Extraction of PCDDs from Marine Sediments Using

ANALYTICAL LETTERS Vol. 37, No. 7, pp. 1385–1399, 2004

ENVIRONMENTAL ANALYSIS

Extraction of PCDDs from Marine Sediments Using Polyoxyethylene 10 Lauryl Ether and Oligoethylene Glycol Monoalkyl Ether Surfactants C. Padroń Sanz, Z. Sosa Ferrera, and J. J. Santana Rodrı´guez* Department of Chemistry, Faculty of Marine Sciences, University of Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, Spain

ABSTRACT In this study, the microwave assisted micellar extraction (MAME) methodology has been optimised to extract and determine a mixture of nine polychlorinated dibenzo-p-dioxins (PCDD) from marine sediments. This is a very efficient extraction procedure which considerably reduces the volume of extractant to be used and the analysis time. The PCDDs under study have been extracted using the non-ionic surfactants polyoxyethylene 10 lauryl ether (POLE) and oligoethylene glycol monoalkyl ether (Genapol X-080), which are biodegradable, meaning that the toxical

*Correspondence: J. J. Santana Rodrı´guez, Department of Chemistry, Faculty of Marine Sciences, University of Las Palmas de Gran Canaria, 35017 Las Palmas de Gran Canaria, Spain; E-mail: [email protected]. 1385 DOI: 10.1081/AL-120035905 Copyright # 2004 by Marcel Dekker, Inc.

0003-2719 (Print); 1532-236X (Online) www.dekker.com

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Padroń Sanz, Sosa Ferrera, and Santana Rodrı´guez effects of the method have been avoided. The optimal extraction variables, such as surfactant and salt concentration, together with the radiation time and microwave power, were determined for each surfactant and then compared. To get the benefit of the properties that non-ionic surfactants possess, in order to obtain a better signal of the analytes under study, the Genapol X-080 extracts were then preconcentrated using the cloud-point methodology, and analysed by liquid chromatography with UV detection system under the optimised conditions. The proposed method was applied to marine sediment samples from Gran Canaria and Fuerteventura islands (Canary Islands, Spain). The results were then compared with those obtained using the traditional Soxhlet extraction methodology. Key Words: Polyoxyethylene 10 lauryl ether; Genapol X-080; PCDD; Microwave assisted micellar extraction.

INTRODUCTION Among the different procedures employed on the extraction of organic pollutants from solid samples, the traditional Soxhlet extraction has undoubtedly been the most widely used. However, this method has a series of drawbacks, as it is time consuming (between 24 and 48 hr) and needs large amounts of organic solvents (100 –300 mL)[1] that have to be evaporated before further clean-up steps. Therefore, a more efficient and economical process to extract these compounds from solid matrixes would be advantageous. As early as 1986, Ganzler et al.[2] were the first to use home microwave ovens to extract anti-nutritive compounds from various plant materials. Since then, microwave methodologies have been adapted for other scientific applications, including the extraction of pesticides,[3,4] metals,[5] organochlorinated compounds, and PAHs,[6] etc. Although the advantages of these procedures include reduced solvent usage and shorter analysis time, most of them still make use of organic solvents. Thus, in order to avoid completely the use of these extractants, a new possibility for the application of microwave assisted extraction (MAE) is the use of micellar media as extractants. From the analytical point of view, one of the most important properties of these organised structures is their good capacity to solubilise solutes of different types and nature. Therefore, the micellar media can be applied to the solubilisation and extraction of several compounds present in different environments, with the benefits of low cost, easy handling, and reduced toxic effects.[7,8] On the other hand, the non-ionic surfactants enable the extraction and preconcentration of the analytes in only one step.[9 – 11]

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PCDD Extraction Using POLE and Genapol X-080

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This paper describes the performance and comparison of the methodology for the extraction of nine polychlorinated dibenzo-p-dioxins (PCDD) using two different surfactants: polyoxyethylene 10 lauryl ether (POLE) and oligoethylene glycol monoalkykl ether (Genapol X-080), for their microwave assisted micellar extraction (MAME) and following determination by liquid chromatography with UV detection. The optimised methodology is applied to the extraction and determination of the organochlorinated dioxins under study from marine sediment samples from coastal areas of Gran Canaria and Fuerteventura islands (Canary Islands). PCDDs are common contaminants of chlorinated industrial chemicals, such as polychlorinated biphenyls, chlorophenols, and their derivatives.[12 – 14] But they are mainly formed as by-products of waste incineration and other combustion processes.[15,16] They can therefore reach the atmosphere and then be globally transported and deposited onto land or water. But as dioxins are highly hydrophobic compounds, they will accumulate on sediments,[17] which can lead to human exposure via the food chain, and can also be harmful to wildlife.[18]

EXPERIMENTAL Reagents All the PCDDs were obtained from Accustandard, Inc. (98% purity) (New Haven, USA), and are listed in Table 1, which sets out their identification

Table 1.

