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Rapid Sample Preparation and Bioanalytical Techniques for Efficient Screening of Organic

Pollutants in the Environment

MA L I N L . N O R D I N G

Environmental Chemistry, Department of Chemistry

UMEÅ UNIVERSITY

2006

Dissertation presented at Umeå University to be publicly examined in KB3B1, KBC, Umeå, Friday, September 22nd, 2006 at 10 am, for the degree of Doctor of Philosophy. Rapid Sample Preparation and Bioanalytical Techniques for Efficient Screening of Organic Pollutants in the Environment Malin L. Nording Environmental Chemistry, Department of Chemistry, Umeå University, 901 87 Umeå, Sweden ISBN 91-7264-109-6

Abstract Large numbers of samples often need to be prepared and analysed in surveys of organic pollutants in the environment, but while the methods commonly used in such surveys can provide abundant detail they are generally costly, time-consuming and require large amounts of resources, so there is a need for simpler techniques. The work underlying this thesis assessed the potential utility of more convenient sample preparation and bioanalytical techniques for rapidly screening various environmental matrices that could be useful complements to higher resolution methods.

Initially, the utility of a simplified extraction technique followed by an enzyme-linked immunosorbent assay (ELISA) for detecting polycyclic aromatic hydrocarbons (PAHs) in authentic (i.e. unspiked) contaminated soils was explored. The results showed that there are relationships between the structure and cross-reactivity among compounds that often co-occur with target PAHs. However, their potential contribution to deviations between estimates of total PAH contents of soils obtained using ELISA and gas chromatography-mass spectrometry (GC-MS) based reference methods were limited. Instead, the cross-reactivity of target PAHs and the failure to extract all of the PAHs prior to the ELISA determinations were the main reasons for these deviations.

Polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) were detected in food and feed matrices, as well as in authentic contaminated soils using different bioanalytical techniques – ELISA and two cell-based bioassays: CAFLUX and CALUX (chemically activated fluorescent/luciferase gene expression) assays. In addition, enhanced sample preparation techniques based on accelerated solvent extraction (ASE) were developed. ASE with integrated carbon fractionation (ASE-C) in combination with CAFLUX produced estimates of PCDD and PCDF contents in fish oil and fish meal that agreed well with results obtained using reference methods. Furthermore, results from ELISA and GC-high resolution MS analyses of extracts of PCDD- and PCDF-contaminated soil samples obtained using an adjusted ASE-C technique were strongly correlated.

Finally, the thesis reports the first experiments in which the results of CAFLUX, CALUX, and ELISA determinations of PCDDs and PCDFs in extracts of authentic contaminated soil samples were evaluated and compared to those obtained using a reference method. All of the bioanalytical techniques were found to be sufficiently sensitive, selective, and accurate for use in screening in compliance with soil quality assessment criteria. Overall, the improved sample preparation and bioanalytical techniques examined proved to be useful potential complements to conventional methods, enhancing the analytical framework for PAHs, PCDDs, and PCDFs. However, further validation has to be undertaken before they are applied on a large-scale. Keywords: accelerated solvent extraction, CAFLUX, CALUX, cell-based bioassay, chemically activated fluorescent gene expression, chemically activated luciferase gene expression, dioxin, ELISA, enzyme-linked immunosorbent assay, feed, food, immunoassay, PAH, PCDD, PCDF, pressurized liquid extraction, soil © Malin L. Nording 2006 Cover by Richard Lindberg ISBN 91-7264-109-6

To Alvar

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CONTENTS

LIST OF PAPERS ........................................................................................iii

ABBREVIATIONS ........................................................................................iv

1. INTRODUCTION ................................................................................1 Objectives ...................................................................................................3

2. POLLUTANTS CONSIDERED .............................................................5 Polycyclic aromatic hydrocarbons.................................................................6

Dioxins ........................................................................................................9

3. THE CONTAMINATED ENVIRONMENT ........................................13

Dioxins and polycyclic aromatic hydrocarbons in soil ..................................14

Dioxins in food and feed.............................................................................15

4. STEPS REQUIRED TO DETECT POLLUTANTS ...............................17 Sampling ...................................................................................................18

Extraction and clean-up .............................................................................18

Instrumental separation and detection ........................................................20

