ple with integrated clean up followed by alternative ...141751/fulltext01.pdf · currently standard...

PLE with integrated clean up followed by alternative detection steps for cost-effective

analysis of dioxins and dioxin-like compounds

Erik Spinnel

Akademisk avhandling

som med vederbörligt tillstånd av Rektorsämbetet vid Umeå universitet för avläggande av doktorsexamen i kemi kommer att offentligen försvaras i sal N360,

Naturvetarhuset, Umeå universitet. Fredagen den 13 juni 2008, kl. 13.00

Fakultetsopponent: Professor Tuulia Hyötyläinen, Department of Chemistry, University of Helsinki, Finland.

Department of Chemistry

Umeå University, Sweden

2008



Erik Spinnel

Akademisk avhandling

som med vederbörligt tillstånd av Rektorsämbetet vid Umeå universitet för avläggande av doktorsexamen i kemi kommer att offentligen försvaras i sal N360,

Naturvetarhuset, Umeå universitet. Fredagen den 13 juni 2008, kl. 13.00

Fakultetsopponent: Professor Tuulia Hyötyläinen, Department of Chemistry, University of Helsinki, Finland.



2008

UMEÅ UNIVERSITY DOCTORAL DISSERTATION

Department of Chemistry SE-901 87 UMEÅ Date of publication

2008-05-19Erik Spinnel

PLE with integrated clean up followed by alternative detection steps for cost-effective analysis of dioxins and dioxin-like compounds

AbstractPolychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) are two structurally related groups of chemicals, generally referred to as `dioxins´. These are of great concern due to their high toxicity and global spread. Other groups of compounds with similar chemical structure and toxicity mechanisms are the brominated analogues polybrominated dibenzo-p-dioxins (PBDDs) and polybrominated dibenzofurans (PBDFs), and the dioxin-like polychlorinated biphenyls (PCBs). Numerous studies have been undertaken to investigate sources and transport routes of dioxins. However, much remains to be done, including analytical, inventories of dioxin-like compounds, such as PBDD/Fs, and the development of more convenient analytical methods. The currently standard procedure for analyzing dioxins (and dioxin-like compounds) is to use Soxhlet extraction followed by multi-step clean-up and gas chromatography - high resolution mass spectrometry (GC-HRMS) for detection. Unfortunately, this method is very solvent, labor and time-consuming, making it very expensive.

The main aim of the studies this thesis was to develop pressurized liquid extraction (PLE) with integrated clean up techniques for fast, convenient preparation of dioxin samples. PLE with integrated clean-up has previously been used for extracting dioxins from biological samples, but in these studies the possibility of extending its use to abiotic samples was explored. The results show that PLE with an integrated carbon trap is suitable for analyzing dioxins in various types of soil samples, sediment and flue gas samples. The results also showed that it has potential for analyzing dioxins in fly ash. The thesis focuses on developments of the methodology for dioxin analysis, but also includes results obtained from PBDDs and dioxin-like PCB analyses. In addition, the possibility of using various other kinds of detection techniques rather than GC-HRMS, such as enzyme-linked immunosorbent assays (ELISAs) or two-dimensional gas chromatography with micro electron capture detection (GCxGC-µECD) was explored. The results indicate that ELISA and GCxGC-µECD could serve as complementary detection systems in some cases. However, it is not yet possible to fully replace GC-HRMS.

A further refinement of the PLE with in-cell clean-up technique is the modular approach developed in these studies. With this technique it is possible to include various steps for both clean-up and fractionation. For example, sulphuric acid impregnated silica could be combined with active carbon for the simultaneous removal of lipids (along with other interferences) and fractionation of PCBs and PCDD/Fs. It was shown that the method could provide data that agreed reasonably well with both reference values and values obtained using traditional methods. In general PLE proved to have high extraction efficiency and to yield very similar congener profiles to the reference method. In addition, it was shown that it allowed one-step extraction and clean-up of a salmon sample. Such single-step procedures are the ultimate goals for any extraction technique, and it would be highly desirable to develop one-step methods that could be extended to other types of samples. For the rest of the matrices tested (soil, sediment, mussel and crab tissue and flue gas) the method was successful, however a final polishing step is currently required, involving either dilution or clean-up using miniaturized multilayer silica columns, to obtain extracts that are pure enough for GC-HRMS analysis.

Using the developed modular-PLE system substantial costs could be saved. It was estimated that the method could reduce the cost of preparing samples by up to 90%, which would greatly facilitate large-scale inventories.

Key Words: Pressuirzed liquid extraction (PLE), PLE-C, modular PLE, ELISA, dioxin, dioxin.like compounds, GCxGC, analytical methods

Language: English ISBN: 978-91-7264-584-4 Number of pages: 37 + 5 papers



Erik Spinnel



2008



Erik Spinnel



2008

Detta verk skyddas enligt lagen om upphovsrätt (URL 1960:729)

ISBN: 978-91-7264-584-4

Printed in Sweden by VMC, KBC, Umeå University

i

Contents

List of papers ................................................................................................................................. ii

Abbreviations ................................................................................................................................ iii

Abstract .......................................................................................................................................... v

Summary in Swedish .................................................................................................................... vi

Introduction ................................................................................................................................... 1

Objectives ....................................................................................................................................... 3

Dioxins and dioxin-like compounds ............................................................................................. 4

Toxicity and toxic pathways ....................................................................................................... 4

Sources, levels and exposure to dioxins and dioxin-like compounds ......................................... 6

Procedures for analyzing dioxins ................................................................................................. 8

Sampling ...................................................................................................................................... 8

Sample preparation ...................................................................................................................... 9

Pre-treatment .............................................................................................................................. 9

Extraction ................................................................................................................................. 10

Clean-up techniques ................................................................................................................... 13

Concentration ............................................................................................................................ 14

Detection and quantification ..................................................................................................... 14

Gas chromatography – high resolution mass spectroscopy (GC-HRMS) ............................................... 14

Two dimensional gas chromatography (GC×GC) ............................................................................. 15

Biological detection methods ........................................................................................................ 15

PLE for extraction and clean-up of dioxins .............................................................................. 18

Screening of soil samples (Paper I) ........................................................................................... 19

Quantification of abiotic samples (Papers II-III) ...................................................................... 20

Modular-PLE (Papers IV-V) ..................................................................................................... 23

The cost-saving potential of modular-PLE ............................................................................... 29

Conclusions and further developments ..................................................................................... 30

Acknowledgements ...................................................................................................................... 32

References .................................................................................................................................... 34

ii

List of papers This thesis is based on the following papers, which will be referred in the text by their respective roman numerals (Paper I-V).

I. Nording M.; Nichkova M.; Spinnel E.; Persson Y.; Gee SJ; Hammock BD; Haglund P (2006). Rapid screening of dioxin-contaminated soil by accelerated solvent extraction/purification followed by immunochemical detection, Analytical & Bioanalytical Chemistry 385 (2), 357-366

II. Spinnel E; Danielsson C; Haglund P (2008)“Rapid and cost effective analysis of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans in soil, fly ash and sediment-CRMs using pressurized liquid extraction with an integrated carbon trap”, Analytical & Bioanalytical Chemistry 390 (1), 411-417

III. Danielsson C; Spinnel E; Haglund P “Analysis of dioxins in soil and fly ash

using pressurized liquid extraction with an integrated carbon trap followed by comprehensive two-dimensional gas chromatography with electron capture detection”, Submitted to Analytical & Bioanalytical Chemistry

IV. Spinnel E; Fick J; Andersson P,L; Haglund P “Streamlined combustion gas

measurement for improved national dioxin inventories” Submitted to Environmental Science & Technology

V. Spinnel, E; Andersson P,L; Haglund P “Modular Pressurized Liquid Extraction

for Combined Extraction and clean up of dioxin-like PCBs, PBDD and PCDD/Fs” Manuscript

Published papers (Paper I-II) are reprinted with the kind permission of Springer Link

iii

Abbreviations 2,4,5-T 2,4,5-trichlorophenoxy acetic acid AhR Aryl hydrocarbon receptor ASE Accelerated solvent extraction BCF Bioconcentration factor CAFLUX Chemically activated fluorescent gene expression CALUX Chemically activated luciferase gene expression CR Cross reactivity CRM Certified reference material CRV Certified reference value DCM Dichloromethane DDT Dichloro-diphenyl-trichloroethane DMSO Dimethylsulfoxide DRE Dioxin responsive element ECD Electron capture detector ELISA Enzyme linked immunosorbent assay FID Flame ionization detector GC-HRMS Gas chromatography - high resolution mass spectroscopy GCxGC Two dimensional gas chromatography LOI Loss-on-ignition MAE Microwave assistant extraction MSW Municipal solid waste OCDD Octachlorinated dibenzo-p-dioxin PAH Polycyclic aromatic hydrocarbon PBDD Polybrominated dibenzo-p-dioxin PBDF Polybrominated dibenzofuran PCA Principal component analysis PCB Polychlorinated biphenyls PCDD Polychlorinated dibenzo-p-dioxins PCDF Polychlorinated dibenzofurans PCP Pentachlorophenol PLE Pressurized liquid extraction PLE-C Pressurized liquid extraction with integrated carbon trap POP Persistent Organic pollutant PUFP Polyurethane foam plug REP Relative effective potency RSD Relative standard deviation SEPA Swedish environmental protection agency SFE Supercritical fluid extraction Sox-T Soxhlet followed by traditional clean up SPE Solid phase extraction TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin

iv

TEF Toxic equivalency factor TEQ Total toxic equivalency TIC Total ion chromatogram TOF-MS Time of flight mass spectrometry UNEP United nation environmental program USEPA United states environmental protection agency WHO World health organization

v

Abstract Polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) are two structurally related groups of chemicals, generally referred to as `dioxins´. These are of great concern due to their high toxicity and global spread. Other groups of compounds with similar chemical structure and toxicity mechanisms are the brominated analogues polybrominated dibenzo-p-dioxins (PBDDs) and polybrominated dibenzofurans (PBDFs), and the dioxin-like polychlorinated biphenyls (PCBs). Numerous studies have been undertaken to investigate sources and transport routes of dioxins. However, much remains to be done, including analytical, inventories of dioxin-like compounds, such as PBDD/Fs, and the development of more convenient analytical methods. The currently standard procedure for analyzing dioxins (and dioxin-like compounds) is to use Soxhlet extraction followed by multi-step clean-up and gas chromatography - high resolution mass spectrometry (GC-HRMS) for detection. Unfortunately, this method is very solvent, labor and time-consuming, making it very expensive. The main aim of the studies this thesis was to develop pressurized liquid extraction (PLE) with integrated clean up techniques for fast, convenient preparation of dioxin samples. PLE with integrated clean-up has previously been used for extracting dioxins from biological samples, but in these studies the possibility of extending its use to abiotic samples was explored. The results show that PLE with an integrated carbon trap is suitable for analyzing dioxins in various types of soil samples, sediment and flue gas samples. The results also showed that it has potential for analyzing dioxins in fly ash. The thesis focuses on developments of the methodology for dioxin analysis, but also includes results obtained from PBDDs and dioxin-like PCB analyses. In addition, the possibility of using various other kinds of detection techniques rather than GC-HRMS, such as enzyme-linked immunosorbent assays (ELISAs) or two-dimensional gas chromatography with micro electron capture detection (GCxGC-µECD) was explored. The results indicate that ELISA and GCxGC-µECD could serve as complementary detection systems in some cases. However, it is not yet possible to fully replace GC-HRMS. A further refinement of the PLE with in-cell clean-up technique is the modular approach developed in these studies. With this technique it is possible to include various steps for both clean-up and fractionation. For example, sulphuric acid impregnated silica could be combined with active carbon for the simultaneous removal of lipids (along with other interferences) and fractionation of PCBs and PCDD/Fs. It was shown that the method could provide data that agreed reasonably well with both reference values and values obtained using traditional methods. In general PLE proved to have high extraction efficiency and to yield very similar congener profiles to the reference method. In addition, it was shown that it allowed one-step extraction and clean-up of a salmon sample. Such single-step procedures are the ultimate goals for any extraction technique, and it would be highly desirable to develop one-step methods that could be extended to other types of samples. For the rest of the matrices tested (soil, sediment, mussel and crab tissue and flue gas) the method was successful, however a final polishing step is currently required, involving either dilution or clean-up using miniaturized multilayer silica columns, to obtain extracts that are pure enough for GC-HRMS analysis. Using the developed modular-PLE system substantial costs could be saved. It was estimated that the method could reduce the cost of preparing samples by up to 90%, which would greatly facilitate large-scale inventories.