Analytes under study and chromatographic parameters.

Compound Dibenzo-p-dioxin 1-Chlorodibenzo-p-dioxin 2,3-Dichlorodibenzo-p-dioxin 2,3,7-Trichlorodibenzo-p-dioxin 2,3,7,8-Tetrachlorodibenzo-p-dioxin 1,2,3,7,8-Pentachlorodibenzo-p-dioxin 1,2,3,6,7,8-Hexachlorodibenzo-p-dioxin 1,2,3,4,6,7,8-Heptachlorodibenzo-p-dioxin 1,2,3,4,6,7,8,9-Octachlorodibenzo-p-dioxin a

Retention time. Determination UV wavelength.

b

lb Identification tra (min) (nm) Abbreviation number DD 1DD 2DD 3DD 4DD 5DD 6DD 7DD 8DD

1 2 3 4 5 6 7 8 9

3.9 4.9 6.9 10.6 12.8 14.3 16.0 17.6 19.2

224 228 228 232 232 240 240 246 246

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Padroń Sanz, Sosa Ferrera, and Santana Rodrı´guez

numbers in the tables and figures. Stock solutions of each PCDD were prepared at 10 mg mL21 by dissolving 1 mg in methanol. The non-ionic surfactants POLE and Genapol X-080 were obtained from Sigma (St. Louis, MO) and prepared in ultra-high quality water. HPLC-grade methanol, toluene, and NaCl were obtained from Panreac Quimica, S.A. (Barcelona, Spain), and were used as received. All solvents were filtered through a 0.22 mm nylon membrane filter, and ultra-high quality water was used throughout.

Apparatus The microwave system used to perform the MAE process was a Multiwave (Perkin Elmer, Madrid, Spain), with a rotor 6EVAP and 6 MF100 vessels (Perkin Elmer, Madrid, Spain). The HPLC system was equipped with Millenium chromatography manager software, a Waters 515 pump (Waters Associates, Milford, MA), fitted with a Rheodyne 7725i injector valve, and a Waters 996 photodiode array detector (Waters Associates). The stationary phase column was a Waters Nova-Pack C18 150 mm 3.9 mm, 4 mm particle diameter (Waters Associates).

Procedure Spiking Procedure A 5 g of sediment sample were spiked with the PCDDs mixture and kept in darkness overnight in order to obtain a dry and well conditioned sample. It was well shaked after the spiking, to ensure the uniform absorption of the analytes on the sediment, and once again before the analysis. MAME Procedure Spiked samples were introduced in Teflon vessels and solutions of varying concentrations of salt (NaCl) and the non-ionic surfactant at the optimized concentrations for each method were added. The vessels were introduced in the microwave oven and irradiated at the optimised conditions and were then allowed to cool to room temperature. The surfactant extract was carefully removed and introduced in hermetically closed vials. This procedure was repeated two times for each of the samples and the extracts were filtrated and joined into the hermetically closed vials, so the final extracted volume was 10 mL.

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CPE Procedure In order to enhance the chromatographic signal of the PCDDs extracted with Genapol, the cloud-point extraction procedure[19] was applied after the MAME process and then directly analysed in the chromatographic system under the optimised conditions. Soxhlet Procedure PCDDs present in 10 g of the spiked sediment samples were extracted with 35 mL of toluene during 24 hr at 60% power.

Chromatographic Analysis A 20 mL of the extracted solutions were analysed in the LC-UV system, with different wavelengths being recorded in each case (Table 1). The optimised conditions for the separation and identification of all the analytes under study were a mobile phase of methanol : water (85 : 15) and a flow rate of 1.0 mL min21 to 3 min and gradient to 100 : 0 (methanol : water) to 13 min, followed by isocratic 100 : 0 (methanol : water) until the end of the analysis (20 min). The obtained retention times are also shown in Table 1. The same chromatographic conditions were applied for the Soxhlet extracts.