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5. BIOANALYTICAL TECHNIQUES ......................................................23 Immunoassays...........................................................................................23

Cell-based bioassays ..................................................................................27

6. PRACTICAL USE OF THE SCREENING CONCEPT .........................31 Summary of Papers I-V...............................................................................33

An immunoassay for analysing PAHs in contaminated soil ...................34

Integrated extraction/purification of contaminated food and

feedstuffs for detecting dioxins by CAFLUX ..........................................34

Integrated extraction/purification of contaminated soil samples

for detecting dioxins by ELISA .............................................................35

Comparison of ELISA, CALUX, and CAFLUX for analysing

dioxins in contaminated soil samples ...................................................36

7. CONCLUDING REMARKS.................................................................37 A bright perspective for the future ..............................................................38

ACKNOWLEDGEMENTS ............................................................................39

SUMMARY IN SWEDISH ...........................................................................40

REFERENCES ............................................................................................41

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List of papers This thesis is based on the following Papers, which are referred to in the text by their respective Roman numerals.

I. Malin Nording, Peter Haglund. (2003). Evaluation of the structure/cross-reactivity relationship of polycyclic aromatic compounds using an enzyme-linked immunosorbent assay kit.

Analytica Chimica Acta 487:43-50. II. Malin Nording, Kristina Frech, Ylva Persson, Mats Forsman, Peter

Haglund. (2006). On the semi-quantification of polycyclic aromatic hydrocarbons in contaminated soil by an enzyme-linked immunosorbent assay kit.

Analytica Chimica Acta 555:107-113.

III. Malin Nording, Sune Sporring, Karin Wiberg, Erland Björklund, Peter Haglund. (2005). Monitoring dioxins in food and feedstuffs using accelerated solvent extraction with a novel integrated carbon fractionation cell in combination with a CAFLUX bioassay. Anal. Bioanal. Chem. 381:1472-1475. IV. Malin Nording, Mikaela Nichkova, Erik Spinnel, Ylva Persson,

Shirley J. Gee, Bruce D. Hammock, Peter Haglund. (2006). Rapid screening of dioxin-contaminated soil by accelerated solvent extraction/purification followed by immunochemical detection.

Anal. Bioanal. Chem. 385:357-366. V. Malin Nording, Michael S. Denison, David Baston, Ylva Persson,

Erik Spinnel, Peter Haglund. Analysis of dioxins in contaminated soils using the CALUX and CAFLUX bioassays, an immunoassay and gas chromatography/high-resolution mass spectrometry.

Submitted manuscript

Reprinted Papers are presented with the kind permission of the publishers.

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Abbreviations AHH Aryl hydrocarbon hydroxylase AhR Aryl hydrocarbon receptor Arnt AhR nuclear translocator ASE Accelerated solvent extraction ASE-C ASE with an integrated carbon trap CAFLUX Chemically activated fluorescent gene expression CALUX Chemically activated luciferase gene expression CR Cross reactivity DDE 1,1-dichloro-2,2-bis(4-dichlorodiphenyl)ethylene DDT 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane DR-CALUX Dioxin responsive chemically activated luciferase gene expression DRE Dioxin-responsive element ECD Electron capture detector EGFP Enhanced green fluorescent protein ELISA Enzyme-linked immunosorbent assay EROD Ethoxyresorufin-O-deethylase EU European Union GC Gas chromatography HR High resolution IARC International Agency for Research on Cancer Kow Octanol-water partitioning coefficient LC Liquid chromatography MFO Mixed function oxidase MS Mass spectrometry PAC Polycyclic aromatic compound PAH Polycyclic aromatic hydrocarbon PCB Polychlorinated biphenyl PCDD Polychlorinated dibenzo-p-dioxin PCDF Polychlorinated dibenzofuran POP Persistent organic pollutant QA Quality assurance QC Quality control SD Standard deviation SEPA Swedish Environmental Protection Agency TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin TEF Toxic equivalency factor TEQ Toxic equivalency TOF Time of flight USEPA United States Environmental Protection Agency WHO World Health Organization