vi

Summary in Swedish Polyklorerade dibenso-p-dioxiner (PCDD) och polyklorerade dibensofuraner (PCDF) är två grupper av likartade kemikalier som oftast går under samlingsnamnet `dioxiner´. De är mycket giftiga och har hittats i miljön över hela jorden. Det finns även andra ämnen med liknande struktur och toxisk verkningsmekanism, t.ex. polybromerade dibenso-p-dioxiner (PBBD) och polybromerade dibensofuraner (PBDD) samt 12 dioxinlika PCBer. Ett stort antal studier har gjorts för att finna källor till dessa ämnen i miljön samt förstå dess spridningsvägar. Många källor och spridningsvägar är dock fortfarande okända och det finns ett stort behov av att identifiera dessa. För att detta ska kunna göras krävs ett stort antal kemiska analyser . Det vanliga tillvägagångssättet då dioxiner ska analyseras är att extrahera provet i en Soxhletutrustning och sedan rena extraktet med en flerstegs reningsmetod. Dioxinhalten i extraktet analyseras sedan med gaskromatografi kopplat till högupplöst masspektroskopi (GC-HRMS). Detta analysförfarande är robust och exakt men tyvärr också väldigt tidskrävande och dyrt. Det finns därför behov av snabbare och billigare metoder. Syftet med studierna i denna avhandling var att undersöka möjligheten att använda trycksatt vätskeextraktion (pressurized liquid extraction = PLE) med integrerad rening för snabb och enkel beredning av dioxinprover. PLE med integrerad rening har tidigare används för extraktion av dioxiner från biologiska prover men i studierna inom denna avhandling undersöktes möjligheterna att också inkludera icke-biologiska prover. Resultaten visade att PLE med en integrerad kolfälla är tillämpbar för analys av olika typer av jordprover, sediment och rökgasprover. De visade också att metoden har potential för analys av dioxiner i flygaska. Vidare undersöktes två alternativa detektionsmetoder, en immunoassay och tvådimensionell gaskromatografi (GC×GC) och resultaten visade att dessa utgör värdefulla komplement till GC-HRMS – framförallt för snabb analys (screening) och prioritering av prover för vidare GC-HRMS analys. Det är i dagsläget dock inte möjligt att fullt ut ersätta GC-HRMS. Slutligen utvecklades ett modulsystem för PLE med integrerad rening vilket gör det möjligt att inkludera olika renings- eller fraktioneringssteg i samma extraktionsprocess. Det var exempelvis möjligt att kombinera silika impregnerad med svavelsyra och aktivt kol för att, i ett första steg, eliminera störande ämnen (ex. lipider eller humusämnen) och i ett andra steg fraktionera PCBer och dioxiner. Tidigare har detta varit omöjligt eftersom de aromatiska lösningsmedel som krävs för att få loss dioxinerna från kolet reagerar med svavelsyra. Modulsystemet gör det möjligt att ta bort prov och svavelsyraimpregnerad silika efter det första extraktionssteget. Det visade sig vara möjligt att utföra en enstegs extraktion/upprening av ett laxprov. Analysresultatet stämde väl med de från traditionell extraktion och upprening. För övriga matriser fungerade metoden också bra med det visade sig vara nödvändig att inkludera ett extra reningssteg för att få extrakten rena nog för GC-HRMS analys. Mycket talar dock för att det är möjligt att eliminera detta steg genom ytterligare metodutveckling. En kostnadsberäkning gjordes för den nya kombinerade extraktions- och uppreningsmetoden. Jämfört med den traditionella metoden kan modul-PLE systemet spara upp till 90 % av analyskostnaden. Genom att kombinera metoden med ett alternativt detektionssteg (ex. en immunoassay) skulle ytterligare besparingar kunna göras.

1

Introduction Persistent organic pollutants (POPs) have been defined by the United Nations Environment Program (UNEP) as compounds that are persistent, bioaccumulative and pose risks to humans and wildlife, both near and far away from their sources. Twelve of these POPs are of special concern and subject to a series of proposals, regulations and limits set by the Stockholm Convention in order to protect the environment and human health. Well-known examples of POPs covered by the convention include dichloro-diphenyl-trichloroethane (DDT) and polychlorinated biphenyls (PCBs). DDT was used as an insecticide and PCBs have been mostly used in transformer oil. These compounds have been found in environmental matrices throughout the world, and shown to cause adverse effects in fauna. Hence, use of these POPs has been banned in most countries, but they can still be found in quite large amounts in environmental compartments all over the world. Among the most toxic compounds included in the convention are the polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofuran (PCDFs), which are commonly referred to simply as dioxins. Dioxins are often considered to be the most toxic man-made compounds ever produced, and they can cause severe effects such as cancer, reproductive toxicity, endocrine disturbances and liver damage. The toxicity of dioxins can be estimated using the toxic equivalence (TEQ) concept, in which the toxicity of each congener is related to that of the most toxic one, 2378-tetrachlorodibenzo-p-dioxin (TCDD) (1). Other groups of compounds with similar chemical properties mediate their toxicity in similar ways to the dioxins (as discussed in more detail later sections). For example, 12 PCB congeners known as the dioxin-like PCBs have similar molecular structures to the dioxin congeners, and their toxicity has also been related to that of TCCD. There are also other compounds that have similar structures, but have not yet been encompassed by the TEQ concept. Such candidates include polybrominated dibenzo-p-dioxins and furans (PBDD/Fs), in which the chlorine atoms are replaced with bromine atoms. Due to the high toxicity of dioxins, the tolerable weekly intake suggested by the European Commission is 14 pg WHO-TEQ/kg body weight (2). The limit value for fish has been set to 4 pg TEQ g-1 fish; a level that is normally exceed in fatty fish from the Baltic Sea. Hence, according to EU legislation, fish from the Baltic Sea should not be sold outside Sweden and Finland (3). Limit values have also been set for various abiotic materials. For example, the limit for dioxins in flue gases emitted from industrial stacks is 0.1 ng WHO-TEQ m-1. Soil intended for “sensitive” use has a recommended limit value of 10 ng kg-1 dry substance and soil intended for less sensitive use has a limit value of 250 ng g-1(4). There are diverse sources of dioxins. For example, they have been released as impurities in organochlorine formulations used as fungicides or insecticides, they are formed during pulp bleaching, combustion of municipal solid waste and the chloralkali process (5-7). In addition, burning of material containing brominated flame retardants is a known source of PBDD/Fs (8). It

2

is also known that dioxins can be transported long distances since they have been found in all compartments of the world, even the Arctic (9). However, although dioxin sources have been identified in many studies, much remains to be learnt about their dispersal, effects, fate and remediation. Furthermore, various sources (especially in developing countries) have not yet been explored (10). In order to help estimate dioxin releases, a flexible toolkit has been developed by UNEP that enables developing countries to acquire estimates of potential dioxin sources and to compile a national dioxin inventory (which otherwise requires large numbers of dioxin analyses). However, there are still large uncertainties associated with such estimates and there is still a need for complementary measurements to improve the data derived using the toolkit. Unfortunately, dioxin analysis is very expensive. Due to the low limit values the analytical methods applied have to meet challenging requirements. A suitable analytical method should be able to detect levels of approximately 1 pg g-1 (10-12 g), and thus capable of distinguishing components accounting for 0.0000000001% of the mass of a sample from the rest of the matrix. Dioxin analysis is often performed by Soxhlet extraction followed by multi-step cleaning using open columns and a final detection step using gas chromatography coupled to high resolution mass spectroscopy (GC-HRMS). However, this analytical procedure is very complex, and requires quite large amounts of ultra-pure organic solvent, skilled laboratory personnel and quite large amounts of time. Consequently, the cost for a single analysis is very considerable. Handling large amounts of organic solvent also poses risks to the laboratory personnel and the environment. Hence, there is a need for more efficient analytical solutions, for economic, safety and environmental reasons, which prompted the studies underlying this thesis.

3

Objectives The objectives of the studies this thesis is based upon were to develop pressurized liquid extraction (PLE) with in-cell clean-up techniques for the rapid preparation of samples prior to dioxin analysis. PLE with in-cell clean-up has previously been used for analysis of dioxins in biological samples, but in these studies the suitability of applying PLE with in-cell clean-up was extended to abiotic samples, and the possibility of further streamlining the procedure for applications to biological samples, were assessed. The ultimate goal was to develop a one-step extraction and clean-up method suitable for analyzing various types of dioxin-like compounds. Possibilities for combining PLE in-cell clean-up with detection techniques other than GC-HRMS were also addressed. The results of this project have been presented in five papers (Papers I-V). In Paper I, PLE with an integrated carbon trap (PLE-C) was evaluated as a tool for screening soil samples. Ten different samples were prepared in parallel with traditional Soxhlet-based extraction and PLE-C. In this study an alternative detection step using an enzyme-linked immunosorbent assay (ELISA) was also evaluated. In Paper II, PLE-C was validated for soil and sediment certified reference materials (CRMs). Promising indications that the method could also be useful for analyzing fly ash were also obtained, although further optimization might be needed. The soil and fly ash CRMs were extracted with PLE-C and a traditional set-up in the investigations reported in Paper III, and the potential utility of detecting analytes using two-dimensional gas chromatography with electron capture detection (GC×GC-µECD) systems was evaluated. A further improvement of the PLE with in-cell clean-up method, presented in Paper IV, was a coupling cartridge that allows the sample, adsorbents or retainers to be added and/or removed at appropriate stages of the extraction. In addition, PLE-C with the coupling cartridge was combined with a device for sampling flue gas and presented, in Paper V, as a tool for streamlined analysis of dioxins in flue gas. The full potential of modular-PLE was assessed in the studies reported in Paper V, which also addressed the possibility of including the dioxin-like PCBs and PBDDs in the analysis.

