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Simultaneous determination of tetrachloro dibenzo-p-dioxin and poly-aromatic chlorinated biphenyls in aqueous environment using liquid phase microextraction
Physics and Chemistry of the Earth 50–52 (2012) 98–103
Contents lists available at SciVerse ScienceDirect
Physics and Chemistry of the Earth journal homepage: www.elsevier.com/locate/pce
Simultaneous determination of tetrachloro dibenzo-p-dioxin and poly-aromatic chlorinated biphenyls in aqueous environment using liquid phase microextraction Phumile Sikiti, Titus A.M. Msagati ⇑, Ajay K. Mishra, Bhekie B. Mamba University of Johannesburg, Faculty of Science, Department of Applied Chemistry, Doornfontein Campus, Johannesburg 028, South Africa
a r t i c l e
i n f o
Article history: Available online 6 October 2012 Keywords: Dioxins Poly-aromatic chlorinated biphenyls Liquid phase microextraction Gas chromatography–mass spectrometry
a b s t r a c t Among the most notable and notorious persistent organic pollutants in many aquatic environments are the dioxins and poly-aromatic chlorinated biphenyls (PCBs). These compounds are nuisances in the environment due to their toxicities which come mainly as a result of their tendencies to bio accumulate because of their lipophilic nature. Dioxins and PCBs belong to the group of compounds known as persistent organic pollutants (POPs). Since these compounds are problematic when they are discharged in the environment, strict regulations and guidelines with regard to their use and discharge has been put in place. Of the dioxin congeners, 2,3,7,8-TCDD (2,3,7,8-Cl4DD) is the most toxic while a number of PCB congeners such PCB-1, PCB-28 and PCB 101 are also known to cause pollution when present in the environment. In this work, the analytical monitoring strategies for dioxins and PCBs employing extraction and purification of samples using liquid phase microextraction as well as gas chromatography and mass spectrometry for the separation and detection of the extracts was employed. The extraction results were validated by various statistical tests such as linearity, accuracy, precision, reproducibility and repeatability data. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction In recent years, much attention has been paid to pollution due to the persistent organic pollutants such as dioxins and PCBs due to their toxicity, high lipophilicity and low water solubility. The compounds are primarily bound to particulate and organic matter in soil, sediment, in biota and they are concentrated in fatty tissues of aquatic organisms. These compounds are environmental contaminants that are detectable in all almost all comportment of the global ecosystem in trace amount (Fiedla et al., 1999). Dioxins and PCBs are chemically similar in terms of their physicochemical properties, and their toxicity responses (Van der Berg et al., 1998; Alcock et al., 1999) and they are suspected to cause disease conditions such as dermal disorder, hepatic damages (Safe, 1990; Safe et al., 1991), endocrine disruption, hypovitaminosis A, hypothyroidism carcinogenicity, neutrotoxicity (Safe et al., 1985; Safe, 2000) and reproductive negative effects in humans wild life (Kennedy et al., 1996). They also do not decompose readily in the environment (Giesy and Kannan, 1998) due to their low half life. Moreover these chemical are hydrophobic and resist metabolism, resulting in their bioaccumulation in fatty tissues in animals and humans. The dioxins-like compounds and PCBs are discharged into the ecosystem through a combination of natural phenomena such ⇑ Corresponding author. Tel.: +27 11 559 6216; fax: +27 11 559 6425. E-mail address: [email protected] (T.A.M. Msagati). 1474-7065/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pce.2012.09.001
as weathering reactions, volcanic emission, and anthropogenic process which include mining, chemical and pesticide manufacturing, pulp and paper bleaching processes, burning of household trash, forest fires, and burning of industrial and medical waste products (Im et al., 2002). Besides the man made sources that are known to produce dioxins, enzyme-mediated formation of dioxins from 2, 4, 5 and 3, 4, 5-tri-chlorophenol has been demonstrated in vitro to be responsible for 63 biogenic formation, e.g., in sewage sludge, compost (Oberg et al., 1990). Human exposure may occur mainly through consumption of contaminated food (such as sea-foods) or water (WHO, 2002). The United States Environmental Protection Agency (EPA) has set maximum contaminants level (MCL) for dioxins in drinking water as 0.00003 lg/L (0.03 pg/L) (EPA, 1994) to protect the possible risk exposure of dioxins-like compounds to human. The MCL for dioxins have been set at very low values because the presence of dioxins-like compounds in water causes a health risk at even lower concentration. However, the concentration levels of dioxins and PCBs in environmental water systems are very low due to dilution and the fact that their solubility in water is low. This then brings in some analytical challenges where quantitative analysis is required for the individual dioxins and furans compounds because of the low detection limits required. Therefore very sensitive and selective clean up and enrichment steps are often required before the detection step of these compounds in order to improve their limits of detection. This makes the development of sensitive analytical
P. Sikiti et al. / Physics and Chemistry of the Earth 50–52 (2012) 98–103
Fig. 1. Selection criterion for the organic solvent suitable for the simultaneous extraction of dioxins and PCBs.