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1. INTRODUCTION Anthropogenic activities have affected natural environments and wildlife throughout human history. Before the Industrial Revolution, the effects of these activities were largely local, but since then they have rapidly expanded, causing serious environmental problems of global scale. Donald Hughes outlines a series of ominous consequences of these developments: "…polluted water and air, acid precipitation, diminution of the ozone layer, global warming, …" in An Environmental History of the World - Humankind's Changing Role in the Community of Life. Previous use of natural resources to sustain communities has been expanded to ruthless exploitation, and the effects are potentially devastating. Cutting down a tree diminishes the forest. However, using pesticides for timber protection can have far-reaching consequences, affecting not just the forest, but the whole world, as illustrated by the effects of widespread spraying of the organochlorine insecticide DDT (1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane), particularly in the 1960s. Many kinds of organisms were affected, as well as the target insects, e.g. carnivorous birds such as the Peregrine falcon, in which DDT accumulated and reduced its reproductive success (Connell, 1997). In 1958, Rachel Carson received a letter expressing a bitter view of an existence deprived of wildlife, prompting her to write a book entitled Silent Spring, referring to a future devoid of bird song due to the effects of anthropogenic substances in the environment. Silent Spring had a huge impact on people's attitudes, and played a major role in the birth of modern environmentalism. Today, at the turn of the millennium, growing environmental awareness has resulted in measures like total bans on certain pesticides, exhaust emission controls, and guidance limits for levels of harmful substances in the environment. The United Nations Environmental Programme has developed three Conventions (the Basel, Rotterdam, and Stockholm Conventions) to provide an international framework for the environmentally sound management of hazardous compounds. For instance, 12 persistent organic pollutants (POPs) are regulated by the Stockholm Convention, adopted in 2001.

Moreover, the European Union (EU) has adopted a proposal for new legislation called REACH (Registration, Evaluation, and Authorisation of CHemicals), a regulatory framework for chemicals intended "…to improve the

1 . I N T R O D U C T I O N

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protection of human health and the environment…". Although measures are being imposed to control and eliminate the production of harmful substances, POPs of historical origin will remain in the environment for many years. For instance, the use of various substances several decades ago at wood treatment sites have caused locally elevated levels of dioxins, amongst other chemicals, which may eventually spread to other environmental compartments. Therefore, a prerequisite for environmental protection is knowledge about the harmful substances, their occurrence, fate, and toxicological effects, as well as important biological processes in affected ecosystems. These and related issues occupy researchers in a broad field of subjects. The work underlying this thesis provides information on recently developed screening techniques for classifying potentially contaminated samples that are more time- and cost-efficient than comprehensive quantitative analyses. Such analytical procedures are desirable in many environmentally relevant circumstances, a few of which are: • mapping the distribution of pollutants at industrially contaminated

sites; • supervision of the progress of soil remediation programs; • monitoring programs, in which rapid identification of a pollution

source is crucial, for instance to prevent distribution of contaminated food; and,

• control of detoxification processes. In efforts to carry out these tasks, versatile analytical tools are of great value. Furthermore, a sound approach for collecting basic data for decision-making is always necessary. For instance, if too few samples are analysed erroneous conclusions regarding the scale of contamination and measures that need to be taken to address it may be drawn. These problems can be overcome by increasing the capacity of the analyses. To expand a given analytical framework, the techniques applied have to be inexpensive, rapid, and preferably field-adapted; features typical of the screening techniques considered in this thesis. A basic overview of two analytical approaches (field-adapted screening and quantitative instrumental analysis) is provided in Figure 1. Since this figure is only intended for orientation, analyte-specific treatments at each stage have not been presented. Rather, the potential simplicity of each stage of the screening approach is illustrated. An explanation of the pollutants studied is provided in Chapter 2, and a description of the contaminated environments can be found in Chapter 3. In Chapter 4, analytical procedures to detect the pollutants are considered. An in-depth background and discussion of immunoassays and bioassays for use in the detection step are provided in Chapter 5. Practical uses of the screening concept, with illustrative examples from studies the thesis is based upon, are presented in Chapter 6, and a summary of conclusions can be found in Chapter 7.