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Dioxins and dioxin-like compounds PCDDs and PCDFs are chemically similar groups of compounds with high melting points and low vapor pressures that are nonpolar and lipophilic. There are 75 possible PCDDs and 135 possible PCDFs, each differing in numbers and positions of chlorine atom(s). Most of these congeners are easily metabolized, and only those with chlorines in the 2378 positions (seven PCDDs and 10 PCDFs) are considered toxic. These congeners are very stable in organic tissues and their bioconcentration factors (BCFs) in fish are very high, > 500000 for 2378-TCDD and > 390000000 for octachlorinated dibenzo-p-dioxins (OCDD), and the half-life of 2378-TCDD in human tissues is estimated to be more than seven years (11).

Figure 1: General structure of polychlorinated dibenzofuran (PCDFs) (left) and polychlorinated dibenzo-p-dioxins (PCDDs) (right) There are also other POPs that have similar properties to the PCDD/Fs. For example, there are PCB congeners without ortho chlorines that have similar molecular structures and properties to the PCDD/Fs, and hence are called dioxin-like PCBs. Other dioxin-like compounds include various compounds such as polychlorinated naphthalenes, polybrominated and polychlorinated diphenylethers, polybrominated biphenyls, and mixed brominated and chlorinated dioxins (1).

Toxicity and toxic pathways

The most toxic of all the dioxin congeners is 2378-TCDD. The LD50 of this compound in guinea pigs has been estimated to be as low as 0.06 µg/kg (12). However, there are wide ranges in its toxicity both within and between different species. For example, the LD50 for hamster is about 5000 times higher than that of guinea pigs (13). For obvious reasons, the LD50 has not been established for humans. However, estimates obtained from people accidentally exposed to high doses indicate that the LD50 for humans is much higher than that of guinea pigs. In an extreme case almost 144 ng/g was found in the blood stream of a living person (14). People who have been exposed to high doses of dioxins have often developed chloracne, a skin disease which looks like

5

severe “normal” acne. One of the most famous recent cases of chloracne is that of the Ukrainian president Victor Yushchenko, who was poisoned by dioxins in 2004. Other direct effects observed in people exposed to dioxins include nausea, headache and vomiting (15,16). In the longer term the chronic effects have proven to be much more severe, since dioxins are known inter alia to promote cancer and cause liver damage, endocrine disruptions and reproductive disturbances (1). The toxicity of dioxins is believed to be mediated through the Aryl hydrocarbon Receptor (AhR), a protein located in the cytoplasm (17). Dioxin molecules bind to the AhR, forming a complex that is translocated into the nucleus via binding to other proteins (18). In the nucleus, the AhR-dioxin complex binds to specific DNA sequences called dioxin responsive elements (DRE) and induces transcription of the CYP1A gene, which encodes the protein cytochrome P4501A1. Enhanced levels of this protein may be indicators of dioxin exposure. The AhR-ligand complex may also bind to DREs in the nucleus expressing other proteins, which may be the causes of effects of dioxins, such as cancer (19). The AhR-mediated toxicity hypothesis has been corroborated by Fernandez-Salguego et al., who found AhR-deficient mice to have resistance to 2378-TCDD (20). The toxic effects of all of the 2378-substituted congeners seems to be mediated through the AhR and their binding affinity to the AhR seems to correlate with their toxicity (notably, 2378-TCDD has the highest binding affinity and is the most potent dioxin). In 1986, Eadon et al. (21) suggested a concept in which the toxicity of each congener is compared to that of the 2378-TCDD, which was assigned a “toxic equivalency factor” (TEF) of 1. The remaining 16 dioxins were assigned relative values between 0.1 and 0.0001. The concentration of each congener could then be multiplied by its assigned TEF value to obtain a TCDD equivalent (TEQ) value, i.e. an indicator of the equivalent amount, in terms of toxicity, of pure 2378-TCDD. The TEQs of each congener present in a sample can be summed to obtain an estimate of the total TEQ for the sample. Some PCBs were also subsequently shown to bind to the AhR (22). Dioxin-like PCBs with no chlorines in ortho-positions seemed to have similar toxicity to the PCDD/F congeners, and some PCBs with one chlorine atom in the ortho position (mono-ortho PCBs) seemed to have dioxin-like toxicity. Hence, a number of non-ortho and mono-ortho PCBs were included in the TEF concept. The TEF-values have been evaluated and reevaluated several times (23,24). The most recent reevaluation was coordinated by the WHO in 2005 and the results were presented by van den Berg et al. (1). As more information becomes available, new reevaluations will be performed to assign compounds new TEF values and possibly assign values to new compounds, e.g. polychlorinated naphthalenes, polybrominated diphenylethers, polybrominated biphenyls, and mixed brominated and chlorinated dioxins (1). Two additional groups of halogenated aromatic compounds are polybrominated dibenzo-p-dioxins (PBDDs) and dibenzo furans (PBDFs) or `brominated dioxins´ which are compounds with similar properties to the chlorinated dioxins. Birnbaum et al. (25) suggested that the PBDD/Fs may have similar toxicity to the PCDD/Fs. This hypothesis was confirmed by Olsman et.al. (26), who showed that brominated and mixed brominated/chlorinated dibenzo dioxins and furans have similar mechanisms of action in the cell (mediated through the AhR). Some of the compounds even seemed to have higher potencies than 2378-TCDD.

6

Sources, levels and exposure to dioxins and dioxin-like compounds

Several accidents and incidents have caused severe human dioxin poisoning. During the Vietnam War in the 1960s and 1970s the American air force used a compound known as Agent Orange for tactical defoliation and crop destruction (27). Agent orange consisted of a mixture of esters of 2,4,5-trichlorophenoxyaceticacid (2,4,5-T) and 2,4-dichlorophenoxyacetic acid. Unfortunately, it was also heavily contaminated with 2378-TCDD. In exposed populations of Vietnam various adverse effects have been observed and there are still large amounts of dioxins in the soils of southern Vietnam (28,29). In Japan, in 1968 almost 1900 people suffered from a disease called “Yusho” which is Japanese for “oil disease”. It was shown that the people had eaten from the same batch of rice oil that had been polluted with PCBs, and contained quite large amounts of PCDF contaminants. In Taiwan an almost identical incident called the “Yu-Cheng” incident occurred in 1979, in which approximately 2000 were affected. In both incidents people suffered from symptoms such as chloracne, increased eye discharges and dermal pigmentation (30,31). Several years after the poisoning adverse effects were still seen in the population (32). Furthermore, in 1976 there was an explosion in a factory producing 2,4,5-T in Seveso, Italy, resulting in the release of quite large amounts of 2378-TCDD into the nearby residential area and many deaths amongst both wild and domesticated animals. There were no reported human deaths, but more than twenty years after the explosion dioxin effects were still being seen in the exposed population (33). There are some known natural sources of environmental dioxins, such as forest fires, volcanoes and burning of material containing common salt, such as sea-weed (5). However, most of the dioxins in the environment have anthropogenic origins, even though dioxins have never been produced intentionally. Incineration of municipal solid waste (MSW) has been a known source of PCDD/Fs ever since Olie et al. found dioxins in MSW fly ash in 1977 (6). In developing countries it has been suggested that back-yard burning, forest fires and fires at dump sites are major dioxin sources (34). If the incinerated material contains brominated materials such as brominated flame retardants, PBDDs may also be formed during combustion (8). In the 1980s, Rappe et al. found that dioxins are formed during the pulp bleaching process when Cl2 is used as a bleaching agent (35,36). The chlorakali process was also shown to be a source of dioxins. Sludge from chloralkali graphite electrodes has been found to contain PCDFs at levels up to 650000 pg g-1sludge (36,37), leading to high levels of PCDFs at sites where this sludge has been deposited. Other known sources include impurities in organochemical products, such as PCPs (7), which have been used as wood preservatives, and the incautious use of these products has led to contamination at many old sawmill sites. The Swedish EPA estimates that in Sweden alone there are approximately 500 sites with a historical record of CP usage that has probably resulted in

7

heavy dioxin contamination (4). Persson et al. have investigated several sawmill sites and found dioxins at levels ranging from 0.14 to 3000 µg kg-1 dry soil on a WHO-TEQ basis (38). Since the dioxins are very persistent and lipophilic there is a considerable risk that they will eventually end up in biological tissues. Furthermore, if the compounds enter the food chain they biomagnify and may eventually pose threats to the top predators and humans. Dioxins from contaminated sawmill sites may enter the food chain via terrestrial organisms such as earthworms and frogs (39,40). From the contaminated sites the dioxins may leach out into water recipients via colloids (41), and if they leach into nearby waters they may enter the food chain, e.g. via fish (42). Most human dioxin intake is via the food, and major contributors to the dietary intake include fish and other seafoods (43). High levels of dioxins have been found in fish and fish-eating predators. PBDDs have also been found in marine biota such as blue mussels (44,45), and it has been suggested that some of these PBDDs may be biosynthesized (44). For these reasons, van den Berg et al. (1) noted that “If the presence of PBDDs and PBDFs in human food as well as in people is more extensively demonstrated, there would be a clear need for assigning TEFs to these compounds”. Hence, it is possible that the PBDD/Fs will be assigned TEFs in the near future, especially since they have been found in quite large amounts in marine biota. The use of former industrial sites for housing, playgrounds etc. might pose threats related to exposure to dioxins, especially for small children, who may ingest high quantities of soil. Unfortunately, it is very expensive to remediate a contaminated site completely. Even a full quantification survey of a contaminated site has been estimated to cost between 100,000-200,000€ (4), and full remediation of the site would cost several millions. Hence, it is important to remediate contaminated sites properly, but due to the high costs involved it is also important to avoid remediation of soil or land that poses little risk. Although levels of dioxins from known sources seem to have declined (5) there are still unknown sources and contamination pathways. Many sources have not yet been identified, especially in developing countries (10). Hence, there is still a need for large-scale surveys to identify possible dioxin sources.

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Procedures for analyzing dioxins Dioxin analysis is a very complex process that is labor, solvent and time intensive and, hence, very expensive. There are several approaches for analyzing POPs such as PCDD/Fs from environmental matrices (46-48). However, all of them can be divided into two parts; sample preparation and detection (or three, if sampling is considered an analytical step) (Figure 2). Advances introduced at various times have improved the methods by making them more integrated, rapid and cheaper. These advances are discussed in this chapter.