methods for pre-concentration and enrichment of dioxins-like compounds in environmental matrices to be a necessity in order to comply with existing and new environmental regulations. Moreover, there is a general concept that the sample preparation is the most important step in the analytical procedures (Mitra, 2003). The common approach for sample preparation that has been reported in many analytical fields for handling and analysis of complex sample is the Soxhlet extraction that was originally inverted in 1879, and was used to extract dioxins from soil samples. In the 1990s, Richter and co-workers introduced extraction procedure that involved elevated temperature and pressure, named accelerated solvent extraction (ASE) or pressurised liquid–liquid extraction (Richter et al., 1996). ASE and the Soxhlet extraction has been successfully used for various purposes including the extraction of PAHs, PCBs, and dioxins from reference material and contaminated solids samples (Richter et al., 1997). However, Soxhlet as a sample preparation method is not economical as it uses lots of organic solvents as well as high temperatures and it is also time consuming and laborious. Using harmful chemicals and large amount of solvents causes environmental pollution, and extra operational costs for waste treatment. Therefore other alternative methods to the Soxhlet extraction were introduced and these include supercritical fluid extraction, and micro-assisted extraction (Pedersen-Bjergaard and Rasmussen, 1999). However, these methods are also known to have shortcomings in terms of economic and the environmental concerns, hence the solids phase extraction approach for sample preparation has become a candi-
99
date of choice in many analytical fields for handling and analysis of complex samples (Stevenson, 1999). Solid phase extraction (SPE) is attractive as the sample preparation technique for analyte in complex matrix due to the following benefits: it is very selective, effective with a variety of matrices, concentration effect, with high recovery and reproducibility. However, the solids phase extraction has a number of drawbacks like: greater complexity/difficult to master, lengthy method development, and costly. Ideally, sample preparation techniques should be fast, easy to use, inexpensive and compatible with a range of analytical instruments. This has encouraged the current trend towards simplification and miniaturisation of the sample-preparation steps and minimisation of quantities of organic solvents. Liquid phase hollow fibre supported liquids membrane microextraction technique is proposed in this study as alternative techniques which will render sample preparation that is simple, less demanding, cost effective and give highly consistent, quantifiable results for very low concentration of analytes as well as high recovery and high enrichment factors (Safe et al., 1985). The extracts from the sample pre-treatment procedures are normally in mixtures and need a separation step before allowing them to go to the detection devices. So far, most of the developed methods have been based on combining a separation technique with specific detection techniques (Rezaee et al., 2006). Various hyphenated techniques such as high resolution gas chromatography coupled to high resolution mass spectrometry have been applied to determine traces of dioxins-like compounds from environmental samples, and high resolution gas chromatography with electron capture detector (Pyell, 2001). A number of separation methods have been used for the separation of dioxins-like compounds from the environment. These methods include electrophoresis, whereby, separation of the compounds is obtained via differential migration of solutes of charged species in an electric field performed in narrow-bore capillaries filled only with buffer (Jiang and Lucy, 2002). Separation by electrophoresis is becoming an advantageous tool for determination of organic compounds such as dioxins-like compounds and many more due to its flexibility in manipulating various parameters on the column in order to obtain the separation. Although separation time is very small with very little sample and solvent, the detection sensitivity of capillary electrophoresis compare to gas chromatography and HPLC has not been satisfactory due to the low UV sensitivity attributed to its path length and low injection volume (Dien Liem, 1999). The detection of dioxins and PCBs with different techniques have been reported widely and the majority of these methods employ the use of gas chromatography with various detectors (Focant
Fig. 2. Optimisation of sample pH.
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P. Sikiti et al. / Physics and Chemistry of the Earth 50–52 (2012) 98–103
2,2',4',4-Tetrachlorobiphenyl 240000
2.1. Materials and methods
230000
Standards 2,3,7,8 TCDD and PCB-1 (2-monochlorobiphenyl), PCB-28 (2,4,40 -tri-chlorobiphenyl), PCB-101 (2,2,4,5,5-pentachlorobiphenyl) and the organic solvent isooctane were purchased from Riedel-de-Haën (Seelze-Hannover, Germany). Pesticidequality solvents (n-hexane and toluene) and calcium chloride were purchased from Panreac (Barcelona, Spain). Sodium hydroxide pellets (98%) was obtained from Saarchem (Krugerdorp, Africa). Stock standard solutions of individual compounds (with concentrations of 1000 mg/L) were prepared. Ttriton x-100 and hydrochloric acid were purchased from N.T. Laboratory Supplies, (Johannesburg, South Africa). A multicompound working standard solution (1 lg/mL concentration of each compound) was prepared by appropriate dilutions of the stock solutions (1000 mg/L) with hexane:toluene (1:1) and stored under refrigeration (4 °C). The hollow fibre tubing used in the extractions were Q3/2 Accurel polypropylene (200 lm wall thickness, 600 lm inner diameter, 0.2 lm pore size) obtained from Membrana (Wuppertal, Germany). An Agilent Technologies 10 lL microsyringe was used in the hollow fibre supported liquid membrane (HFSLM) extraction.
220000
Ee
2. Experimental
210000 200000 190000 180000 170000 5
10
15
20
25
30
Extraction Time (mins) Fig. 3. Optimisation of extraction time.
2,2'4',4-Tetrachlobiphenyl 150000000 140000000
2.2. Gas chromatographic (GC–ECD) analysis of HF-LPME extracts
130000000
The HP Agilent 6890 GC/5973 (Santa Clara, CA 95051 USA) was used for the analysis of dioxins and PCBs. The column, DB-5 (30 m 0.25 mm i.d. 0.25 lm film thickness) from Supelco (Bellefonte, PA, USA), was used for all separations. The flow rate of the carrier gas (nitrogen) was 1.5 mL/min and the injection volume was 0.5 lL. The glass liner was equipped with a plug of carbofrit (Resteck, Bellefonte, PA, USA). The GC temperature program used for the separation of the compounds had the initial oven temperature set at 40 °C and held for 1 min. The temperature was then ramped at 9 °C/min to 285 and held for 10 min.
Ee
120000000 110000000 100000000 90000000 80000000 70000000 100
150
200
250
300
Stirring Speed (rpm)
3. Results and discussion Fig. 4. Optimisation of the stirring speed.
et al., 2005). These methods are attractive in terms of speed, cost and sample turn over (Focant et al., 2005). In this work, a gas chromatograph equipped with an electron capture detector has been employed in the analysis of dioxins and PCBs.
3.1. Optimisation of parameters in the liquid phase microextraction of dioxins and PCBs A number of extraction parameters had to be optimised to enhance the extractability of dioxins and PCBs. Factors studied
Fig. 5.1. GC–ECD chromatogram for PCB101 standard.