RESULTS AND DISCUSSION Optimization of the MAME Procedure Effect of Surfactant Volume In order to check if the volume of extractant to be added would affect the extraction of the analytes due to possible evaporation losses, a preliminary study was made. In this way, measurements of the analyte recoveries were performed using 5, 10, and 20 mL of a POLE solution (5%, w/v), with no influence of the volume of surfactant being observed. At the same time, the internal temperature of the vessels in the aforementioned cases was measured, with an average value of 608C being obtained. A volume of 5 mL was chosen for the following studies.

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Effect of Surfactant Concentration In order to determine the effect of surfactant concentration on the recovery percent, several samples containing different POLE and Genapol X-080 concentrations 1, 3, 5, and 8% (w/v) were analyzed. The spiked sediments were extracted with 5 mL of each surfactant solution in the microwave system at 200 W during 10 min. The results obtained in general show that the recovery percent increases slightly with the POLE concentration to 5% after which a little decrease is observed, while a smooth effect on the recovery of PCDDs is appreciated when Genapol X-080 is employed. This behaviour can be seen in Fig. 1 for 4DD and 7DD cases. On the other hand, the extraction percentages obtained when POLE surfactant is used are greater for the dioxins that contain a higher number or Cl atoms, while the opposite was observed when Genapol X-080 was used. An amount of 5% (w/v) was fixed as the concentration to be used for both surfactants in posterior studies. Effect of Salt Addition To study the effect of the NaCl addition on the recovery percent, different salt amounts were added to the surfactant solution prior to the radiation in the microwave oven. Samples with NaCl concentrations ranging from 1% to 8% (w/v) were extracted using the POLE (5%, w/v) and Genapol X-080 (5%, w/v) in the microwave oven at 200 W during 10 min. In the case of POLE, the obtained results (Fig. 2) show how the recovery percentages increase

Figure 1. Effect of the surfactant concentration: POLE (—) and Genapol X-080 (– –) on the extraction of PCDDs from spiked sediments (5 g). Volume of surfactant, 5 mL; microwave power, 200 W; radiation time, 10 min.

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Figure 2. Effect of NaCl concentration on the extraction of PCDDs from spiked sediments (5 g) using different surfactants POLE (—) and Genapol X-080 ( – – ). Volume of surfactant, 5 mL; concentration of each surfactant, 5% (v/v); microwave power, 200 W; radiation time, 10 min.

with the NaCl concentration to 5% (w/v) of salt, at which point a sharp drop in the percentages is noted. As far as the Genapol X-080 is concerned, the percentages increase to 3% (w/v) NaCl and then decrease slightly or stabilise. It can also be observed how Genapol X-080 is the better extractant for those dioxins with a fewer number of Cl atoms in its molecule. In accordance with these results, values of 4% and 3% (w/v) of NaCl, for POLE and Genapol X-080, respectively, were selected for later studies.

Effect of the Microwave Radiation Time In this study, using the previously optimised conditions for each surfactant, different radiation intervals from 30 sec to 10 min at the same power (200 W) were tested. The results obtained show how that when POLE is used, the majority of the components suffer a sharp increase in the recovery percentages during the first minute of microwave radiation and then stabilise. On the other hand, when Genapol X-080 is used, the analytes with a greater number of Cl atoms behave differently and their recovery percentages increase slightly during the first 5 min and then stabilise. In the case of the dioxins with fewer Cl atoms, the recovery percentages sharply increase during the first minute and then continue increasing very slightly or then stabilise. On the other hand, it can be seen that the percentages obtained when POLE is used are lower in the majority of the cases than those obtained with Genapol X-080. Finally, radiation times of 5 and 8 min were chosen, respectively, to ensure the reproducibility of the radiation conditions on the extraction.