Figure 2. General schematic picture of the dioxin analysis procedure

Sampling Sampling is the step in which samples are collected and, as with all environmental analysis of POPs, appropriate sampling is essential in order to obtain a representative sample. For obvious reasons sampling procedures differ greatly depending on the matrix to be sampled. For example, the procedures for taking fish samples and soil samples differ enormously, as do the procedures for sampling flue gases and sediments. Several issues must be considered when sampling, because (inter alia) contamination of the collected material during sampling is a common source of error in the analytical procedure. It is also very important to perform the sampling at the right time and place. For example, when evaluating an old sawmill site with possible dioxin contamination it is important to know where polluting activities could have been performed. Hence, large amounts of planning and experience are often required to obtain appropriate samples (49).

Pre-treatment Extraction Clean-up Concentration

.

DetectionSample PreparationSampling

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Sample preparation After sampling the samples need to be prepared for chemical analysis in order to transfer the analytes into a medium (extraction) that can be introduced to the detection system, to remove compounds that might interfere with the analytes (clean-up) and finally concentrate the analytes (if necessary) so they can be detected by the detection system. For some analytes, sample preparation procedures may be very simple and merely involve straightforward steps such as dilution, filtration and/or centrifugation (50). However, for POP analysis sample preparation is often the bottleneck, and may require more than 60% of the total analysis time (51). Traditional dioxin sample preparation also often involves many manual steps, raising risks of analyte losses or contamination. The large numbers of manual steps also means that dioxin analysis is labor-intensive, which contributes to the high costs of analysis. The requirement for many manual steps also increases risks to the laboratory personnel (and the environment) since quite large amounts of organic solvent are used. Hence, skilled and experienced personnel are needed to perform high-quality dioxin analysis and there is a need for more efficient sample preparation procedures. Ideally sample preparation should be fast, efficient, safe, relatively cheap and requiring as few manual steps as possible. Sample preparation procedures have two major stages, extraction and clean-up. Prior to extraction a sample must undergo pre-treatment and after extraction and clean up, the final step of sample preparation is to concentrate the sample (Figure 2). An internal standard is often added before the extraction to be used during the final quantification.

Pre-treatment Pre-treatment is the first step of sample preparation. Pre-treatment varies depending on the matrix and it has several purposes. If there is water in the sample it has to be removed since it might otherwise reduce the effectiveness of the extraction of POPs. This can be done by several methods, such as air drying, freeze-drying and/or mixing with anhydrous sodium sulfate. In addition, some samples may be ground to reduce the size of particles in them, increase the surface area and enhance the diffusion rate of the analytes/solvent. Since POPs such as dioxins tend to adsorb to organic materials or lipids it could also be important to determine the fat or organic carbon or fat content of a sample. This latter is generally done by extracting them from the sample with an organic solvent, then evaporating the solvent and measuring the residual lipids gravimetrically. The amount of organic compounds in some matrices, e.g. soil samples, may be determined by measuring the loss-on-ignition (LOI), i.e. the weight of the sample after heating it to 550ºC minus its weight after drying it at 105ºC.

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Extraction The main purpose of extraction is to transfer the analyte(s) from the matrix to a suitable medium for introduction into the analytical instrument for analysis or further clean-up prior to analysis. There are four major factors governing the extraction efficacy: solute-matrix interactions, solute solubility and diffusion-, and viscosity of the solvent. Thus, the most important issue when extracting POPs from a solid matrix is to choose an appropriate solvent. Efficient extraction requires use of a solvent in which the analyte has good solubility and that efficiently overcomes solute-matrix interactions. Samples with high percentages of polar surface functional groups or organic matter, in particular soot carbon, are particularly difficult to extract and thus require “strong” solvents. Generally, abiotic samples bind pollutants more tightly than biological tissue samples. Binary mixtures of polar and nonpolar solvents are often required to overcome solute-matrix interactions, especially for polar solutes, and to achieve good analyte solubility. For example, Fitzpatrick and Dean found dichloromethane (DCM) to be suitable for extracting DDT and its metabolites, while a mixture of acetonitrile and DCM was required to extract chlorophenols efficiently (52). A mixture of solvents with different properties could further improve the extraction efficiency. For example, wet samples are more efficiently extracted by a mixture of polar and nonpolar solvents since the polar solvent (e.g. acetone) can improve penetration of thin water layers surrounding particles (53). Kirgushi et al. showed a mixture of toluene and acetone to be more suitable for extracting PCDD/Fs from soil samples than pure toluene (54). Diffusion is another important issue to consider in extraction. If the diffusion rate is high the solvent can more easily enter pores and cavities where analytes might be located. The viscosity of the solvent is also important. A reduction in viscosity has the same general effect as an increase in the diffusion rate. Another key variable influencing diffusion rates in extractions is the temperature, the influence of which on diffusion rates can be predicted using the Stokes Einstein equation (55,56). For instance, increasing the temperature from 50ºC to 150ºC raises the solubility of anthracene in DCM 13-fold (55). If the temperature and diffusion rates are increased and viscosity decreased pores and cavities in a matrix will be more easily accessed by the solvent and thus more readily reach the analytes. Furthermore, the strengths of chemical bonds are weakened by increases in temperature, which increases the tendency of analytes to leave the matrix (55,56). Extraction efficiency is also enhanced if a high chemical gradient is maintained, e.g. by constantly introducing fresh solvent to the sample. If this is not done there is a risk that the solvent will become saturated, preventing any further extraction of analytes. Extraction in which a constant flow of fresh solvent is introduced to the matrix is referred to as “dynamic”, while in “static” extraction the solvent is not exchanged. To optimize the efficiency of extraction a balance must be reached between the volume of solvent used and the time that is needed for diffusion of the analytes to the bulk solvent.

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The extraction of POPs from aqueous samples is simpler, since such samples can be merely shaken directly with a water-immiscible organic solvent. This is called liquid-liquid extraction, since the analytes are partitioned between the resulting two liquid phases, in proportions that depend on their chemical properties (50). A modern approach used to extract POPs from water is solid phase extraction (SPE), which has proven to be excellent for extracting various substances, e.g. dioxins, from water samples (57). Further aspects of extraction and sorption mechanisms are reviewed by Pawliszyn (58).

Classical solid-liquid extraction methods There are several ways to extract POPs from a solid sample. Cold column extraction is a common method for extracting various analytes from biological tissues (59). However, this method requires quite large amounts of ultra-pure solvent, thus entailing high costs and both environmental and occupational risks. Nevertheless, it is a robust, widely accepted method so cold column extraction was used for the reference analyses described in Paper V. Several other extraction techniques are well established, such as reflux extraction, ultrasonication and shake flask extraction (50). However, the most common of the “classical” extraction techniques is Soxhlet extraction. This method was developed in 1879 by Franz von Soxhlet and has been used with only minor modifications ever since. Due to the slow diffusion rates of the solvent into the sample, the Soxhlet process often has to continue for several hours and consumes relatively large amounts of solvents. Nevertheless, since the Soxhlet method has been used for so long it is often regarded as the reference method when developing new methods (e.g. USEPA method 1613) (60). Soxhlet extraction can be used with a wide range of solvents, but for dioxin analysis the most commonly used solvent is toluene (46). In summary, the Soxhlet method is robust and reliable but time and solvent consuming.

Modern solid-liquid extraction techniques In this thesis, three extraction techniques are referred to as “modern”: microwave-assisted extraction (MAE), supercritical fluid extraction (SFE) and pressurized liquid extraction (PLE). Results obtained with these methods and Soxhlet extraction have been compared in several studies, and in most cases their extraction efficiency has equaled or exceeded that of Soxhlet extraction (61-63). Microwave-assisted extraction was first described in 1986 by Gantzler, who used a household microwave oven to extract organophosphate pesticides and crude fat from soil, seed, food and feed (64). MAE has been used for extracting organic compounds such as Polycyclic Aromatic Hydrocarbons (PAHs) and PCBs from soil and sediments (47,65), and PCDD/Fs from sewage sludge (66). The MAE technique requires lower amounts of solvent than Soxhlet extraction and offers higher sample throughputs due to the shorter extraction times. However, it has been applied in few studies to PCDD/Fs in environmental matrices, especially since PLE and SFE (see below)

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systems have become available. A major advantage of MAE is the possibility it provides to extract several samples in parallel, while its main disadvantages are the requirements for intense clean-up prior to detection after its use, and the lack of possibilities for automation it offers. SFE is a technique that was first described by Stahl in 1977 (67). It is an extraction method that utilizes gases/liquids in a supercritical phase, reached at high pressures and temperatures, in which the substance is in an intermediate state between a gas and a liquid, having the density of a liquid, the viscosity of a gas and a diffusion rate somewhere between that of a gas and a liquid. This is commonly performed with water or CO2 (which has a number of advantageous characteristics, including low polarity and non-toxicity). CO2 dissolves the analytes in the sample which are subsequently trapped in either an organic solvent or a solid adsorbent such as carbon. However, since supercritical CO2 is a relatively nonpolar solvent polar compounds are poorly extracted by it. The solution for this is to add a modifier or co-solvent; often a polar solvent that is added either to the sample matrix or to the compressed extraction gas. The modifier increases the solubility of polar analytes and compounds in the nonpolar CO2. Key advantages of using SFE are the low amounts of organic solvent required and the possibility to adjust the extraction settings for extracting different target compounds, e.g. PCDD/Fs, PCBs and pesticides (68). Its main disadvantages are the advanced instrumentation and technical skill required and its matrix dependency. SFE has been used for analyzing dioxins in a wide range of matrices such as soil, fly ash and sediment (69-71). However, the most widely used modern extraction method is pressurized liquid extraction (PLE), also known as accelerated solvent extraction (ASE) or pressurized fluid extraction. It was first presented by Richter et al. (65) in 1996, who showed that it can be used to extract PAHs, PCBs and total petroleum hydrocarbons from a wide range of solid matrices. In PLE high pressure and temperature are used to promote efficient extraction. Due to the high pressure, the temperature can be kept above the boiling point of the solvent, which further increases the extraction efficiency. The elevated temperature also increases the diffusion rate and reduces the viscosity of the solvent, while the high pressure forces the solvent into pores and cavities in the matrix that it would not otherwise enter, thereby exposing more analytes to the solvent and allowing them to partition into the solvent. The high pressure/temperature settings of PLE allow samples to be extracted more rapidly, efficiently and with lower solvent consumption than other extraction methods. When performing a PLE extraction, the choice of solvent used is also of great importance. For example, Zuloaga et al. found that methanol extracted ca. 30% less PAHs from soil than dichloromethane (65). In addition, a thin layer of water covering particles in the sample may adversely affect the extraction efficiency by preventing the solvent from reaching the analytes, but this can be countered by extracting with a mixture of polar and nonpolar solvents (54), or drying the sample prior to extraction. Since 1996 many applications have been presented for PLE (72,73). It has been used for extracting (inter alia): PCBs from soil (54,74), sediment (55,75,76) and sewage sludge; PAHs from soil (55,74) sediment (55,77) and fly ash (78,79); and pesticides

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from feed samples (80). The first study in which PCDD/Fs were extracted by PLE was published in 1997 by Richter et al. (81), who used a Dionex ASE 200 to extract soil, sediment, fly ash and urban dust, with similar recoveries to Soxhlet extraction. Since then PLE has been used for extracting PCDD/Fs from many different matrices and several alternative approaches have been applied (78,79,81,82).