P. Sikiti et al. / Physics and Chemistry of the Earth 50–52 (2012) 98–103
Peak area
(a) 5000
3.3. Optimisation of sample pH
y = 2078.4x + 160.4 R2 = 0.9931
4500 4000 3500 3000 2500 2000 1500 1000 500 0 0
0.5
1
1.5
2
2.5
Concentration/ppm Fig. 5.2a. Calibration curve for PCB-1.
Peak area
(b) 2500000 2000000 1500000 1000000 500000 0 0.5
1
1.5
The pH of a mixture of sample solution was optimised by keeping all other factors constant (using 5% TOPO in isooctane). These factors include pH of the stripping solution, stirring speed, organic solvent and extraction time. The sample pH range investigated ranged from pH 6 to 10. The extraction time was 15 min and at a stirring speed 300 rpm. The results of optimisation are shown in Fig. 2. The optimal sample pH was found to be between 7 and 8 (Fig. 2). The compounds were mostly extractable at pH near or around neutral which displayed the most efficient extraction. This optimal pH range was used for all the subsequent extraction. 3.4. Optimisation of time of extraction
y = 1E+06x + 14891 R2 = 0.996
0
101
2
2.5
Concentration/ppm Fig. 5.2b. Calibration curve for PCB-28.
The time of the extraction of dioxins and PCBs was varied as it affects sample enrichment. It is expected that the longer the contact time the higher the extraction as there will be a longer period for mass transfer of the analyte into the fibre. The results for the optimisation are shown in Fig. 3. The results obtained from Fig. 3 show that the enrichment factors increased with extraction timeup to the optimum point at 15 min. However, the extraction efficiency began to decrease after 15 min presumably due to the saturation of the receiving solution. 3.5. Optimisation of stirring speed of the extraction
Peak area
(c)
800000 700000 600000 500000 400000 300000 200000 100000 0
y = 328011x + 38732 R2 = 0.9801
0
0.5
1
1.5
2
2.5
Concentration/ppm Fig. 5.2c. Calibration curve for 2,3,7,8-TCDD.
The effect of stirring speed on the efficiency of the extraction of dioxins and PCBs was also investigated. The results of optimisation are shown in Fig. 4. From the results it is evident that an increase in the stirring speed increases the efficiency of the extraction. The motion of the solution in which the fibre is placed increases the rate of mass transfer. The optimum speed was at 100 rpm. Speeds higher than this showed a negative effect on the extraction. Higher stirring speeds reduce the contact of the analyte with the membrane, thus reducing the amount extracted. 3.6. GC–ECD analyses of extracts
(d) 1400000
y = 606782x + 54374 R2 = 0.9915
Peak area
1200000
The extracts were analysed using a gas chromatograph equipped with an electron capture detector and the identification was done by means of comparison of the retention times of standards and the samples extracts (Fig. 5.1).
1000000 800000 600000 400000 200000 0 0
0.5
1
1.5
2
2.5
concentration/ppm Fig. 5.2d. Calibration curve for PCB-101.
included, the type of organic solvent, sample pH, extraction time, and the stirring speed.
3.2. Optimisation of organic liquid membrane The organic liquid membrane in HFSLM extraction influences the efficiency of the extraction and the selectivity of the enrichment of the analytes. The type of organic solvent chosen should be stable, with a low volatility. It should not be soluble in water or have a very low solubility (Mitra, 2003). In the extraction of dioxins and PCBs, the organic liquids tested were octanol and isooctane. The results of optimisation depicted in Fig. 1 shows that isooctane gave the highest enrichment factors.
3.7. Quantitation The optimum conditions for the extraction process were used in the extraction of a mixture of all the four compounds. The optimum conditions of extraction was with sample pH of 7, stirring speed 100 rpm, extraction time of 15 min and organic solvent was isooctane. Calibration curves of peak areas vs. concentration for each of the compounds was plotted and used to quantify the amount extracted (Figs. 5.2a–5.2d). 3.8. Statistical validation of the extraction process To validate the results obtained for during the extraction process, a number of statistical tests were performed. These include the test for linearity in the extraction process to ensure that there were no external causes of loss of extracts. Repeatability and reproducibility experiments were set and the results obtained were used to confirm that the method is robust and reliable.
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y
sample independent of time. Mathematically the two variables can be represented by the following equations:
y = 10112x + 1265.6 R2 = 0.9607
12000
Peak area
10000
Reproducibility ¼ 2:77ðr2 t þ r2 =nÞ1=2
8000 6000
Repeatability ¼ 2:77ðr2 Þ1=2
4000 2000 0 0
0.2
0.4
0.6
0.8
1
1.2
Concentration/ppb Fig. 6. Linearity studies during the liquid phase extraction process for 2,3,7,8-TCDD.
Table 1 Testing series. 1st Day
2nd Day
3rd Day
4th Day
PCB1-1a PCB1-1b PCB28-1a PCB28-1b
TCDD-1a TCDD-1b PCB101-1a PCB101-1b
PCB1-2a PCB1-2b PCB28-2a PCB28-2b
TCDD-2a TCDD-2b PCB101-2a PCB101-2b
Table 2 Average enrichment factors and standard deviations from HFSLM. Pesticide
Average enrichment factors
Standard deviation
PCB101 TCDD PCB28 PCB1
0.712 1.012 8.576 6.446
0.121 0.477 0.0962 0.7264
Table 3 Comparison between repeatability and reproducibility of HFSLM (enrichment factors). Pesticide
Repeatability
Reproducibility
PCB101 TCDD PCB28 PCB1
0.0051 0.0102 0.5600 1.4600
0.0024 0.0090 0.3512 0.9961
3.9. Linearity studies during the liquid phase extraction process Linearity experiments were performed at optimal conditions by extracting a spiked water sample at an increasing concentration of individual compounds. Fig. 6 shows a good linearity for 2,3,8,9TCDD with r2 of 0.9 which is an indicative of a good performance of the extractability of this compound. The other compounds as well gave r2 values of 0.9 thus proving that the developed method worked well with all the compounds.