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Effect of the Microwave Radiation Power Once the variables were optimised at a particular power, the last study to be performed was the effect of different radiation powers on the extraction. Following the same procedure as the one set out above powers ranging from 100 to 800 W were applied to different samples at the optimised values for each parameter. The extracts were finally analysed showing a smooth increase in the recovery with the radiation power to 300 W for POLE, after which the recoveries remain unaffected, while a maximum in the recovery percentages is observed at 200 W for Genapol X-080 surfactant. It can once again be noted that the best recovery percentages under the same experimental conditions were obtained when Genapol X-080 was used as extractant. Radiation powers of 300 and 200 W were then selected for each surfactant, respectively. Efficiency of the Extraction It has been previously reported that the efficiency of the extraction may be improved applying this methodology several times to the same sediment sample.[20,21] Several successive extraction steps were therefore carried out, and it was observed that the results are enhanced considerably when two extractions steps are applied, with recoveries between 60% and 100% being obtained for most of the compounds.

Optimization of the HPLC Analysis The separation and determination of PCDDs was carried out by directly injecting the obtained extracts in an HPLC/UV system under the following chromatographic conditions: mobile phase of methanol : water (85 : 15) at a flow rate of 1.0 mL min21 to 3 min and gradient to 100 : 0 (methanol : water) to 13 min followed by isocratic 100 : 0 (methanol : water) to the end of the analysis (20 min). The specific wavelength for each analyte was recorded (Table 1). Under these conditions a good separation and identification of the peaks is observed (Fig. 3). These conditions were also used for the analysis of Soxhlet extracts.

ANALYTICAL PARAMETERS The corresponding calibration curves were obtained, by injecting standard solutions containing the surfactant POLE or Genapol X-080

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Figure 3. Chromatogram of a standard solution containing the PCDDs mixture (500 ng mL21) in Genapol X-080 (5%, v/v). Injected volume, 20 mL; mobile phase, methanol : water under optimized gradient; UV wavelength, 240 nm.

and a known concentration of the PCDDs mixture into the chromatographic system. The results revealed a linear relationship in the interval 10 –500 ng mL21 for both surfactants with high correlation coefficients (0.999) in all cases. In order to study the reproducibility, the optimised extraction methods were applied to six samples containing a mixture of the PCDDs under study and analysed in the HPLC-UV system at the established chromatographic conditions. The relative standard deviation values are listed in Table 2, where values equal and lower than 2.8% and 4.5% were obtained for POLE and Genapol X-080, respectively. The limits of detection were also calculated for each analyte using the signal to noise (s/n ¼ 3) ratio[22] once the MAE method was fully applied. The results obtained are also listed in Table 2.

ANALYTICAL APPLICATIONS Both surfactants were then applied to several natural sediment samples collected from Gran Canaria and Fuerteventura islands (Canary Islands, Spain) at several meters depth. The analysis of these marine sediments showed the absence of PCDDs. Therefore they were spiked with 100 ng g21 of PCDDs, before the application of the extraction procedures using the two different non-ionic surfactants under the optimised conditions. The results showed satisfactory recoveries

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Analytical parameters for POLE and Genapol X-080 surfactants. POLE

Compound DD 1DD 2DD 3DD 4DD 5DD 6DD 7DD 8DD

Genapol X-080

R.S.D.a (%)

L.O.D.b (ng mL21)

R.S.D.a (%)

L.O.D.b (ng mL21)

L.O.D.c (ng mL21)

2.79 2.08 2.8 2.18 2.02 2.41 1.71 0.68 1.99

14.7 30.1 3.9 1.0 8.5 3.2 14.8 5.1 14.5

0.87 —d 0.53 0.17 0.92 4.45 2.92 0.46 1.19

27.8 —d 11.9 9.2 10.4 7.9 43.4 1.3 6.5

—d 3.2 0.9 0.7 0.8 0.6 3.3 0.1 0.5

a

Relative standard deviation (n ¼ 6). Limits of detection. c Limits of detection after cloud-point preconcentration. d High interference of surfactant signal. b

(Table 3) for both kind of sediments. It can be appreciated how the results are very similar for each surfactant when it is applied to different sediments, but when dioxins present in the same sediment are extracted with different surfactants some differences can be observed. In general, the results obtained when Genapol X-080 was used were better than those using POLE, therefore we would like to propose the first one as the most suitable to extract PCDDs from marine sediments. In this way, in order to enhance the detection limits of the analytes under study, when they are extracted with Genapol X-080, they were preconcentrated by applying the CPE procedure[19] before their HPLC-UV analysis. Extracts were taken after the MAME procedure and heated in a controled bath at 1108C for 10 min. The surfactant extract was then directly analysed in the HPLC system, which revealed satisfactory identification and recoveries for most of the PCDDs under study (60 – 95%) (Fig. 4). On the other hand, the interferent peak at the retention time of 1DD that it is obtained when using Genapol X-080 under MAME conditions is solved with the preconcentration because the signal of the surfactant is moved to the begining of the chromatogram and then, 1DD can therefore be measured. Under these conditions, using MAME and CPE procedures, a preconcentration factor of 13 times and lower LOD are obtained (Table 2).