Clean-up techniques When extracting a sample to analyze dioxins, many other compounds are often co-extracted. Notably, when extracting a biological sample lipids is co-extracted with the analytes, and when extracting a sample of an abiotic matrix, e.g. soil, a wide range of macromolecules are co-extracted, such as humic substances. These co-extracted compounds will interfere in subsequent analyses, reducing the detection system’s sensitivity and possibly co-eluting with the analytes. Hence, interfering compounds should be removed, in the so-called clean-up phase, prior to the detection step.

Classical clean-up methods

The normal clean-up procedure in dioxin analysis is performed using several open columns (59). One common option is to use a multi-layer silica column containing layers of silica and acid- and/or base-modified silica, which removes compounds such as lipids. Another adsorbent that could be used is aluminum oxide, which is commonly used for separating the bulk of the PCBs from the non-ortho PCBs and PCDD/Fs. In addition, active carbon can be used for shape-selective separation of PCBs and PCDD/Fs (83), which is an important step sometimes since PCBs are abundant in many, especially biotic, samples and can interfere with subsequent dioxin analysis by GC-HRMS. Florisil can also be used for separating PCBs and PCDD/Fs (60,84).

Modern cleanup methods Two systems for clean-up tailored for dioxin analyses are commercially available that prepares extracts of samples in readiness for chemical analysis, are commercially available: the Dioxin Prep System and PowerPrepTM. The “Dioxin Prep System” is a manual system produced by Supelco that consists of a multi-layer silica column coupled in series to a dual-layer carbon cartridge (85). It has been further developed by the addition of a florisil column, which enables the 12 dioxin-like PCBs to be included in the analysis (86). However, it is still quite labor-intensive and solvent consuming. The PowerPrepTM (Fluid Management System, Inc.) has been utilized for samples of several abiotic matrices(87), and also biological matrices (88). The system can be left unattended and a sample can be run in less than three hours. However, it is expensive since it utilizes quite large amounts of ultra-pure solvent and disposable Teflon columns. The sample throughput is also low, with a maximum of six parallel samples.

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Concentration After extraction and clean-up, a volume correction (recovery) standard is normally added and the sample volume is reduced by evaporation in order to lower the limits of detection. A high boiling solvent such as tetradecane or dimethyl sulfoxide (a “keeper”) is sometimes added to avoid evaporation losses.

Detection and quantification The final step in an analysis is detection, in which levels of the analytes are measured. Generally, a chemical analytical technique is applied, and the instruments used are often very expensive and have high maintenance and labor requirements. For determining individual dioxin congeners only one option used to be available (GC-HRMS), but the recently developed two-dimensional GC (GC×GC) with micro electron capture detection (µECD) now offers a second option. However, in many cases it is sufficient to determine TEQ levels, which can be done using various bioanalytical methods.

Gas chromatography – high resolution mass spectroscopy (GC-HRMS) In the GC the analytes are introduced into a column containing a stationary phase that may have various properties, depending on the target analytes, ranging from charged to nonpolar. The analytes are eluted from the column by the application of a temperature gradient that separates them according to their boiling points and affinity to the stationary phase. After they have eluted from the column the analytes are introduced to the HRMS where they are charged and/or fragmented using electrons (electron impact) or charged molecules (chemical ionization), yielding ions with characteristic mass-to-charge (m/z) ratios. The generated ions are then separated in the mass spectrometer according to their m/z ratios by electromagnetic fields that only allow ions with specific m/z ratios to reach the detector at any given time. A HRMS can typically distinguish between ions with differences in m/z of 0.01 and detect analytes in quantities of less than one picogram. Dioxins and dioxin-like PCBs are analyzed in single ion recording (SIR) mode, in which samples are scanned for ions with m/z ratios of specific ions (compounds). This selective mode maximizes the selectivity, and thus sensitivity, of the instrument, which is essential for analyzing the typically low amounts of these compounds. The GC-HRMS technique has high sensitivity but is very expensive and requires highly skilled technicians. The throughput of samples is also very low since it takes approximately an hour per sample. However, it is possible to run several sample in series and the system can be left unattended. Using GC-HRMS the amounts of dioxins present in a sample can be quantified by the isotope dilution method (60), in which 13C12-labeled compounds are added to the sample at the beginning and end of the sample preparation process. The method is based on the assumption that 13C12-

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labeled dioxins behave identically to the native congeners. The native and labeled compounds can be distinguished in HRMS mode and, hence, it is possible to compensate for losses of analytes during the sample preparation procedures. They are subsequently quantified by comparing the peak areas of compounds detected in the sample and a reference standard with known amounts of each congener.

Two dimensional gas chromatography (GC×GC) When complex samples are being analyzed, use of a single GC column and temperature program may be insufficient to separate all 2378-congeners fully. Often, two or even three columns may be required to separate fully all of the coeluting peaks. An upcoming method for detecting different types of analytes is comprehensive two dimensional GC (GC×GC). In this approach two coupled GC columns (usually with widely varying properties, e.g. a polar column and a nonpolar column) are used to separate the analytes more comprehensively than is possible with single GC. The analytes can be detected after separation using flame ionization detection (FID), electron capture detection (ECD) or time-of-flight mass spectroscopy (TOF-MS). Due to the high detection limits of FID it is not appropriate for dioxin analysis, but analyses using both ECD and TOF-MS have been presented (89). GC×GC combined with ECD has been used for analyzing dioxins in matrices such as fly ash, food and feed (90,91). In the investigations reported in Paper III, GC×GC connected to a micro-ECD (µECD) system was used for analyzing dioxins in soil and fly ash. The advantage with GC×GC-µECD is that congener-specific data can be acquired from a single injection, even for very complex samples, while at least two injections are often needed for a full separation of all 2378-congeners by GC-HRMS. The disadvantage with GC×GC-µECD is that 13C-labeled standards interfere with quantification of the analytes. Hence, other native compounds with similar properties have to be used instead. However, such compounds could be difficult to find since possible candidates may be abundant in complex environmental samples.

Biological detection methods Bioanalytical methods such as bioassays or immunoassays could provide high throughput, low cost alternatives to instrumental analysis. Bioanalyses are not likely to replace instrumental analysis completely in the near future. However biological detection methods (or bioanalytical methods) are currently used for screening and prioritizing samples that are contaminated and for distinguishing those that are uncontaminated. Bioanalytical methods are relatively simple to apply and quite large numbers of samples can be processed in parallel using them. For dioxin analysis bioanalytical methods do not yield congener-specific results but can provide indications of total TEQ values. A recent review of various bioassay and biomarkers techniques has been presented by Behnisch et al. (92).

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Cell-based Bioassays (CALUX and CAFLUX) In chemically activated luciferase gene expression (CALUX) bioassays, genetically modified mouse or rat hepotoma cells containing a gene that expresses luciferase following exposure to AhR-agonists are used for measuring AhR-inducing toxicants (93). The levels of luciferase expressed by the cells are correlated to the amount of AhR-agonists present in the sample, and thus the levels of dioxin and dioxin-like compounds, i.e. the TEQ value. Chemically activated fluorescent gene expression (CAFLUX) assays are similar system to CALUX assays, but instead of luciferase an enhanced green fluorescent protein (EGFP) is expressed (94). Unlike CALUX cells, the cells in a CAFLUX assay do not need to be harvested prior to measurement since the fluorescent light is measured in the intact cells. Both CALUX and CAFLUX assays have been used for analyzing dioxins in diverse matrices, e.g. soil, fly ash, and food and feed stuff (18,95-98). In order to estimate a TEQ value for a sample using a bioassay, binding affinities of individual congeners – relative effect potencies (REPs) – need to be determined since they may differ from the TEF-values (96,99). Relative potencies of individual compounds are calculated based on a standard curve obtained from cells exposed to known concentrations of 2378-TCDD (TEF =1.0).

Immunoassays Immunoassays have been used to predict total TEQ values of many types of samples (100-103). Instead of using recombinant cells, in immunoassays antibodies that bind to dioxins or dioxin-likes compounds, e.g. 2378-TCDD, are used. Other congeners will also bind to the antibodies, but with lower affinity, referred to as their cross reactivity (CR), which is comparable to the individual REPs determined in CALUX and CAFLUX assays. Using CRs the total TEQ in a sample can be predicted. However, CR values may differ from TEF values, which might lead to an under- or over-estimation of the total TEQ. For example, the CR of 1,2,3,4,6,7,8-HpCDF is 0.0006 (101), while its TEF value is 0.01 (1). This congener is often present in relatively large amounts at sites contaminated with PCPs, and if such samples are analyzed using an immunoassay their true TEQ-values may be underestimated. However, this problem can be solved using safety or site-specific factors. In Paper I, an enzyme-linked immunosorbent assay (ELISA) was evaluated as an alternative to GC-HRMS for detecting dioxins when screening soil samples. The advantages and disadvantages of bioassays and immunoassays are often very similar. Both offer the capacity to run many parallel samples and they can detect compounds with dioxin-like activity since they respond to all compounds that bind to the AhR. This can be both advantageous and disadvantageous. It is advantageous since compounds with dioxin-like activity such as PBDDs are detected, which might be missed if only GC-HRMS is used. However, there are non-dioxin-like compounds that could interfere with the bioassay or immunoassay and bind to the AhR, and thus induce a false positive or negative dioxin signal. Since there are many compounds that might interfere with such assays, rigorous clean-up is needed and thus the benefits of using a bioanalytical method are reduced. A major disadvantage with bioanalytical methods is the lack of

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scope to measure recoveries since 13C-labeld standards interfere with the assay. In addition, the cell lines are living materials that require a sterile laboratory environment due to the risk of infection. One convenient application of bioanalytical methods is for measuring samples before and after treatments designed to remove PCDD/Fs, e.g. thermal treatment of fly ash (104,105). In such cases degradation products with dioxin-like activity that might be present, and may be missed by GC-HRMS, may be detected by the bioassay. They can also be used for following a remediation process over time and as simple tools for prioritizing samples for further analysis by GC-HRMS.

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PLE for extraction and clean-up of dioxins Dionex presented a strategy for in-cell clean-up already in 1996, in which acidic alumina was used to retain lipids when PCBs in fish samples were analyzed (106). Since then PLE with in-cell clean-up has been used to extract diverse contaminants in biological and food samples (107), such as acrylamide (108), sulfonamides (109), musk compounds (110) and PCBs (111-113). It has been applied less often to abiotic samples, although PAHs have been extracted from sediments and soil, while polar sample residues have been removed using in-cell silica gel purification (114,115). A further development was described by Lundstedt et al. who presented a method for simultaneous extraction and fractionation of PAHs and their oxygenated derivatives from soil samples (116). PLE combined with in-cell clean-up has also been used in PCB and PCDD/F analysis. A method that was first presented by Sporring and Björklund (113), in which PLE with sulphuric acid modified silica was used for simultaneous fat removal and extraction of PCBs, was later shown to be suitable for extracting PCDD/Fs by Wiberg et al. 2007, using an ASE®300 with large extraction cells to maximize the sample capacity. The PCBs and PCDD/Fs were subsequently separated using a carbon column prior to analysis by GC-HRMS. This shape-selective fractionation procedure was later combined with PLE. Nording et al. (98) loaded activated carbon into specially designed assemblies for combined extraction and fractionation of dioxins in fish meal and fish oil. The extracts were further purified by passage through a miniaturized multi-layer silica column and were detected with a CAFLUX bioassay. These special inserts were further used by Haglund et al. (117), who also examined the feasibility of including the dioxin-like PCBs in the analysis. Two fractions were collected, the first of which contained fat and mono-ortho PCBs while the second contained the non-ortho PCBs, PCDD/Fs and fat residues. The second fraction was ready for GC-HRMS after it had been passed through a miniaturized multi-layer silica column.