3.10. Repeatability and reproducibility during the liquid phase microextraction To determine the reliability of the liquid phase microextraction technique, reproducibility and repeatability in the extraction process was evaluated. Reproducibility refers to the difference in the results obtained between two quantities of the same sample (number of repeated measurements made on the same sample). Repeatability refers to the difference of results obtained on the same
For these set of experiments, the four pesticide solutions, each of 1 mg/L, were extracted twice, one after the other, over period of 4 days. The experiments are shown in Table 1. The results obtained (Tables 2 and 3) suggest that PCB1 and PCB28 gave better reproducibility and repeatability than TCDD and PCB101. This could be attributed by the fact that, TCDD and PCB101 are more hydrophobic than PCB1 and PCB28 thus posing relatively more difficulties in terms of their extractability than PCB1 and PCB28. 4. Conclusion A method for the simultaneous analysis of 2,3,7,8 TCDD and three PCB compounds in water using liquid phase microextraction was established. Factors affecting the efficiency of the extraction were optimised by varying the sample pH as well as the extraction time, stirring speed and organic liquid solvent. However, the attractive features of this method are the use of minimal organic solvent, low cost, simplicity and fewer sample handling steps. Acknowledgement Financial support from NIC-Mintek is gratefully appreciated. References Alcock, R.E., Gemmil, R., Jones, K.C., 1999. Improvement to the UK PCDD/F and PCB atmospheric emission inventory following an emission measurement programme. Chemosphere 38 (4), 759–770. Dien Liem, A.K., 1999. Important development in methods and techniques for the determination of dioxins and PCBs in foodstuffs and human tissues. TrAC Trends Anal. Chem. 18, 449–507. Fiedler, H., Hutzinger, O., Timms, C., 1999. Dioxins: sources of environmental load and human exposure. Toxicol. Environ. Chem. 29, 157–234. Focant, J.F., Pirard, C., De Pauw, E., 2005. Recent advances in mass spectrometric measurement of dioxins. J. Chromatogr. A 1067, 265–275. Giesy, J.P., Kannan, K., 1998. Dioxins-like and non-dioxins-like toxic effects of polychlorinated biphenyl: implications for the risk assessment. Crit. Rev. Toxicol. (28), 511–569. Im, S.H., Kannan, K., Giesy, J.P., Matsuda, M., Wakimoto, T., 2002. Concentration and profiles of polychlorinated dibenzo-p-dioxins and dibenzofurans in soil from Korea. Environ. Sci. Technol. 36 (17), 3700–3705. Jiang, J., Lucy, C.A., 2002. Determination of alkylphosphoric acids using micellar electrokinetic chromatography with laser-induced fluorescence detection and high salt stacking. J. Chromatogr. A 966, 243–244. Kennedy, S.W., Lorenzen, A., Norstrom, R.J., 1996. Chicken embryo hepatocyte bioassay for measuring cytochrome P5014A-based 2, 3, 7, 8tetrachlorodibenzo-dioxins equivalent concentrations environmental samples. Environ. Sci. Technol. 30, 706–715. Mitra, S., 2003. Sample Preparation Techniques in Analytical Chemistry. WileyInterscience, Ney York. Oberg, LG., GLas, B., Swanson, SE., Rappe, C., Pual, K.P., 1990. Peroxidase-catalyzed oxidation of chlorophenol to polychlorinated dibenzo-p-dioxins and dibenzonofurans. ERCH Environ. Contam. Toxicol. 19, 93–938. Pedersen-Bjergaard, S., Rasmussen, K.E., 1999. Anal. Chem. 71, 2650. Pyell, U., 2001. Micellar electrokinetic chromatography from theoretical concept to real sample (review). Fresenius J. Anal. Chem. 371, 691–703. Rezaee, M., Assadi, Y., Millani, M.R., Aghaee, E., Ahmadi, F., Berijani, S., 2006. J. Chromatogr. A 1116, 1. Richter, B.E., Jones, B.A., Ezzell, J.L., Porter, N.L., Avdalovic, N., Poll, C., 1996. Accelerated solvent extraction: a technique for sample preparation. Anal. Chem. 68, 1033–1039. Richter, B.E., Ezzell, J.L., Knowles, E., Hoefler, F., Mullalat, A.K.R., Schuntwinkel, M., Waddel, D.S., Gobran, T., Khurans, V., 1997. Extraction of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans from environmental samples using accelerated solvent extraction (ASE). Chemosphere (34), 975– 987. Safe, S., 1990. Polychlorinated-biphenyl (PCBS), dibenzo-para-dioxins (PCDDs), dibenzofurans (PCDFs), and related-compounds environmental and
P. Sikiti et al. / Physics and Chemistry of the Earth 50–52 (2012) 98–103 mechanistic consideration which support the development of toxic equivalent factors (TEFs). Crit. Rev. Toxicol. 21, 51–88. Safe, SH., 2000. Endocrine disruptors and human health – is there a problem? An update. Environ. Health Perspect. 108, 487–493. Safe, S., Bandiera, S., Sawyer, 1985. Effect of structure on binding to the 2, 3, 7, 8TCDD receptor protein and AHH induction halogenated biphenyl. Environ. Health Perspect. 61, 21–33. Safe, S., Astroff, B., Harris, M., 1991. 2, 3, 7, 8-Tetrachloridibenzo-para dioxin (TCDD) and related-compounds as antiestrogens characterization and mechanism of action. Pharmacol. Toxicol. 69, 400–409.
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Stevenson, D., 1999. Molecularly imprinted polymers for solid-phase extraction. TrAC. Trends Anal. Chem. 18, 154–158. Van der Berg, M., Birnbuam, L., Bostveld, ATC., Brunostorm, B., 1998. Toxic equivalent factors (TEFs) for PCBs, PCDDs, PCDFs for humans and wildlife. Environ. Health Perspect. 106, 775–792. WHO, 2002. Polychlorinated dibenzodioxins, polychlorinated dibenzofurans, and coplanar polychlorinated biphenyls. In: Safety Evaluation of Certain Food Additives and Contaminants. Geneva, World Health Organization (WHO Food Additives Series, No. 48).
