Introduction
Marine organisms accumulate many contaminants from their environment. Field and laboratory studies have shown that contaminant concentrations in some marine organisms reflect concentrations in their environment (
FAO/IOC/IAEA, 1993;
Marc et al., 2006). Polychlorinated biphenyls (PCBs) are among the immense variety of toxic organic components that can be found in marine organisms. The PCBs are a class of 209 congeners synthesized by chlorination (1-10 molecules of chlorine) of both biphenyl rings (
Hutzinger, 1974). In the previous decades, PCBs were produced and were widely used in such industrial processes as dielectrics and heat transfer systems mainly because they are highly stable. Due to their persistence, bioaccumulation, and adverse effects on plant and animal life, including humans, the production and use of these chemicals was banned in developed countries during the early 1970s (
Bommanna and Kurunthachalam, 1994). In China, it was reported that approximately 8000 tons of PCBs were produced during the 1960s-70s (
Yang and Matsuda, 2006).
In recent years, although surveys and studies on PCBs in various environment phases has increased in China (
Wu et al., 1999;
Zhou et al., 2000,
2001), few studies have been performed on the analysis of PCBs in marine food. Shellfish are PCBs accumulators and may present special problems if eaten by humans, such as neurotoxicity and dermatological and pulmonary diseases (
Orlinskii et al., 2001). Currently, there is no rapid and reliable PCBs detection method for shellfishes. Most methods are based on simultaneous extraction and determination procedures. The traditional Soxhlet extraction technique needs a considerable time investment (8-36 h) and uses a large amount of various chemical solvents (
Gabriela et al., 2008). Ultrasound-assisted extraction (UAE) has demonstrated good extraction efficiency compared with that of Soxhlet extraction for PCBs in food (
Knorr and Zenker, 2004;
Patist and Bates, 2008). The preferred technique for the determination of PCBs is capillary gas chromatography (GC). For the particular case of organochlorine compounds, mass spectrometry (MS) has been widely used due to its high sensitivity and selectivity (
Alfonso DM et al., 1997;
Patel et al., 2005). In this study, an efficient extraction method by UAE was developed and optimized for the extraction of PCBs from marine shellfish. After extraction, the PCBs are determined by GC/MS.
Materials and methods
Reagents and materials
All chemicals used were of analytical grade. Anhydrous Na2SO4, concentrated H2SO4, and n-hexane were purchased from Tianjin Chemical Reagents Company, China. Silica gel for thin-layer chromatography (80-100 mesh) was purchased from Qingdao Haiyang Chemical Reagents Company, China. A mixed standard of PCBs including PCB 28, 52, 101, 138, 153, and 180 was purchased from Dr. Ehrenstorfer (Augsburg, Germany). The standard stock solution of target compounds (1 mg·L-1 of each PCBs) was prepared in n-hexane, and the solutions were stored in the dark at 4°C. Nitrogen and helium (Persee, Beijing) were respectively used for the final evaporation of the extracts and as a GC carrier gas.
Instrumentation
A 40 kHz and 300 W US-bath (Jiangsu Kunshan Instruments, Jiangsu, China) was used for assisting the back-extraction process. Rotary evaporator (Shanghai Yarong Biochemistry Instrument Factory, Shanghai, China) was obtained for the concentration of samples. GC7890/MS5975 was purchased from USA (Agilent, USA) with autosampler. The centrifuge was obtained from Anke Instruments (Shanghai Anting, China). The transmembrane equipment was purchased from Hoefer (USA). The tissue grinder equipment was from JiuYang (Shandong, China).
GC/MS analysis
The GC/MS analysis of samples was performed using an Agilent GC/MS equipped with an HP-5MS capillary column (30 m × 0.25 mm, 0.25 μm film thickness). The following temperature program was applied: 100°C (2 min) - 15°C/min - 180°C (0 min) - 3°C/min -240°C (0 min) - 10°C/min - 270°C (2 min). Helium was employed as carrier gas at 1mL/min. The injector, ion source, and transfer line were respectively set at 250°C, 250°C, and 280°C. One microlitre of sample was injected in splitless mode. The ionization was made by electronic impact at 70 eV.
Standard and sample preparation
Ultrasound assisted extraction
The shellfish samples were collected from the coastal areas of Qinhuangdao City, Hebei Province, China. The collected specimens were cleaned and preserved in deep freeze until analysis.