In the studies reported in Papers I-IV a PLE method with in-cell extraction, clean-up and fractionation (PLE-C) was refined and applied for analyzing PCDD/Fs in soil, sediment, fly ash and flue gas. PLE-C is a two-step extraction method in which the cell is packed with a carbon/ Celite mixture (1:3, w/w) followed by the sample mixed with sodium sulfate. In the first step the cell is extracted with pure n-heptane followed by a mixture of n-heptane/acetone (1:2.5 v/v). In this step all of the PCDD/Fs are extracted from the sample and retained in the carbon trap while interfering compounds such as PCBs are flushed through. In the next step, the cells are inverted and the PCDD/Fs are extracted with toluene. Finally, the extracts are passed through a miniaturized multi-layer silica column prior to analysis.

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Screening of soil samples (Paper I) The purpose of the first study (Paper I) was to examine whether the PLE-C method previously used for extracting biological samples was also suitable for extracting more complex soil samples from heavily polluted industrial sites. To determine if remediation of a dioxin-contaminated site is required, many analyzes may be needed to locate polluted hotspots, both vertically and horizontally. Complementary post-remediation analyses are also needed to assess the success of remediation procedures. In the studies underlying Paper I, PLE-C was performed on nine different soil samples and ELISA was evaluated as an alternative to costly chemical analyses by GC-HRMS for detecting the analytes. Portions of each of the nine samples were prepared using both PLE-C and Soxhlet followed by traditional clean-up (Sox-T), and then analyzed by both ELISA and GC-HRMS. The samples were taken from a small scale industrial combustion site in Uruguay that had been used for reclaiming metal from waste cable, a Swedish chloralkali site and from old wood treatment sites. Thus, they covered a range of soil types with widely differing congener profiles and concentrations. The results from the study are displayed in figure 3.

Figure 3. TEQ values obtained for nine samples of soil using four combinations of methods: ELISA or GC-HRMS after soxhlet extraction with classical clean-up (Sox-T) and pressurized liquid extraction (PLE-C). (Wood = Wood treatment site, Chlor = Chlor alkali site, Comb = Combustion site) The results indicate that PLE-C has similar extraction efficiency to Sox-T, since linear regression coefficients for the TEQ values obtained by the two extraction methods, prior to GC-HRMS and ELISA, were 0.78 and 0.99, respectively. Hence, the results from this study indicate that PLE-C can be used instead of Sox-T for preparing samples for dioxin analysis. However, the correlations between results obtained using the two detection methods was rather poor for some samples,

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Wood 1 Wood II Wood III Chlor I Chlor II Comb I Comb II Comb III Artificial

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PLE-C + ELISA

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especially those from wood impregnation and combustion sites, for which ELISA seems to underestimate dioxin contents. A possible reason for this is that the CRs for the highly chlorinated CDD/Fs are generally lower than the corresponding TEFs. Hence, there is a risk that the ELISA may give false negative results, especially when samples with high proportions of highly chlorinated dioxin congeners, e.g. from wood treatment sites, are investigated. The ideal use of PLE-C coupled to ELISA would be to extract many samples and split each extract into two portions, one to be concentrated in DMSO and analyzed by an ELISA while the other is stored. A few of the split samples could then be analyzed by GC-HRMS to obtain data that could be used to calculate a site-specific correction factor. It would then be possible to use ELISA to classify the samples into three categories: those with levels much higher than, close to, and substantially lower than a certain threshold value. Only samples with levels close to the threshold would have to be subjected to GC-HRMS. By applying this procedure it would be possible to screen many samples with relatively small resources and thus increase the numbers of samples that could be screened within a limited budget.

Quantification of abiotic samples (Papers II-III) The screening study (Paper I) indicated that PLE-C has potential utility for extracting samples of abiotic matrices such as soil, but the methods needed to be validated. This was done using three certified reference materials (CRMs): a soil, sediment and a fly ash. The results were compared in terms of both total TEQs and individual congeners. The values obtained using PLE-C, using Sox-T and the certified reference values (CRV) are compared in Table 1.

Table 1. TEQ values obtained following Soxhlet extraction with classical clean-up (Sox-T) and pressurized liquid extraction with an integrated and carbon trap (PLE-C), and corresponding certified reference values (CRVs) for soil, fly ash and sediment CRMs.

TEQ (µg/kg) CRV PLE-C nPLE-C Sox-T nSox-T

Soil 7.2±1.0 7.9±0.33 7 7.9±0.61 5Fly ash 3.6±0.40 3.2±0.26 4 3.6±0.22 5

Sediment 0.065±0.021 0.079 2 0.077±0.047 3

The TEQ values for the soil samples ranged from 7.6 to 8.3 ng g-1 with a mean of 7.9 ng g-1. The mean of the PLE-C TEQ data was slightly higher than but still in range of the CRV data and almost identical to the Sox-T data. For the individual dioxin congeners, the PLE-C values tended to be higher than both the CRVs and Sox-T values. However, according to statistical analysis using the procedures recommended by the Institute for Reference Materials and Measurements of the European Union (118), the PLE-C derived estimate for only one congener was significantly above the corresponding CRV (12378-PeCDD). The soil sample was extracted in seven replicates,

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and the relative standard deviations (RSDs) of the individual congener values obtained following PLE-C ranged between 7-26%; comparable to the corresponding Sox-T values, which ranged between 3-30%, and similar to both the RSDs of the CRVs (7-26%) and the indicative values (8-19%) (119). Clearly, the RSDs of results obtained using the PLE-C method were within acceptable limits, similar to those yielded by the Sox-T procedure and the CRV data. The PLE-C results for the sediment samples were also promising, although based on only two samples, due to contamination problems. Both TEQ values were within the range of the CRVs. The results for individual congeners were also in good agreement, the estimate for only one congener (1234678-HpCDD) significantly differing from the corresponding CRV. However, the results of applying PLE-C to the fly ash indicate that this material was less efficiently extracted than the soil and sediment samples. The TEQ value obtained was slightly lower than the given CRV, but the difference was not statistically significant. The results for the individual congeners showed similar trends; the PLE-C values were generally lower than the Sox-T values, and values for four congeners were significantly lower than the respective CRVs. Fly ash is a complex matrix, to which dioxins are bound very tightly, and it is well known that fly ash is more difficult to extract than the two other matrices studied (78,120). The overall conclusion that could be drawn from the validation study was that PLE-C is suitable for soil and sediment samples, while for fly ash it needs further improvement. In Paper III, the possibility of combining PLE-C with an alternative detection technique, GC×GC with electron capture detection (GC×GC-µECD), was assessed. PLE-C was used for extracting three CRMs: the fly ash and soil sample used in the previous validation study and a clay soil sample (BCR 530) with high, known amounts of non-2378-substitued PCDD/Fs. Duplicate portions of each sample were also extracted by Sox-T, then both PLE-C and Sox-T extracted samples were analyzed by GC x GC-µECD. Generally, the levels of analytes found in the fly ash following PLE-C were in the range of the CRVs, with only that of 12378-PeCDF deviating more than ±25% (+36%) from its CRV. For the sandy soil sample all values were within ±25% of the corresponding CRVs, except those for four congeners, which ranged from 133% to 270% of the CRVs. It should be noted that levels of 12378-PeCDD, which were overestimated by GC-HRMS (Papers II and V) were also overestimated by GC×GC-µECD. The clay soil material is a very difficult material to analyze, and in the validation study only seven congeners could be given certified values. Due to the presence of many interfering compounds, measured levels of only two of the congeners were of the same magnitude as the CRVs. The remaining compounds were either impossible to detect or clearly overestimated by the GC×GC-µECD. On a TEQ basis, the results were in agreement with or slightly higher than the corresponding CRV-TEQs for the sandy soil and fly ash. Due to the interferences and the analytical difficulties, no TEQ value was calculated for the clay soil.

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Overall, the results from the studies reported in Papers I-III indicate that PLE-C could be a useful tool for preparing samples for analyzing PCDD/Fs in abiotic samples. It has previously been shown that PLE with in-cell clean-up (using either carbon or acid-modified silica) is suitable for preparing biological samples and the results presented in Papers I-III suggest that it is also suitable for preparing samples of various abiotic matrices. The method also seems to have potential for fly ash samples, albeit with some minor modifications. Results presented in Papers I and III indicate that it is also possible to use an alternative detection technique, either GC×GC-µECD or ELISA bioassays, in combination with PLE-C as a complement to conventional GC-HRMS. However, the PLE in-cell clean-up technique also has some disadvantages. Notably, in the PLE-C method, the analytes pass through the sample matrix when they are back-flushed with toluene and there is thus a chance that some of the analytes may be adsorbed to the sample matrix. However, the major concern is that the toluene, which is a strong aromatic solvent, will co-extract components from the matrix that might interfere with the analysis of the dioxins in the detection step. Hence, modifications that allowed the sample to be removed after the first extraction step, and to add or remove different adsorbents or matrix retainers during different stages of the extraction would be highly desirable. A simple way of doing this was developed and assessed in the studies reported in Papers IV and V.

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Modular-PLE (Papers IV-V) For the studies described in Papers IV and V a cartridge was made in-house that allowed extraction cell segments containing sample, adsorbents and/or matrix retainers to be added or removed at appropriate stages of the extraction procedure. The cartridge, which consists of two cylindrical stainless steel guides and a polyetheretherkentone (PEEK) seal joined together with three screws, couples two cells together and when compressed in the ASE oven the two cells are pressed into the PEEK disc and sealed. When the original ASE PEEK disc was used in the coupling there were problems with leakage, but using thicker discs (3 mm) instead solved the problem and made the cell assembly leak-free. It is possible to freely combine 1, 5, 11 and 22 ml cells, as long as the total length does not exceed that of a 33-ml cell. A detailed drawing of the coupling cartridge is provided as supplementary material to Paper IV.