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Extraction Clean-up DetectionPre-treatmentSampling

A vastamount of samples

None Agitation with solvent

FiltrationBioanalyticaltechniques

ImmunoassaysBioassays

A selectionof samples

Drying and sieving

Soxhlet Multi-stepcolumns

Instrumental techniques

GC-MS

Semi-quantitative

data for many samples

Quantitative data for a

few samples


A vastamount of samples

None Agitation with solvent

FiltrationBioanalyticaltechniques

ImmunoassaysBioassays

A selectionof samples

Drying and sieving


Instrumental techniques

GC-MS

Semi-quantitative

data for many samples

Quantitative data for a

few samples

Figure 1. Overview of two analytical procedures. Above, the field-adapted screening approach; below, conventional instrumental analysis.

Objectives

Briefly the objectives of the work outlined in this thesis were to investigate and develop rapid sample preparation and bioanalytical techniques for use in efficient screening strategies to identify organic pollutants in environmental samples, including soil, food and feed, thereby facilitating classification of potentially contaminated samples. To that end, specific issues addressed were: • the specificity of an antibody for detecting polycyclic aromatic

hydrocarbons (Paper I); • the extent and causes of uncertainties in immunoassay determinations of

polycyclic aromatic hydrocarbons (Paper II); • simultaneous extraction and fractionation of dioxin-contaminated food

and feed in combination with a cell-based bioassay (Paper III); • simultaneous extraction and fractionation of dioxin-contaminated soil

in combination with immunodetection (Paper IV); and • comparison of the performance of an immunoassay, and two cell-based

bioassays for analysing dioxin-contaminated soil samples (Paper V).


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2. POLLUTANTS CONSIDERED An environmental pollutant can be defined as a toxic anthropogenic compound found in natural ecosystems, focusing here on POPs. Such chemicals remain in the environment for extended periods since they are resistant to degradation processes. Compounds with a high tendency to bioaccumulate, which is strongly correlated to their lipid solubility, are especially alarming since they may accumulate in fatty tissues of living organisms. A common measure of lipophilicity is the octanol-water partition coefficient (Kow), which for instance increases with increasing numbers of chlorine atoms in polychlorinated biphenyl (PCB) molecules (Hawker and Connell, 1988). Hence, the most lipid-soluble PCBs (and thus those with the highest propensity to bioaccumulate) are highly chlorinated. However, Kow is not the only factor that influences bioaccumulation. Resistance to metabolic activities also favours bioaccumulation. In contrast, limited bioavailability may inhibit bioaccumulation. A bioavailable compound that bioaccumulates may migrate through the food web and increase in concentration at higher trophic levels, a process called biomagnification, as in the example of DDT in the Peregrine falcon. In Table 1, log Kow values of selected pollutants are shown that are of major environmental concern, partly because they are lipophilic (log Kow > 2). However, although they are all lipophilic, they differ greatly in aqueous solubility and vapour pressure. Hence, they are expected to behave differently when released into the environment. For instance, triphenyl phosphate, which has the highest aqueous solubility (1.9 mg/L), may be less persistent than some of the others since it is susceptible to degradation processes when free in solution. Furthermore, phenanthrene, which has the highest vapour pressure (0.02 Pa), is semi-volatile and vaporizes relatively rapidly at temperatures in the higher end of typical environmental ranges, e.g. during the summer at high latitudes. Pesticides like DDT exemplify early pollutants that were deliberately dispersed in the environment before their harmful effects were known. In this case, not only the parent compound, but also its metabolite DDE (1,1-dichloro-2,2-bis(4-dichlorodiphenyl)ethylene) is toxic. DDT and DDE are both persistent and currently DDE levels in Sweden generally exceed those of

2 . P O L L U T A N T S C O N S I D E R E D

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DDT, since the use of DDT was banned decades ago (Bernes, 1998). Products of today's intensively chemical-dependent society include organophosphorus flame retardants (e.g. triphenyl phosphate), which have only recently been identified in environmental matrices (Marklund, 2005), and there is still limited knowledge concerning their effects on wildlife and human health. Besides pesticides and emerging pollutants, combustion-related compounds like polycyclic aromatic hydrocarbons (PAHs), technical mixtures including, inter alia, PCBs used in contained systems, and unintentionally produced chemicals like dioxins, may increase the risk for adverse effects on wildlife and human health. Furthermore, there is reason to believe that as yet unidentified pollutants also occur in the environment. The investigations underlying this thesis focused mainly on two classes of organic compounds: PAHs, and dioxins (polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans or, simply, PCDDs and PCDFs). Table 1. Illustrative physical and chemical properties of selected organic pollutants. Dioxins are represented by 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD), pesticides by 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT), polychlorinated biphenyls by 2,2´,4,5,5´-pentachlorobiphenyl (PCB 101), PAHs by phenanthrene, and phosphorus flame retardants by triphenyl phosphate (Mackay et al., 1997; Marklund, 2005).