Contents lists available at SciVerse ScienceDirect
Physics and Chemistry of the Earth journal homepage: www.elsevier.com/locate/pce
Simultaneous determination of tetrachloro dibenzo-p-dioxin and poly-aromatic chlorinated biphenyls in aqueous environment using liquid phase microextraction Phumile Sikiti, Titus A.M. Msagati ⇑, Ajay K. Mishra, Bhekie B. Mamba University of Johannesburg, Faculty of Science, Department of Applied Chemistry, Doornfontein Campus, Johannesburg 028, South Africa
a r t i c l e
i n f o
Article history: Available online 6 October 2012 Keywords: Dioxins Poly-aromatic chlorinated biphenyls Liquid phase microextraction Gas chromatography–mass spectrometry
a b s t r a c t Among the most notable and notorious persistent organic pollutants in many aquatic environments are the dioxins and poly-aromatic chlorinated biphenyls (PCBs). These compounds are nuisances in the environment due to their toxicities which come mainly as a result of their tendencies to bio accumulate because of their lipophilic nature. Dioxins and PCBs belong to the group of compounds known as persistent organic pollutants (POPs). Since these compounds are problematic when they are discharged in the environment, strict regulations and guidelines with regard to their use and discharge has been put in place. Of the dioxin congeners, 2,3,7,8-TCDD (2,3,7,8-Cl4DD) is the most toxic while a number of PCB congeners such PCB-1, PCB-28 and PCB 101 are also known to cause pollution when present in the environment. In this work, the analytical monitoring strategies for dioxins and PCBs employing extraction and purification of samples using liquid phase microextraction as well as gas chromatography and mass spectrometry for the separation and detection of the extracts was employed. The extraction results were validated by various statistical tests such as linearity, accuracy, precision, reproducibility and repeatability data. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction In recent years, much attention has been paid to pollution due to the persistent organic pollutants such as dioxins and PCBs due to their toxicity, high lipophilicity and low water solubility. The compounds are primarily bound to particulate and organic matter in soil, sediment, in biota and they are concentrated in fatty tissues of aquatic organisms. These compounds are environmental contaminants that are detectable in all almost all comportment of the global ecosystem in trace amount (Fiedla et al., 1999). Dioxins and PCBs are chemically similar in terms of their physicochemical properties, and their toxicity responses (Van der Berg et al., 1998; Alcock et al., 1999) and they are suspected to cause disease conditions such as dermal disorder, hepatic damages (Safe, 1990; Safe et al., 1991), endocrine disruption, hypovitaminosis A, hypothyroidism carcinogenicity, neutrotoxicity (Safe et al., 1985; Safe, 2000) and reproductive negative effects in humans wild life (Kennedy et al., 1996). They also do not decompose readily in the environment (Giesy and Kannan, 1998) due to their low half life. Moreover these chemical are hydrophobic and resist metabolism, resulting in their bioaccumulation in fatty tissues in animals and humans. The dioxins-like compounds and PCBs are discharged into the ecosystem through a combination of natural phenomena such ⇑ Corresponding author. Tel.: +27 11 559 6216; fax: +27 11 559 6425. E-mail address: [email protected] (T.A.M. Msagati). 1474-7065/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pce.2012.09.001
as weathering reactions, volcanic emission, and anthropogenic process which include mining, chemical and pesticide manufacturing, pulp and paper bleaching processes, burning of household trash, forest fires, and burning of industrial and medical waste products (Im et al., 2002). Besides the man made sources that are known to produce dioxins, enzyme-mediated formation of dioxins from 2, 4, 5 and 3, 4, 5-tri-chlorophenol has been demonstrated in vitro to be responsible for 63 biogenic formation, e.g., in sewage sludge, compost (Oberg et al., 1990). Human exposure may occur mainly through consumption of contaminated food (such as sea-foods) or water (WHO, 2002). The United States Environmental Protection Agency (EPA) has set maximum contaminants level (MCL) for dioxins in drinking water as 0.00003 lg/L (0.03 pg/L) (EPA, 1994) to protect the possible risk exposure of dioxins-like compounds to human. The MCL for dioxins have been set at very low values because the presence of dioxins-like compounds in water causes a health risk at even lower concentration. However, the concentration levels of dioxins and PCBs in environmental water systems are very low due to dilution and the fact that their solubility in water is low. This then brings in some analytical challenges where quantitative analysis is required for the individual dioxins and furans compounds because of the low detection limits required. Therefore very sensitive and selective clean up and enrichment steps are often required before the detection step of these compounds in order to improve their limits of detection. This makes the development of sensitive analytical
P. Sikiti et al. / Physics and Chemistry of the Earth 50–52 (2012) 98–103
Fig. 1. Selection criterion for the organic solvent suitable for the simultaneous extraction of dioxins and PCBs.
