd
<0.12
6.96
<1.25
1.34
1.98
1.1
0.14
<0.12
SW2008/201b
0.21
<0.15
nd
<0.15
3.34
nd
1.73
nd
4.45
0.24
0.16
SW2008/203
nd
<0.12
nd
nd
1.19 <1.19
2.32
1.38
3.56
0.21
0.14
nd: not detected. The “less than” values are method limits of quantification. They have been calculated for each sample
individually and are related to the exact sample weight taken in each case and so can be different for each sample.
Both anti- (a-DP) and syn-Dechlorane Plus (s-DP) were detected at low concentrations (up to
0.36 g /kg wet weight) in the blubber samples.
For the BDEs, BDE47 was in most cases the dominant congener, averaging 43 % of the sum of
BDEs. BDE183 was found at low concentrations (0.63 to 1.7 g/kg wet weight). (Data not shown,
more details can be found in [7]).
6. Conclusion
In this study, mainly alternative BFRs have been identified that are either not being used in large
volumes in products placed on the UK market or in UK manufacture, or are being used reactively
and so are less likely to leach out of materials. It is likely, however, that other alternative flame
retardant products are being used as replacements for the PBDE formulations.
The presented screening method including pressurized solvent extraction using SpeedExtractor
E-916 and subsequent concentration of the extraction without sample transfer using Syncore®
Analyst is a reliable and fast tool for monitoring the aBFR in biota samples as in marine mammals.
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7. Acknowledgement
The study was founded by the UK Department for Environment, Food and Rural Affairs (DEFRA)
as part of its program of marine environmental research. Post mortem investigations which
yielded the blubber samples used in this study were carried out within the Cetacean Strandings
Investigation Programme, which is funded by Defra and the UK's devolved administrations as part
of the UK Government's commitment to a number of international conservation agreements.
We gratefully thank S. Losada, P. Bersuder and J.L. Barber from CEFAS (Centre for Environment,
Fisheries and Aquaculture Science), Lowestoft, UK for sharing their data and for the fruitful cooperation.
8. References
[1] Bergman A., Rydén A., Law R.J., de Boer J., Covaci A., Alaee M., Birnbaum L., Petreas M.,
Rose M., Sakai S., Van den Eede N., van der Veen I. “A novel abbreviation standard for
organobromine, organochlorine and organophosphorus flame retardants and some
characteristics of the chemicals” Envir. Internat. 2012, 49: 57-82
[2] Covaci, A., Harrad, S., Abdallah, M.A.-E., Ali, N. , Law,, R.J., Herzke, D., de Wit, C.A. “Novel
brominated flame retardants: A review of their analysis, environmental fate and behaviour”
Environ. Internat. 2011, 37: 532–556
[3] Papachlimitzou A., Barber, J.L, Losada, S., Bersuder, P, Law, R.J. “A review of the analysis
of novel brominated flame retardants” J. Chrom. A 2012, 1219: 15-28.
m R., Ebinghaus R. “Brominated flame retardants and dechlorane
plus in the marine atmosphere from southeast Asia toward Antarctica” Environ. Sci. Technol.
2012, 46: 3141−3148
[5] Yang R., Wei H., Guo J., Li A. “Emerging brominated flame retardants in the sediment of the
great lakes” Environ. Sci. Technol. 2012, 46, 3119−3126
[6] Ezechiaš M., Svobodova K., Cajthaml T. “Hormonal activities of new brominated flame
retardants” Chemosphere 2012, 87: 820–824
[7] Law R.J., Losada S., Barber J.L, Bersuder P., Deaville R., Brownlow A., Penrose R., Jepson
P.D. “Alternative flame retardants, Dechlorane Plus and BDEs in the blubber of harbour
porpoises (Phocoena phocoena) stranded or bycaught in the UK during 2008”, Environ.Int.
2013,60: 81–88
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