Property 2,3,7,8-TCDD DDT PCB 101 Phenathrene Triphenyl phosphate

Molecular weight (g/mol) 322 354 326 178 326

log Kow 6.80 6.36 6.38 4.57 4.59

Aqueous solubility (mg/L) 8×10-6 0.003 0.01 1.2 1.9

Vapor pressure (Pa) 2×10-7 2×10-5 0.001 0.02 8.4×10-4

Polycyclic aromatic hydrocarbons

Polycyclic aromatic compounds (PACs) encompass a wide range of chemicals, for instance PAHs. The PAHs consist of carbon and hydrogen atoms, with varying numbers of fused aromatic rings (Figure 2). Related heterocyclic aromatic rings often co-exist with PAHs, in which carbon atoms are substituted by nitrogen, sulfur or oxygen atoms. Alkylated PAHs are also common co-pollutants. Despite the vast number of PACs, the levels of only 16 priority-pollutant PAHs, listed by the United States Environmental Protection Agency (USEPA), are routinely monitored at PAC-polluted sites. This is because they represent a significant fraction of occurring PACs, and provide good indications of the overall PAC contamination load. However, other PACs, like dibenzofuran and 9-fluorenone, can occasionally be found at comparable levels (Lundstedt et al., 2003).


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The PACs are ubiquitous since they derive from the widespread, incomplete combustion of organic material in, inter alia, motor engines and wood fires, all of which contribute to the elevated PAC levels in the environment. Levels harmful to human health are mainly found in working, urban, and industrial environments (close to the sources), but low concentrations can be found virtually everywhere due to e.g. extensive atmospheric transport (Bernes, 1998).

N

Phenanthrene Benzo[a]anthracene Carbazole 1-Methylanthracene

Figure 2. A selection of unsubstituted PAHs (phenanthrene and benzo[a]anthracene), heterocyclic PAHs (carbazole), and alkylated PAHs (1-methylanthracene). Several PAHs are known to be toxic and carcinogenic (as reviewed, for example, by Pickering 1999, and Delistraty, 1997). The International Agency for Research on Cancer (IARC) has classified PAH compounds and mixtures into three categories, depending on the evidence for their carcinogenicity (IARC, 1983). For instance, coal tars, mineral oils, and tobacco smoke belong to group 1, embracing human carcinogenic compounds and mixtures based on sufficient human evidence. Group 2a includes benzo[a]anthracene, benzo[a]pyrene, creosotes, and dibenz[a,h]anthracene, all which are possibly carcinogenic to humans based on sufficient animal evidence and limited human evidence. The last group, 2B, mainly includes nitro-PAH derivatives. These are possibly carcinogenic to humans, but there is insufficient human and/or animal evidence to classify them definitively as such. For the PAHs to exert their carcinogenicity, they have to be metabolized and thereby activated (Pickering, 1999). In this respect, the cytochrome P450 enzyme family plays an essential role in catalyzing mixed function oxidase (MFO) reactions, for instance PAH conversion. Other MFO reactions catalyzed by cytochrome P450 enzymes include those involving aryl hydrocarbon hydroxylases (AHHs) and ethoxyresorufin-O-deethylase (ERODs), which are discussed in Chapter 5. In general, lipophilic compounds may be converted to water-soluble species and consequently more easily excreted by the organism. In this process, the compounds are detoxified or, worse, activated to toxic metabolites by cytochrome P450 and related enzymes through MFO reactions (Guengerich 1993). For instance, mutagenic PAH-intermediates might form, possibly inducing cancer (IARC, 1983). As an


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example, the metabolic activation of benzo[a]pyrene is shown in Figure 3. The original molecule is initially converted to short-lived epoxides, which may rearrange to phenols, with the formation of water-soluble end products that may eventually be excreted by the organism. Another route is involved if the epoxides are hydrolyzed to dihydrodiols, which may undergo cytochrome P450-catalyzed transformation into dihydrodiol epoxides, possibly causing mutations and consequently maybe also cancer (Pickering, 1999; IARC, 1983).