methods for pre-concentration and enrichment of dioxins-like compounds in environmental matrices to be a necessity in order to comply with existing and new environmental regulations. Moreover, there is a general concept that the sample preparation is the most important step in the analytical procedures (Mitra, 2003). The common approach for sample preparation that has been reported in many analytical fields for handling and analysis of complex sample is the Soxhlet extraction that was originally inverted in 1879, and was used to extract dioxins from soil samples. In the 1990s, Richter and co-workers introduced extraction procedure that involved elevated temperature and pressure, named accelerated solvent extraction (ASE) or pressurised liquid–liquid extraction (Richter et al., 1996). ASE and the Soxhlet extraction has been successfully used for various purposes including the extraction of PAHs, PCBs, and dioxins from reference material and contaminated solids samples (Richter et al., 1997). However, Soxhlet as a sample preparation method is not economical as it uses lots of organic solvents as well as high temperatures and it is also time consuming and laborious. Using harmful chemicals and large amount of solvents causes environmental pollution, and extra operational costs for waste treatment. Therefore other alternative methods to the Soxhlet extraction were introduced and these include supercritical fluid extraction, and micro-assisted extraction (Pedersen-Bjergaard and Rasmussen, 1999). However, these methods are also known to have shortcomings in terms of economic and the environmental concerns, hence the solids phase extraction approach for sample preparation has become a candi-
99
date of choice in many analytical fields for handling and analysis of complex samples (Stevenson, 1999). Solid phase extraction (SPE) is attractive as the sample preparation technique for analyte in complex matrix due to the following benefits: it is very selective, effective with a variety of matrices, concentration effect, with high recovery and reproducibility. However, the solids phase extraction has a number of drawbacks like: greater complexity/difficult to master, lengthy method development, and costly. Ideally, sample preparation techniques should be fast, easy to use, inexpensive and compatible with a range of analytical instruments. This has encouraged the current trend towards simplification and miniaturisation of the sample-preparation steps and minimisation of quantities of organic solvents. Liquid phase hollow fibre supported liquids membrane microextraction technique is proposed in this study as alternative techniques which will render sample preparation that is simple, less demanding, cost effective and give highly consistent, quantifiable results for very low concentration of analytes as well as high recovery and high enrichment factors (Safe et al., 1985). The extracts from the sample pre-treatment procedures are normally in mixtures and need a separation step before allowing them to go to the detection devices. So far, most of the developed methods have been based on combining a separation technique with specific detection techniques (Rezaee et al., 2006). Various hyphenated techniques such as high resolution gas chromatography coupled to high resolution mass spectrometry have been applied to determine traces of dioxins-like compounds from environmental samples, and high resolution gas chromatography with electron capture detector (Pyell, 2001). A number of separation methods have been used for the separation of dioxins-like compounds from the environment. These methods include electrophoresis, whereby, separation of the compounds is obtained via differential migration of solutes of charged species in an electric field performed in narrow-bore capillaries filled only with buffer (Jiang and Lucy, 2002). Separation by electrophoresis is becoming an advantageous tool for determination of organic compounds such as dioxins-like compounds and many more due to its flexibility in manipulating various parameters on the column in order to obtain the separation. Although separation time is very small with very little sample and solvent, the detection sensitivity of capillary electrophoresis compare to gas chromatography and HPLC has not been satisfactory due to the low UV sensitivity attributed to its path length and low injection volume (Dien Liem, 1999). The detection of dioxins and PCBs with different techniques have been reported widely and the majority of these methods employ the use of gas chromatography with various detectors (Focant
Fig. 2. Optimisation of sample pH.
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2,2',4',4-Tetrachlorobiphenyl 240000
2.1. Materials and methods
230000
Standards 2,3,7,8 TCDD and PCB-1 (2-monochlorobiphenyl), PCB-28 (2,4,40 -tri-chlorobiphenyl), PCB-101 (2,2,4,5,5-pentachlorobiphenyl) and the organic solvent isooctane were purchased from Riedel-de-Haën (Seelze-Hannover, Germany). Pesticidequality solvents (n-hexane and toluene) and calcium chloride were purchased from Panreac (Barcelona, Spain). Sodium hydroxide pellets (98%) was obtained from Saarchem (Krugerdorp, Africa). Stock standard solutions of individual compounds (with concentrations of 1000 mg/L) were prepared. Ttriton x-100 and hydrochloric acid were purchased from N.T. Laboratory Supplies, (Johannesburg, South Africa). A multicompound working standard solution (1 lg/mL concentration of each compound) was prepared by appropriate dilutions of the stock solutions (1000 mg/L) with hexane:toluene (1:1) and stored under refrigeration (4 °C). The hollow fibre tubing used in the extractions were Q3/2 Accurel polypropylene (200 lm wall thickness, 600 lm inner diameter, 0.2 lm pore size) obtained from Membrana (Wuppertal, Germany). An Agilent Technologies 10 lL microsyringe was used in the hollow fibre supported liquid membrane (HFSLM) extraction.
220000
Ee
2. Experimental
210000 200000 190000 180000 170000 5
10
15
20
25
30
Extraction Time (mins) Fig. 3. Optimisation of extraction time.
2,2'4',4-Tetrachlobiphenyl 150000000 140000000
2.2. Gas chromatographic (GC–ECD) analysis of HF-LPME extracts
130000000
The HP Agilent 6890 GC/5973 (Santa Clara, CA 95051 USA) was used for the analysis of dioxins and PCBs. The column, DB-5 (30 m 0.25 mm i.d. 0.25 lm film thickness) from Supelco (Bellefonte, PA, USA), was used for all separations. The flow rate of the carrier gas (nitrogen) was 1.5 mL/min and the injection volume was 0.5 lL. The glass liner was equipped with a plug of carbofrit (Resteck, Bellefonte, PA, USA). The GC temperature program used for the separation of the compounds had the initial oven temperature set at 40 °C and held for 1 min. The temperature was then ramped at 9 °C/min to 285 and held for 10 min.
Ee
120000000 110000000 100000000 90000000 80000000 70000000 100
150
200
250
300
Stirring Speed (rpm)
3. Results and discussion Fig. 4. Optimisation of the stirring speed.
et al., 2005). These methods are attractive in terms of speed, cost and sample turn over (Focant et al., 2005). In this work, a gas chromatograph equipped with an electron capture detector has been employed in the analysis of dioxins and PCBs.
3.1. Optimisation of parameters in the liquid phase microextraction of dioxins and PCBs A number of extraction parameters had to be optimised to enhance the extractability of dioxins and PCBs. Factors studied
Fig. 5.1. GC–ECD chromatogram for PCB101 standard.
P. Sikiti et al. / Physics and Chemistry of the Earth 50–52 (2012) 98–103
Peak area
(a) 5000
3.3. Optimisation of sample pH
y = 2078.4x + 160.4 R2 = 0.9931
4500 4000 3500 3000 2500 2000 1500 1000 500 0 0
0.5
1
1.5
2
2.5
Concentration/ppm Fig. 5.2a. Calibration curve for PCB-1.