Figure 4; Photo of one assembled and one dissembled PLE assembly using the coupling cartridge

The coupling cartridge concept was first tested in the study reported in Paper IV. The sample was packed in one cell, which was connected to another cell containing carbon. After the first extraction step, the sample was removed and the carbon trap was back-flushed with toluene. In later investigations (Paper V) the modular concept was further developed for combined sample preparation for dioxin, dioxin-like PCB and PBDD analysis. Both sulfuric acid-modified silica and sample were packed into the top cell, which was connected to the carbon-containing cell and extracted. The carbon trap was removed and inverted, and then a new cell containing KOH was attached prior to a second extraction to further reduce the amount of co-extracted material. However, KOH was not used in the final validation since it gave no apparent benefits. A method was also developed, and described in Paper IV, in which polyurethane foam plug (PUFP) sampling of flue gas was combined with PLE-C sample preparation. In PUFP sampling flue gas is led through a PUFP to which the analytes (e.g. dioxins) adsorb. This method has been described in detail by Marklund et al. (121). The aim was to develop a suitable method for compiling inventories of sources of dioxins from combustion, which is the major contributor of

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dioxins in the environment. The relatively simple sampling equipment makes it possible to sample numerous sites and then send the PUFPs to a central laboratory equipped with ASE and GC-HRMS systems. This approach should enable large numbers of possible dioxin sources to be investigated with relatively meager resources, and provide a valuable complement to the UNEP dioxin toolkit. The concept was applied to several types of flue gas samples (Paper IV). In the PLE-C step the PUFP was removed after the first extraction step to avoid unwanted extraction of matrix components. This step also reduces the volume of toluene needed for efficient back-flushing. Since the volume of toluene needed in the previous PLE-C method exceeded the capacity of the collection vial, the number of samples that could be run in parallel was limited (Papers I-III). With this approach it was possible to collect eluates from three “back-flush” cycles in one vial, thereby increasing the number of samples that could be included in a batch, and thus the throughput.

Figure 5. TEQ values from full-scale combustion (PUF 1-6) and laboratory-scale combustion (PUF 7-11) (Paper IV) obtained using PLE with in-cell purification (grey) and traditional Soxhlet extraction with multi-step clean-up (white).

Each PUFP was split into two parts, one of which was extracted by Sox-T and one by PLE-C. The results indicated that the levels of analytes obtained using PLE-C were similar to those obtained using Sox-T (Figure 5). The homologue and congener profiles of the PLE-C extracts were also similar to those obtained using the Sox-T method. The TEQ values acquired for Sox-T extracted samples ranged from 0.35 to 0.95 TEQ m-3 and those for corresponding PLE-C samples from 0.50 to 1.1 m-3. It was also shown in Paper IV that the different types of samples could be separated using PCA. Thus, this is a valuable tool for identifying dioxin sources. In summary, the results from this study indicate that PLE-C could be used for extracting dioxins from PUFPs.

0.00

0.40

0.80

1.20

PUF 1 PUF 2 PUF 3 PUF 4 PUF 5 PUF 6 PUF 7 PUF 8 PUF 9 PUF 10 PUF 11

TEQ (n

g/m

3 )

Sox ‐Trad

PLE‐C

200° C

300° C

Full‐scale incineration (200 °C)

Laboratory‐scale incineration

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The objective of the study described in Paper V was to exploit the full potential of the modular-PLE system by performing one-step extraction and clean-up for dioxin, dioxin-like PCB and PBDD analysis. Samples of a crab powder and a mussel powder that were expected to contain analytes from all three groups were analyzed along with a salmon reference material used in-house with relatively high amounts of PCBs and dioxins (but no detectable PBDDs). Two abiotic samples were also included (the sandy soil and sediment CRMs used in the previous papers) that were expected to contain relatively high amounts of non-2378-PCDD/Fs, besides the 2378-PCDD/Fs. The modular approach was performed as described in figure 6 and the first fraction (PCB-fraction) was collected for analyzing seven regulatory PCBs (indicator-PCBs) and mono-ortho PCBs while the second (dioxin fraction) was collected for analyzing non-ortho PCBs, dioxins and PBDDs. The PCB and dioxin fractions of the salmon sample were pure enough to be directly introduced into the GC-HRMS system (after concentration). Minor fat residues in the mussel and crab dioxin fractions had to be removed prior to analysis by passing the extracts through a miniaturized multi-layer silica column.

Figure 6. Schematic representation of the extraction cell packing and the modular PLE procedure for mono-ortho PCBs, non-ortho PCBs, PCDD/Fs and PBDD extraction and in-cell clean-up (Paper V). In order to achieve this fractionation the adsorbent capacity of the carbon trap and the eluting strength of the first extraction solvent had to be optimized. In previous studies (Papers I-IV) acetone/n-heptane was used for the extractions, but acetone could not be used in this application since it reacts with sulphuric acid under the experimental conditions. Therefore, it was substituted with dichloromethane (DCM), which does not react with the acid and has been found in previous studies to produce similar target compound separations to acetone (117). Mussel samples spiked with 13C12-labeled dioxins and PCBs were extracted with 1:10, 1:5 and 1:1 DCM/n-heptane

Sample

H2SO4 Silica

Carbon in Celite (5%)

Na2SO4 + filter paper

Filter paper

Filter paper

Filter paper

KOH Silica

Fraction 1i-PCB, mono-ortho PCBs

Fraction 2non-ortho PCB, PBDD, PCDD/Fs

Step 1 Step 2 Step 3

Cell 1

Cell 2

Cell 1

Cell 3

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mixtures. Even with the highest percentage of DCM large amounts of mono-ortho-PCBs were retained on the carbon. Therefore, in subsequent trials the 1:1 mixture was used and the percentage of carbon in the carbon trap was reduced from 25% to 15%, 10%, 5%, and 2.5%. The resulting recoveries of both 13C12-PCBs and 13C12-PCDD/Fs in the two fractions are displayed in Figure 7.

Figure 7. Relative proportions of 13C12-labeled PCBs and PCDD/Fs in the two fractions obtained using PLE with 1:1 DCM/n-heptane and a carbon trap with 15, 10, 5 and 2.5% carbon contents in Celite (Paper V)

A clear trend could be seen in the results; leaner carbon mixtures resulted in higher percentages of mono-ortho PCBs in the first fraction. Even with a carbon content of just 5% mono-ortho PCBs were still found in the carbon trap. However, when the carbon percentage was reduced further to 2.5% both non-ortho PCBs and dioxins started to penetrate the trap. A combination of 5% carbon and 1:1 DCM/n-heptane was found to be optimal, since non-ortho PCBs and dioxins were effectively retained while all of the i-PCBs and most of the mono-ortho PCBs passed through with these parameters. The effects of optimizing these variables can be clearly seen in the total ion chromatograms (TICs) obtained from the mussel samples (Figure 8). The upper TIC originates from the first tests (using n-heptane and a carbon content of 20%), the middle TIC from experiments performed using the optimized conditions, and the lower TIC from a traditional analysis using column extraction and a multi-step clean-up. There are many more peaks in the upper chromatogram than in the chromatogram obtained following traditional extraction and clean-up, while the chromatograms obtained after the optimization are very similar to those from the traditional analysis. Hence, modular PLE seems to have similar capacity to the traditional clean-up procedure for removing potential interferences.

0%

20%

40%

60%

80%

100%

Dichloromethane/n‐heptane (1:1, v/v), Fraction 1 15% 10%

5% 2.5%

0%

20%

40%

60%

80%

100%

Toluene, Fraction 2

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Figure 8. Effect of optimizing the modular PLE method is expressed by showing partial total ion chromatograms obtained from mussel samples using: PLE with n-heptane as the first extraction solvent and a 20% carbon mixture (upper chromatogram); PLE with 1:1 dichloromethane/n-heptane and 5% carbon (middle chromatogram); the reference extraction and clean-up method (lower chromatogram).

The optimized conditions were used to validate the modular PLE method for extracting dioxins, dioxin-like PCB and PBDDs. Mussel, crab and salmon samples and the sediment and soil CRMs used in the study reported in Paper II were all extracted in triplicate. The RSDs of the amounts of PCBs and dioxin congeners detected in the mussel samples ranged from 0 to 20% and from 2 to 25%, respectively; comparable to the RSDs obtained using the traditional extraction/clean-up procedure (2-19% and 1-23%, respectively), indicating that the precision of modular-PLE was comparable to that of the traditional technique.

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The results, expressed on a TEQ-basis, are displayed in Table 2. The TEQs obtained using the modular-PLE approach were similar to, or exceeded, those obtained using the traditional methodology and reference values. The results obtained using modular-PLE for the salmon sample were within the 95% confidence intervals of the reference values. The results for the soil and sediment CRMs were slightly higher than the CRVs. Similar trends were seen when these materials were used to validate the PLE-C without the coupling cartridge (Paper II). The TEQs obtained using modular-PLE for the mussel, and especially the crab, samples were considerably higher than those obtained by column extraction followed by traditional clean-up.

Table 2. TEQ values for each sample extracted with optimized modular-PLE, and corresponding RVs

Sample PCB-TEQ Diox-TEQ Tot-TEQ

module-PLE RV module-PLE RV module-PLE RV

Mussel 8.64±0.13 6.27±0.28 1.10±0.09 0.79±0.08 9.79±0.22 7.06±0.37 Crab 0.90±0.05 0.35±0.04 0.91±0.15 0.48±0.07 1.82±0.20 0.83±0.10 Salmon 67.0±7.3 8.0±7.4 22.3±1.0 23.3±2.3 89.3±8.3 81.3±7.8 Soil (ng/g) n.a. n.a. 8.04±0.22 6.76±0.99 8.04±0.22 6.76±0.99 Sediment 12.5±0.22 9.7±3.9 68.6±23.5 58.9±19.6 92.7±6.1 80.2±5.9

The data for individual congener show the same overall trends. This analysis was done according to the methods recommended by IRMM (118), and for comparison purposes results from the traditional approach were considered equal to CRVs. The uncertainties of the values obtained using modular-PLE and the biological samples analyzed by the traditional method were assumed to be equal to the standard deviations. For certified values of the soil and sediment CRMs the uncertainties were determined using t-factors based on the degrees of freedom. For the mussel samples values of eleven PCBs, two PCDD/Fs and six homologue sums were significantly higher than the corresponding RVs. The trends for the crab samples were similar; values for nine PCBs, nine PCDD/Fs and five homologue sums being significantly higher than the corresponding RVs. For the salmon sample the results were generally in better agreement with the RVs. However, the results for four PCBs, all PCDFs and two PCDDs were still significantly higher than the corresponding RVs. For the sediment and soil samples the results obtained using modular-PLE were generally slightly, but significantly, higher than the corresponding CRVs, except that the value for 12378-PeCDD was much higher, possibly due to coeluting compounds. In summary, modular-PLE yields slightly higher values, probably due to higher extraction efficiency. The tendency for values obtained using PLE-C to be slightly higher than those obtained using the traditional extraction methods was seen in all studies (Papers I-V), and only minor differences were seen in the congener profiles of the extracts obtained. Hence, the differences were probably due to the extraction efficiency of the PLE procedures being higher.

