Kuwait Institute For Scientific Research (KISR)

Central Analytical Laboratory (C.A.L)

Pressurized Liquid Extraction of Polybrominated Diphenyl Ethers in Selected Marine Samples

Followed by Automated Power PrepTM Clean-up

Murad I.H. Helaleh*, Amal Al-Rashdan, Ibtisam A.

Kuwait Institute for Scientific Research (KISR),

Central Analytical Laboratory (CAL)

PBDEs uses:

(1)- Electronic equipment (television sets, computers, radios).

(2)- Textiles and plastics.

(3)- Household products and transport (e.g. synthesis building materials and automobiles.

Why they are added?

To prevent fire and slow down burning process.

How PBDEs can enter into the environment:

(1)- Leaching during production and application process.

(2)- Losses during use and disposal.

(3)- Releases from waste such as incinerators (municipal waste) or deposited in landfills.

(4) Volatilization.

Why PBDEs are environmentally concerned:

(1)- Due to their persistence, potential accumulation.

(2)- Widespread distribution via atmospheric transport.

(3)- Possible adverse effects on wildlife and humans.

Are PBDEs banned?

(1)- Penta and Oct-products were listed as persistence organic pollutants (POPs) and their usage was restricted under the stockholm convention in 2009.

(2)- Deca-production are banned by EU from 2008, due to their ubiquitous, high lipophilicity and inert characteristic.

Where PBDEs are found?

They are found in a variety of environmental media such as:

Air marine mammals

Water birds

human serum Soil

Breast milk

Sediment Humans

Fish

What was the major routes of PBDEs?

(1)- Major human exposure route of PBDEs is through diet. The total dietary intake was estimated between 73 – 93%.

(2)- Recent study shows that fish consumption significantly contributed to the total exposure and the most significant source.

(3)- Dust ingestion and inhalation count also another pathway exposure.

Classification of PBDEs:

(1)- Additive-BFRs such as: polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecane (HBCD) is mixed with or dissolved in the polymer material and can migrate out of the product.

(2)- Reactive-BFRs such as: tetrabromobisphenol A, is covalently bound to the polymers, which make them less release to the environment.

GC/MS (NCI) conditions for the analysis of PBDEs

• PBDEs analysis was conducted using an Agilent 5890, Series II, gas chromatography (Agilent, Avondale, USA) interfaced to a mass selective detector (Agilent 5972, Agilent, Avondale, USA), using Negative Chemical Ionization mode (NCI).

• Separation of PBDEs were performed using a 5% phenyl-methyl-silicone (DB-5MS) bonded-phase fused-silica capillary column (Hewlett-Packard, 15 m x 0.25 mm i.d., film thickness 0.25 µm).

GC/MS (NCI) conditions for the analysis of PBDEs:

Acquisition

method

Ax.

Temp.

Injector

Temp.

Column Oven temperature Helium

gas flow

Methane gas

flow

NCI 320 0C 280 0C

DB-5 MS 15 m, 0.25

mm; i.d. x 0.25 µm

film thickness

capillary column

(Chrom Pack).

800C held for 2.0

min, 2200C at

250C/min held for

0.0 min; 315 0C at

50C/min held for

10.0 min.

1.2

ml/min

40

ml/min

Marine samples:

• Marine samples such as fish, squid, bivalves, shell, octopus and shrimp were analyzed in order to evaluate the automated technique PLE system and the automated Power-prepTM clean-up system.

• Fresh samples (5 g) of each species were mixed with anhydrous sodium sulphate and the extraction cell was filled with the samples.

Marine samples:

• A measured aliquot of surrogate standard mixture and mirex (I.SD), were added to each sample.

• Fish local and English names are as follows: (1) Hamoor (orange spotted grouper); (2) Zubaidy (silver pomfret); (3) Sheim (yellowfin seabream); (4) Suboor (hilsa shad); and (5) Rubyan (green tiger shrimp).

• Samples were collected from local market of Kuwait.

Results and discussion:

Effect of PLE parameters

• Temperature: is a critical parameters for PLE extraction due to its role in improving the extraction efficiency by enhancing the solubility of the analytes in the solvent, improving mass transfer from the matrix to the solvent.

• Pressure: The primary function of the high pressure is to keep the solvent in the liquid state in the PLE cell.

Power prepTM system for clean-up:

PLE system for extraction:

Results and discussion:

Effect of PLE parameters

• The solvent in PLE is under its critical conditions, increases salvation power, decreases viscosities and permits higher diffusion rates for the analytes.

• PLE has been recognized as an official EPA method (method 3545) for the monitoring of POPs in a variety of samples.

Methods Of Quality Control/Quality Assurance:

• A set of experiments were run in the laboratory in order to obtain an acceptable reliable data, which include: mirex as an internal standard. Native PBDEs to measure recoveries for quality control purposes.

• The average recovery were required to be within 70-125%. Recoveries obtained were generally over 80%.

• RSD are lower than 25% in spite of considering changes of days.

Method Of Quality Control/Quality Assurance:

• The recovery were considered to be satisfactory, and there were no interference or a serious co-elution encountered during the evaluation process. The background indicates that the blank was not contaminated and exhibits negligible for most of the analytes tested.

Matrix effect:

• A known concentration of PBDEs were spiked to the fresh weight fish, shellfish samples and one un-spiked sample was extracted at the same time along with procedural blank (Na2SO4).