Peak area
(b) 2500000 2000000 1500000 1000000 500000 0 0.5
1
1.5
The pH of a mixture of sample solution was optimised by keeping all other factors constant (using 5% TOPO in isooctane). These factors include pH of the stripping solution, stirring speed, organic solvent and extraction time. The sample pH range investigated ranged from pH 6 to 10. The extraction time was 15 min and at a stirring speed 300 rpm. The results of optimisation are shown in Fig. 2. The optimal sample pH was found to be between 7 and 8 (Fig. 2). The compounds were mostly extractable at pH near or around neutral which displayed the most efficient extraction. This optimal pH range was used for all the subsequent extraction. 3.4. Optimisation of time of extraction
y = 1E+06x + 14891 R2 = 0.996
0
101
2
2.5
Concentration/ppm Fig. 5.2b. Calibration curve for PCB-28.
The time of the extraction of dioxins and PCBs was varied as it affects sample enrichment. It is expected that the longer the contact time the higher the extraction as there will be a longer period for mass transfer of the analyte into the fibre. The results for the optimisation are shown in Fig. 3. The results obtained from Fig. 3 show that the enrichment factors increased with extraction timeup to the optimum point at 15 min. However, the extraction efficiency began to decrease after 15 min presumably due to the saturation of the receiving solution. 3.5. Optimisation of stirring speed of the extraction
Peak area
(c)
800000 700000 600000 500000 400000 300000 200000 100000 0
y = 328011x + 38732 R2 = 0.9801
0
0.5
1
1.5
2
2.5
Concentration/ppm Fig. 5.2c. Calibration curve for 2,3,7,8-TCDD.
The effect of stirring speed on the efficiency of the extraction of dioxins and PCBs was also investigated. The results of optimisation are shown in Fig. 4. From the results it is evident that an increase in the stirring speed increases the efficiency of the extraction. The motion of the solution in which the fibre is placed increases the rate of mass transfer. The optimum speed was at 100 rpm. Speeds higher than this showed a negative effect on the extraction. Higher stirring speeds reduce the contact of the analyte with the membrane, thus reducing the amount extracted. 3.6. GC–ECD analyses of extracts
(d) 1400000
y = 606782x + 54374 R2 = 0.9915
Peak area
1200000
The extracts were analysed using a gas chromatograph equipped with an electron capture detector and the identification was done by means of comparison of the retention times of standards and the samples extracts (Fig. 5.1).
1000000 800000 600000 400000 200000 0 0
0.5
1
1.5
2
2.5
concentration/ppm Fig. 5.2d. Calibration curve for PCB-101.
included, the type of organic solvent, sample pH, extraction time, and the stirring speed.
3.2. Optimisation of organic liquid membrane The organic liquid membrane in HFSLM extraction influences the efficiency of the extraction and the selectivity of the enrichment of the analytes. The type of organic solvent chosen should be stable, with a low volatility. It should not be soluble in water or have a very low solubility (Mitra, 2003). In the extraction of dioxins and PCBs, the organic liquids tested were octanol and isooctane. The results of optimisation depicted in Fig. 1 shows that isooctane gave the highest enrichment factors.
3.7. Quantitation The optimum conditions for the extraction process were used in the extraction of a mixture of all the four compounds. The optimum conditions of extraction was with sample pH of 7, stirring speed 100 rpm, extraction time of 15 min and organic solvent was isooctane. Calibration curves of peak areas vs. concentration for each of the compounds was plotted and used to quantify the amount extracted (Figs. 5.2a–5.2d). 3.8. Statistical validation of the extraction process To validate the results obtained for during the extraction process, a number of statistical tests were performed. These include the test for linearity in the extraction process to ensure that there were no external causes of loss of extracts. Repeatability and reproducibility experiments were set and the results obtained were used to confirm that the method is robust and reliable.
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y
sample independent of time. Mathematically the two variables can be represented by the following equations:
y = 10112x + 1265.6 R2 = 0.9607
12000
Peak area
10000
Reproducibility ¼ 2:77ðr2 t þ r2 =nÞ1=2
8000 6000
Repeatability ¼ 2:77ðr2 Þ1=2
4000 2000 0 0
0.2
0.4
0.6
0.8
1
1.2
Concentration/ppb Fig. 6. Linearity studies during the liquid phase extraction process for 2,3,7,8-TCDD.
Table 1 Testing series. 1st Day
2nd Day
3rd Day
4th Day
PCB1-1a PCB1-1b PCB28-1a PCB28-1b
TCDD-1a TCDD-1b PCB101-1a PCB101-1b
PCB1-2a PCB1-2b PCB28-2a PCB28-2b
TCDD-2a TCDD-2b PCB101-2a PCB101-2b
Table 2 Average enrichment factors and standard deviations from HFSLM. Pesticide
Average enrichment factors
Standard deviation
PCB101 TCDD PCB28 PCB1
0.712 1.012 8.576 6.446
0.121 0.477 0.0962 0.7264
Table 3 Comparison between repeatability and reproducibility of HFSLM (enrichment factors). Pesticide
Repeatability
Reproducibility
PCB101 TCDD PCB28 PCB1
0.0051 0.0102 0.5600 1.4600
0.0024 0.0090 0.3512 0.9961
3.9. Linearity studies during the liquid phase extraction process Linearity experiments were performed at optimal conditions by extracting a spiked water sample at an increasing concentration of individual compounds. Fig. 6 shows a good linearity for 2,3,8,9TCDD with r2 of 0.9 which is an indicative of a good performance of the extractability of this compound. The other compounds as well gave r2 values of 0.9 thus proving that the developed method worked well with all the compounds.