Each sample of shellfish, typically 5 g, was accurately weighed in a 40-mL glass vial, a 20 mL of extraction solvent n-hexane, and 3 g anhydrous Na
2SO
4 were added and mixed. The resulting mixture was immersed into an ultrasonic bath for 60 min at 45°C. The mixture was centrifuged at 4000 r·min
-1 for 5 min to separate the solvent from the sediment (
Gabriela et al., 2008;
Ozcan et al., 2009). This procedure was repeated twice. The solvent was concentrated by Rotary evaporator, and extraction solvent was removed from the bottom of the bottle by using 5 mL n-hexane, decontaminated by 2 mL concentrate H
2SO
4. Then, the sample was loaded into a mixed silica column.
Cleanup procedure
The sample preparation was carried out in glass columns, which were 7.6 cm long with 1.3 cm of inner diameter. The columns were filled from bottom to top, with 1 g of anhydrous Na
2SO
4, 2 g of neutral activated silica, 2 g of sulphuric acid modified activated silica, and 2 g of anhydrous Na
2SO
4. This multilayer silica column was washed with 20 mL of n-hexane prior to use (
Nevado et al., 2010). The samples were then eluted by 60 mL of n-hexane with a peristaltic pump, collecting the eluent in the test tubes. Later, this eluent was evaporated under a gentle nitrogen stream. Finally, samples were reconstituted with 1 mL of n-hexane and analyzed by GC/MS.
Results
Chromatogram of standard and samples (Figs. 1 and 2)
Calibration curve, recovery, and method detection limit
A PCBs mixed standard including PCB 28, 52, 101, 138, 153, and 180 (1 mg·L
-1 of each PCB) was prepared in n-hexane. Working solutions were prepared by dilution of standard stock solution with n-hexane into 300, 200, 100, 50, 25, 10 ng·mL
-1. The method detection limit was calculated using the 3 S/N ratio. Six replicate samples of fan scallop (
Argopectens irradias) spiked with two levels of standard solutions of PCBs (50 ng·mL
-1 and 100 ng·mL
-1), and one unspiked sample were analyzed using the procedures mentioned in the Section 2.4 to calculate recovery percentages and relative standard deviations (RSD) of PCBs (
Stephen et al., 1997;
Fontana et al., 2009;
Jose et al., 2010). The standard curves, recovery, and detection limits are shown in Table 1.
Optimization conditions
To reduce the consumption of both sorbents and eluent, a simplification column was used, as recommended in previous works (
Berzas et al., 2007;
GB/T5009.190-2006). Likewise, in order to reduce the analysis time, we used a peristaltic pump for the elution of the analytes, rather than gravity.
The total GC run time was 33 min, including a postrun step of 3 min at 280°C to remove impurities trapped in the column. For mass spectrometry detection, the filament was switched on, 6 min after the injection, which is approximately 1 min before the elution of the first peak of interest, to diminish damage to the mass detector.
Actual analysis of marine shellfish
Six main species of marine shellfish samples (Argopecten irradians, Gomphina aequilatera, Crassostrea virginica, Pecten yessoensis, Scapharca subcrenata, and Mytilus edulis) collected from the coastal areas of Qinhuangdao city (Hebei Province, China) were analyzed for PCBs according to the procedures mentioned above. The analytical results are shown in Table 2. The results show that all these economic shellfish had been contaminated by PCBs. The Mytilus edulis were most effective in accumulating PCBs congeners, as their total levels were the highest (Table 2). Pecten yessoensis had the fewest congeners, and the lowest total level. PCB 28, 52, and 118 were detected in all the marine shellfish samples, whereas PCB 101, 130, 153, and 180 were only found in some samples. The predominant contaminants were PCB 28, 52, and 118. The total PCBs level ranged from 14.12 to 33.24 μg·kg-1, which was below the edible hygienic criteria of 200 μg·kg-1 (GB9674-88; SC/T-3111-2006). These results indicate that the industrial pollutants are still under control in the coastal areas of Qinhuangdao.
Conclusions and discussion
Using marine shellfish as test organisms, this study established a method for the simultaneous purification and separation of seven PCBs congeners with a complex column using UAE techniques. Furthermore, these seven groups of PCBs in the same sample were determined using GC/MS modes. Consequently, the cost and time required for the pretreatment of biota sample were reduced, and more reliable quantification and identification results were obtained. The economic shellfish in the coastal areas of Qinhuangdao city had been contaminated by PCBs, but the level of total PCBs is lower than the edible hygienic criteria, indicating that the pollutants remain controlled.
Higher Education Press and Springer-Verlag Berlin Heidelberg
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