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Table 3. Recovery percentages of PCDDs from marine sediment samples.a Las Canteras (Gran Canaria)

Jandıá (Fuerteventura)

POLE DD 1DD 2DD 3DD 4DD 5DD 6DD 7DD 8DD

61.8 108.5 106.0 61.6 80.7 83.1 74.8 75.4 78.2

53.9 100.8 94.7 59.7 80.8 87.7 79.9 79.7 86.4

Genapol X-080 DD 1DD 2DD 3DD 4DD 5DD 6DD 7DD 8DD

89.31 —b 63.7 97.3 107.5 91.8 55.1 95.8 70.8

92.6 —b 69.3 90.3 102.9 102.1 69.2 109.0 94.0

a

Mean of three determinations. High interference with surfactant signal.

b

Soxhlet Extraction In order to validate the proposed method, it was compared with the results obtained using the extraction procedure established by the Environmental Protection Agency (Methods 8280A and 8290) based on the Soxhlet technique[23,24] for the extraction of PCDDs from soil/sediment samples. In Fig. 5, it can be appreciated the higher and similar values obtained for Gran Canaria sediment sample when dioxins are extracted following the MAME procedure with Genapol X-080, with the exception of 2DD. In the case of the Fuerteventura sediment sample, the behaviour is very similar. It has to be noticed that the non-chlorinated dioxin (DD) is not extracted under the conditions employed for the Soxhlet extraction procedure, probably due to its high volatility.

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Figure 4. Chromatogram of the PCDDs mixture extracted from a Fuerteventura sediment sample previously spiked (100 ng g21) and preconcentrated under CPE conditions. Injected volume: 20 mL; mobile phase, methanol : water under optimized gradient, UV wavelength, 240 nm.

CONCLUSIONS This study has once again proved the suitability of the non-ionic surfactants as extractants of non-polar compounds as PCDDs in different matrixes. The results obtained for both surfactants are satisfactory being Genapol X-080 the surfactant proposed because it enables better results. The proposed method leads to highly satisfactory and better recoveries than those obtained using the traditional Soxhlet extraction and the former has a series of advantages over the latter. The use of surfactants as extractants

Figure 5. Comparison of the results obtained using two different extraction procedures (MAME and Soxhlet) on a Gran Canaria sediment sample previously spiked with PCDDs (100 ng g21) and analyzed under HPLC optimized conditions.

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combined with the MAE means that the extraction procedure is more rapid, less extractant is needed, thus lowering the costs dramatically. It is relatively simple, insofar as it does not require a high level of handling and the extract can be analysed directly. On the other hand, the use of non-ionic surfactants means that the extracts can be preconcentrated at an earlier stage, which considerably improves the detection of any component extracted in the surfactant. It can therefore be considered that this method may be a good alternative to the traditional technique used to extract PCDDs.

ACKNOWLEDGMENTS This work was supported by funds provided by Autonomous Government of Canary Islands (Spain). Research Project No. PI2000/045 and Spanish Ministry of Science and Technology. Research Project No. PPQ2002/04683.

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22. Lindsay, S. High Performance Liquid Chromatography; John Wiley and Sons: New York, 1992; 337. 23. EPA Method 8280A, The Analysis of Polychlorinated Dibenzo-p-Dioxins and Polychlorinated Dibenzofurans by High Resolution Gas Chromatography/Low Resolution Mass Spectrometry (HRGC/LRMS), Washington D.C., 1996; 1 – 55. 24. EPA Method 8290, Polychlorinated Dibenzo-p-Dioxins (PCDDs) and Polychlorinated Dibenzofurans (PCDFs) by High Resolution Gas Chromatography/High Resolution Mass Spectrometry (HRGC/HRMS), Washington D.C., 1994; 1– 71. Received January 16, 2004 Accepted January 22, 2004