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The modular PLE approach was also used to analyze PBDDs in mussel and crab samples. The problems affecting dioxin analysis caused by interfering compounds during the initial experiments were less severe for the PBDDs. Generally, the values obtained for the crab samples agreed well with the reference values during both the initial experiments and the final validation experiments. However, in the initial analysis of the mussel sample, interferences led to overestimation of the values for the dibrominated congeners. These results indicate that the PBDDs could be included in analyses of dioxins and dioxin-like PCBs, which could be an important feature of the method if the TEQ concept is expanded to include the brominated dioxins.

The cost-saving potential of modular-PLE Since modular-PLE seems to perform as efficiently as other techniques, a critical issue for its implementation in commercial laboratories is the potential cost savings it offers. It is impossible to estimate universally applicable costs, since prices of labor, laboratory equipment and solvents vary widely between countries. However, an attempt to calculate the cost saving potential of the method was presented in Paper IV (table 3), using calculations based on estimated average costs for glass-distilled solvents (50€/l), technicians’ time (75€/h) and Sox-T consumables (40€). The analysis showed that the cost per sample could be reduced almost six-fold if large numbers of samples are analyzed in batches, as they are for instance when national inventories of combustion sources are compiled. This estimate is for a procedure in which miniaturized multi-layer silica column purification is applied. If this final polishing step could be avoided, the results presented in Paper V indicate that costs and time could be substantially further reduced. Another measure that could potentially reduce costs even further is the use of cheaper detection techniques.

Table 3. Cost and time estimates for sample preparation using Sox-T and PLE-C (Paper IV).

Technique Cost type Solvent volume Manual labor / total time and costs 1 sample 6 Samples 24 Samples 96 Samples Sox-T 700 ml 8h / 25h 16h / 33h 64h / 100h 256h / 400h Labor €600 €200 €200 €200 Solvent €35 €35 €35 €35 Consumablesa €40 €40 €40 €40 Total €675 €275 €275 €275 PLE-C 100 ml 2h / 3h 9h / 15h 12h / 36h 40h / 136h Labor €150 €113 €38 €31 Solvent €5 €5 €5 €5 Instrumentb €10 €10 €10 €10 Total €165 €128 €53 €46 Sox-T/PLE-C 4.1 2.2 5.2 5.9 a Costs of carbon and silica-based adsorbents b Depreciation costs based on 100 sample per month

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Conclusions and further developments PLE with integrated clean-up has been previously proven to be an efficient method for extracting dioxins and dioxin-like PCBs from biological matrices prior to their analysis (112,117,122). In the work this thesis is based upon the method has been further developed and applied to samples of both biotic and abiotic matrices such as soil, sediment, flue gas, fly ash mussel, crab, and salmon. The levels of the analytes detected following use of the method are similar to, or slightly higher than, corresponding reference values and results from traditional analyses. There are two possible explanations for the slightly higher values obtained using PLE; either co-eluting compounds extracted using PLE give false positive signals or its extraction efficiency is higher. However, chromatograms generated following PLE with in-cell clean-up are almost identical to those generated following conventional methods. Furthermore, statistical analysis by PCA indicates that the congener profiles of extracts obtained using the two methods are similar. Hence, the most likely explanation for the elevated levels of dioxins and similar compounds detected in the PLE extracts is the latter hypothesis, i.e. that its extraction efficiency is higher. The ultimate goal for any strategy to prepare samples for analyzing dioxins and dioxin-like compounds is to develop a one-step, combined extraction and clean-up procedure. This goal seems achievable, as demonstrated in Paper V, where it was also shown that PBDD/Fs could be analyzed simultaneously with dioxins. The TEQ concept may be expanded to include these brominated compounds in the future (1), but even now there are concerns about them, since they are toxic and relatively abundant in marine biota such as crabs and mussels (44,45). Although the modular-PLE procedure yielded very promising results, most of the samples needed to be cleaned using miniaturized silica columns to remove remaining interfering material. It should be possible to eliminate this post clean-up step if further retainers and adsorbents (e.g. Florisil and basic alumina) are evaluated to identify more effective options and other extraction parameters (e.g. static time, extraction solvent and temperature) are optimized. However, even after further refinements to the method, specific samples and matrices may require special treatments. The modular-PLE procedure is relatively simple to adopt in commercial analytical laboratories since original ASE cells and end caps are used. For samples with low dioxin levels large samples have to be processed in order to detect them, and the cell size may be a limiting factor. Fortunately, PLE-systems with larger cells are now available (e.g. the ASE 300 and ASE 350 systems) and it should be relatively easy to manufacture coupling devices that fit the larger cells. The ASE350 system also provides improved washing options to prevent cross-contamination, which sometimes occurs with the current systems if samples with orders of magnitude differences in analyte concentrations are processed in the same batch. PLE with in-cell clean-up could be very useful in several applications. As shown in Paper I, for instance, PLE-C combined with ELISA has good potential for screening soil samples with

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different congener profiles and characteristics. One option would be to use PLE-C for sample preparation and ELISA or CA(F)LUX assays for prioritizing samples for further GC-HRMS analysis. Using this approach it would be possible to perform many analyses at relatively low cost. A great advantage of bioanalytical methods is that dioxin-like compounds present in the sample can also be detected. This may be very useful in applications such as monitoring remediation processes or investigating combustion sources, especially since potentially highly toxic dioxin-like compounds may not be detected by GC-HRMS analysis. Furthermore, PLE combined with PUFP sampling could be a valuable tool to supplement the UNEP dioxin toolkit. It was estimated that this method would reduce costs of sample preparation by almost 90%. If the full potential of the modular-PLE system is exploited the costs could probably be even further reduced. Finally, PLE could be combined with GC×GC-µECD and replace GC-HRMS in applications requiring full congener-specific data. However, more work is needed to improve the data evaluation process for this approach, which is currently quite time consuming. The modular PLE proved to be capable of extracting and cleaning up samples for analysis of dioxin-like PCBs and PBDDs, indicating that all of these dioxin-like compounds could be analyzed using this novel method, at least after minor modifications. There have been several reports on the use of PLE with in-cell clean-up for sample preparation prior to analysis of other types of pollutants, e.g. sulfonamines, musk compounds, PAHs and oxy-PAHs. Using the modular PLE system it should be possible to further improve these and similar applications. PLE can be performed with diverse adsorbents, matrix retainers or solvents during different stages of the extraction. Liljelind et al. 2003 (123) presented a method for combined analysis of PAHs, PCBs, PCDD/Fs within the same sample. It may be possible to transfer this approach to the modular PLE platform, and possibly extend it further to include all POPs included in the Stockholm convention list. This would be a reasonable first step in efforts to develop PLE systems for a broader range of environmental target compounds.

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Acknowledgements För ett par år sedan sa jag att det finns två saker jag aldrig kommer att göra. Det första var att jag inte skulle börja doktorera och det andra var att jag inte skulle hem till Piteå igen. Nu när jag har doktorerat klart och flyttat tillbaka till Piteå har jag därför nu lovat mig själv att aldrig vara ensam med sju rätt på V75. Det senaste året eller framför allt det senaste halvåret har varit mycket hektiskt och det finns ett stort antal personer både inom och utanför universitetsvärlden som förtjänar ett stort tack eftersom dessa på ett eller annat sätt gjort det möjligt för mig att komma i mål. Till att börja med vill jag tacka mina handledare. Jag börjar med min huvudhandledare Peter Haglund har varit suverän handledare under dessa år. Patrik Andersson, biträdande handledare som till en början mest var en ”administrativ” handledare men i slutet även haft en bidragande del i forskningen. Ni har båda också varit ett stort stöd och ställt upp på ett föredömligt sätt för att jag skulle kunna slutföra avhandlingen trots mitt ganska hastiga ”frånfälle” ifrån universitetsvärlden. Alla nya och gamla miljökemister förtjänar att tackas men några personer förtjänar kanske lite extra. Malin N ska ha ett extra tack eftersom du introducerade mig till miljökemins magiska värld genom att vara en ypperlig handledare till mitt examensarbete. Vill även tack Lisa L som även hon var med på ett hörn på mitt exjobb samt hjälp med provtagning av rökgas på Dåva. Jerker F tack för gott samarbete. Det är fascinerande att en person kan vara så stressad men ändå någonstans verka njuta av det. Per L för fantastisk hjälp varje gång det varit trubbel med masspektrometrarna. Hela verksamheten skulle nog fallera om du försvann. Detta gäller även Lena B som alltid varit en klippa då det gäller de administrativa frågorna. Johanna, lycka till i staterna. Conny D, tack för gott samarbete och lycka till med pokern. Ylva, tack för all hjälp på labb. Sarah, jag överlämnar härmed häftapparaten åt dig med varm hand. Under min studietid i Umeå finns det ett flertal personer utanför kemiområdet som på olika sätt muntrat upp min tillvaro (Johan, Peter, Jonathan, Krille, m fl). Ni förtjänar alla ett STORT tack trots att ni alla övergivit Umeå före jag gjorde det. Det finns dock några tappra själar som hållit sig kvar i stan och dessa förtjänar därför ett litet större tack. Max tack för alla barrundor, rosa hattar, ångestfyllda stunder framför diverse sportevenemang samt spelkvällar där du varit en sann värdemätare i den ständiga kampen om att undvika att komma sist. Lars för alla tennismatcher, fester, välklädda svenska män i Aten, förvaring av vinterdäck samt skapandet av det oförglömliga uttrycket ”en kväll är aldrig förstörd om man fått hångla”.

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Daniel för många luncher på stan där du alltid varit den som kört (nästan) och uppstyrning av innebandytider. Stefan B för alla vulgära skämt samt att du alla slag du stå ut med på innebandyplanen. Henning som stolt stugägare (nåja, den är nästan din) ser jag dig fortfarande Umebo. Tack många intressanta diskussioner över en öl och roliga fester på stugan i Obbola. Det finns många användningsområden för ett mjärde. Det finns också ett flertal andra personer som förtjänar ett tack men som jag enligt devisen ”ingen nämnd, ingen glömd” låter bli att nämna vid namn. Tack i alla fall! Familjen hemma i Piteå, Mamma Ulla, Calle, Pappa Stefan, Kristin, Syster Hanna och Stefan med familj, bror Johan (tack för hjälpen med fotografierna) och Hanna samt Farfar Sigvard. Ni har alla alltid varit ett stort stöd för mig på ett eller annat sätt trots att jag misstänker att ni nog aldrig egentligen förstått vad det är jag sysslat med nere i Umeå. Jag skänker också en extra tanke till farmor Berta samt mormor Connie som båda gick bort under min doktorandtid. En tanke går också till morfar Kurt. Hoppas ni har det bra där ni är. ”Svärföräldrarna” Evy och Sven-Erik ska ha ett stort tack för att man alltid känner sig så välkommen då man hälsat på oavsett om det varit på Silverringen, i Stora sjöfallet eller på Las Dunas. Till sist, Ann-Helen. Du ska ha ett stort tack för att du alltid varit ett sådant stort stöd och för att du visat sådant stort tålamod och förståelse trots alla sena kvällar och helger jag tillbringat på labbet eller framför datorn. Du betyder så otroligt mycket för mig. Jag älskar dig!

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