3.10. Repeatability and reproducibility during the liquid phase microextraction To determine the reliability of the liquid phase microextraction technique, reproducibility and repeatability in the extraction process was evaluated. Reproducibility refers to the difference in the results obtained between two quantities of the same sample (number of repeated measurements made on the same sample). Repeatability refers to the difference of results obtained on the same
For these set of experiments, the four pesticide solutions, each of 1 mg/L, were extracted twice, one after the other, over period of 4 days. The experiments are shown in Table 1. The results obtained (Tables 2 and 3) suggest that PCB1 and PCB28 gave better reproducibility and repeatability than TCDD and PCB101. This could be attributed by the fact that, TCDD and PCB101 are more hydrophobic than PCB1 and PCB28 thus posing relatively more difficulties in terms of their extractability than PCB1 and PCB28. 4. Conclusion A method for the simultaneous analysis of 2,3,7,8 TCDD and three PCB compounds in water using liquid phase microextraction was established. Factors affecting the efficiency of the extraction were optimised by varying the sample pH as well as the extraction time, stirring speed and organic liquid solvent. However, the attractive features of this method are the use of minimal organic solvent, low cost, simplicity and fewer sample handling steps. Acknowledgement Financial support from NIC-Mintek is gratefully appreciated. References Alcock, R.E., Gemmil, R., Jones, K.C., 1999. Improvement to the UK PCDD/F and PCB atmospheric emission inventory following an emission measurement programme. Chemosphere 38 (4), 759–770. Dien Liem, A.K., 1999. Important development in methods and techniques for the determination of dioxins and PCBs in foodstuffs and human tissues. TrAC Trends Anal. Chem. 18, 449–507. Fiedler, H., Hutzinger, O., Timms, C., 1999. Dioxins: sources of environmental load and human exposure. Toxicol. Environ. Chem. 29, 157–234. Focant, J.F., Pirard, C., De Pauw, E., 2005. Recent advances in mass spectrometric measurement of dioxins. J. Chromatogr. A 1067, 265–275. Giesy, J.P., Kannan, K., 1998. Dioxins-like and non-dioxins-like toxic effects of polychlorinated biphenyl: implications for the risk assessment. Crit. Rev. Toxicol. (28), 511–569. Im, S.H., Kannan, K., Giesy, J.P., Matsuda, M., Wakimoto, T., 2002. Concentration and profiles of polychlorinated dibenzo-p-dioxins and dibenzofurans in soil from Korea. Environ. Sci. Technol. 36 (17), 3700–3705. Jiang, J., Lucy, C.A., 2002. Determination of alkylphosphoric acids using micellar electrokinetic chromatography with laser-induced fluorescence detection and high salt stacking. J. Chromatogr. A 966, 243–244. Kennedy, S.W., Lorenzen, A., Norstrom, R.J., 1996. Chicken embryo hepatocyte bioassay for measuring cytochrome P5014A-based 2, 3, 7, 8tetrachlorodibenzo-dioxins equivalent concentrations environmental samples. Environ. Sci. Technol. 30, 706–715. Mitra, S., 2003. Sample Preparation Techniques in Analytical Chemistry. WileyInterscience, Ney York. Oberg, LG., GLas, B., Swanson, SE., Rappe, C., Pual, K.P., 1990. Peroxidase-catalyzed oxidation of chlorophenol to polychlorinated dibenzo-p-dioxins and dibenzonofurans. ERCH Environ. Contam. Toxicol. 19, 93–938. Pedersen-Bjergaard, S., Rasmussen, K.E., 1999. Anal. Chem. 71, 2650. Pyell, U., 2001. Micellar electrokinetic chromatography from theoretical concept to real sample (review). Fresenius J. Anal. Chem. 371, 691–703. Rezaee, M., Assadi, Y., Millani, M.R., Aghaee, E., Ahmadi, F., Berijani, S., 2006. J. Chromatogr. A 1116, 1. Richter, B.E., Jones, B.A., Ezzell, J.L., Porter, N.L., Avdalovic, N., Poll, C., 1996. Accelerated solvent extraction: a technique for sample preparation. Anal. Chem. 68, 1033–1039. Richter, B.E., Ezzell, J.L., Knowles, E., Hoefler, F., Mullalat, A.K.R., Schuntwinkel, M., Waddel, D.S., Gobran, T., Khurans, V., 1997. Extraction of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans from environmental samples using accelerated solvent extraction (ASE). Chemosphere (34), 975– 987. Safe, S., 1990. Polychlorinated-biphenyl (PCBS), dibenzo-para-dioxins (PCDDs), dibenzofurans (PCDFs), and related-compounds environmental and
P. Sikiti et al. / Physics and Chemistry of the Earth 50–52 (2012) 98–103 mechanistic consideration which support the development of toxic equivalent factors (TEFs). Crit. Rev. Toxicol. 21, 51–88. Safe, SH., 2000. Endocrine disruptors and human health – is there a problem? An update. Environ. Health Perspect. 108, 487–493. Safe, S., Bandiera, S., Sawyer, 1985. Effect of structure on binding to the 2, 3, 7, 8TCDD receptor protein and AHH induction halogenated biphenyl. Environ. Health Perspect. 61, 21–33. Safe, S., Astroff, B., Harris, M., 1991. 2, 3, 7, 8-Tetrachloridibenzo-para dioxin (TCDD) and related-compounds as antiestrogens characterization and mechanism of action. Pharmacol. Toxicol. 69, 400–409.
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Stevenson, D., 1999. Molecularly imprinted polymers for solid-phase extraction. TrAC. Trends Anal. Chem. 18, 154–158. Van der Berg, M., Birnbuam, L., Bostveld, ATC., Brunostorm, B., 1998. Toxic equivalent factors (TEFs) for PCBs, PCDDs, PCDFs for humans and wildlife. Environ. Health Perspect. 106, 775–792. WHO, 2002. Polychlorinated dibenzodioxins, polychlorinated dibenzofurans, and coplanar polychlorinated biphenyls. In: Safety Evaluation of Certain Food Additives and Contaminants. Geneva, World Health Organization (WHO Food Additives Series, No. 48).