• The matrix effect was evaluated in order to find out if there was any effect on the analyte concentration.

• The obtained chromatogram matches the un-spiked sample and the blank. The results shows that there was no matrix effect on PBDEs congeners.

Chromatogram of the separated PBDEs using GC-NCI-MS:

Limit of detection (LOD) and limit of quantification (LOQ):

• The LOD and LOQ were determined at the residual level (ng/g), corresponding to signal/noise ratio of 3 for LOD, and 10 for LOQ, respectively.

Concentration of ∑6PBDEs in selected marine

samples:

PBDEs Ham.(n*= 4)

Shell (n= 12)

Zobaidi (n= 5)

Sheim (n= 2)

Shrimp (n= 2)

Sbour (n= 2)

Sponge (n= 5)

Octup. (n= 4)

Squid (n= 2)

Bival.(n= 2)

Total PBDEs

17 2.29 6.67 12.24 2.17 2.86 1.08 0.20 0.91 0.82 1.66 30.89

47 1.15 2.00 1.66 0.22 1.21 0.52 1.15 1.31 0.34 0.37 9.93

100 0.15 0.71 1.09 0.16 0.72 0.47 0.32 0.41 0.35 0.42 4.79

99 0.19 2.11 1.30 0.12 1.45 0.54 0.63 1.15 0.36 0.44 8.30

154 n.d. 0.35 n.d. n.d. 0.23 n.d. n.d. 0.14 n.d. 0.18 0.89

153 n.d. 0.29 n.d. n.d. 0.17 n.d. n.d. 0.10 n.d. n.d. 0.56

∑PBDEs 3.79 12.13 16.54 2.67 6.64 2.60 2.30 4.03 1.86 3.07

* Number of determinations.

Distribution of the total ∑6PBDEs levels in

selected marine samples:

17 47 100 99 154 1530

5

10

15

20

25

30

35

PBDEs congeners

Tot

al c

once

ntr

atio

ns

(ng/

g)

Percentage of the total ∑6PBDEs in selected marine

samples:

PBDEs

congeners

total ∑6PBDEs % of the total

17 30.89 55.79

47 9.93 17.94

100 4.79 8.65

99 8.3 14.99

154 0.89 1.61

153 0.56 1.01

Calibration data of the total ∑6PBDEs:

PBDEsCalibration

Range(pg/µl)

Correlation coefficient (r2)

Limit of detectionLOD (ng/g)

17 16.25-125 0.999 0.05

47 16.25-125 0.997 0.22

100 16.25-125 0.994 0.04

99 16.25-125 0.994 0.13

154 16.25-125 0.993 0.15

15316.25-125 0.991 0.19

PBDEs found in marine samples from other studies (ng/g wet wt):

Location ∑PBDEsa nbSampling

yearReferences Target species

USA 0.011-3.73 13 2006 Schecter et. alHerring, canned tune,

catfish, salmon, trout etc.

Japan 0.018-1.72 6 2002 Ohta et. al Salmon, tuna, yellowtail

Hong Kong

7.29-13.2 22 2008 Cheung et. alYellow croaker, yellowfin

deafin, spotted snakehead, etc.

The Netherlan

d1.2-28 5 2001

De Boer et al. (2003)

Mussels

San Francisco Bay, USA

9-106 3 2002Ores et al.

(2005)Mussels, oysters, clams

Kuwait 1.86-16.54 6 2009Present study.Helaleh et al.

Fish, shell, shrimp, octopus, sponge, bivalves, squid

a) ∑PBDE= Sum of the target PBDE congeners; b) Number of PBDEs congeners analyzed in the samples.

Estimated daily PBDEs intake through selected marine samples by people in Kuwait:

* Estimated daily intake (EDI)= Average concentration (ng/g) x consumption rate (66.4 g/ day).

Average conc. (ng/g

wet wt)

EDI* (ng/ day)

Kuwait USA Belgium Finland Sweden SpainHong Kong

Total Fish 7.39 490.878.94-15.7

14 23 23.1 20.8 514-931

Bivalves, octopus, squid, shell, sponge

2.81 186.85 - - - - - -

ReferencesPresent study

Schecter et al., 2006

Voorspoels et al.,

2007

Kiviranta et al.,

2004

Darnerud et al.,

2006

Domingo et al., 2007

Cheung et al., 2008

Conclusion (1):

• PBDEs concentration in selected marine samples ranged from 1.86-16.54 ng/g wet wt.

• Concentrations of PBDEs were comparable to or higher than those reported from other countries.

• BDEs-17, 47, 99 and 100 were predominant congeners in selected marine samples.

• Daily PBDEs intakes through marine samples consumption estimated in our study were comparable or higher than those reported from other countries.

Conclusion (2):

• The comprehensive method was established, based on automated extraction, automated clean-up and separation of PBDEs from fish and shellfish sample marketed at Kuwait.

• The advantage derived from the use of NCI source along with GC/MS detection is capable of obtaining high selective method, resulting in eliminating the interferences substance from sample matrices efficiently and allowing the detection of PBDEs in fish and shellfish tissue at low ng/g levels.

Conclusion (3):

• The separation of PBDEs showed a good accuracy, precision and linearity in the range studied.

• The cost and time required for the pretreatment of fish and shellfish samples were reduced.

• The effectiveness of the combined power-prep/PLE system with power-prep TM system, make this combination a powerful tool for the analysis of biota samples.

Thank you