O OH

OH

OH

OH

OH

O

Benzo[a]pyrene-7,8-oxideBenzo[a]pyrene

GlucuronideSulphate esterGluthathione conjugate

7-hydroxybenzo[a]pyrene

trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene

BINDING TO DNA

1.

2.

dihydrobenzo[a]pyrene-9,10-oxidetrans-7,8-dihydroxy-7,8-

cyt P450

cyt P450

Figure 3. Bioactivation of benzo[a]pyrene, with route 1 leading to water-soluble and excretable products, while route 2 leads to mutagenic transformation products (IARC, 1983).


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Dioxins

Dioxin is a generic term for 75 structurally very similar PCDD molecules, congeners, and (often) 135 PCDFs. They are lipophilic compounds that characteristically have low vapour pressures (Table 1). Dioxins all have a planar configuration due to their aromatic ring structure (Figure 4). Hydrogen atoms are substituted by various numbers of chlorine atoms, at varying positions in the different congeners. The numbers in the names of the congeners designate the positions of the substituents, while Greek letters denote the degree of chlorination. For instance, highly toxic congeners tend to have chlorines at all of the lateral positions, i.e. 2, 3, 7, and 8, and the most toxic dioxin congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, currently classified as carcinogenic to humans) has chlorines at only those positions (Cole et al., 2003). The lethal dose for guinea pigs can be as low as 3 µg/kg, but there are major between- and within-species variations (Harris et al., 1973). Other adverse effects of 2,3,7,8-substituted dioxins include immunotoxicity, carcinogenicity, and harmful effects on reproduction (Safe, 1986; Bernes, 1998). Dioxin exposure to humans may, for instance, cause chloracne (Rodriguez-Pichardo et al., 1991).

O

O

O

Clx ClyClx Cly

12

346

7

89 1

2

3

46

7

89

Figure 4. The general structure of dioxins (left) and furans (right). The toxic action of dioxins is mediated by the aryl hydrocarbon receptor (AhR), which was first identified in mouse hepatic cytosol by Poland and co-workers in 1976. Subsequent research on the processes involved has revealed the molecular mechanism of action of the nuclear AhR complex (e.g. Whitlock, 1993). More recent reviews by Denison and co-workers deal with the activation of the AhR by a structurally diverse range of chemicals, including PAHs (Denison and Heath-Pagliuso, 1998; Denison et al., 2002; Denison and Nagy, 2003). Ligand binding to the AhR initiates the biochemical events that eventually lead to adverse effects (Figure 5). After a conformation change, the complex is translocated into the nucleus where a dimerization with the nuclear protein Arnt (AhR nuclear translocator) takes place. Consequently, the complex is


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converted to its high affinity DNA-binding form. Subsequent binding to dioxin-responsive elements (DREs) upstream of CYP1A1 (encoding the protein cytochrome P4501A1) and other AhR-responsive genes, initiate transcription and ultimately production of certain proteins. Elevated levels of these proteins can be used as indicators of dioxins and other compounds in bioassays (for details, see Chapter 5). The specific proteins originating from AhR-dependent gene expression responsible for the adverse effects elicited by dioxins and similar compounds have not yet been identified. Hence, the causes of a broad spectrum of species- and tissue-specific toxic effects remain to be elucidated. However, the adverse effects are often delayed, indicating that continuous inappropriate gene expression is a prerequisite for toxic responses. Furthermore, the responsiveness to TCDD is in principal lost in AhR knockout mice (Fernandez-Salguero et al., 1996; Thurmond et al., 1999), which support the hypothesis that AhR mediates the biological events causing dioxin toxicity. Other evidence for the AhR role in dioxin toxicity includes a correlation between similar structural properties to TCDD and high affinity binding to the AhR (Birnbaum, 1994; Safe, 1986).

DRE

AhR ligands

AhRTranslocation

CytochromeP450 1A1

EROD

Luciferase EGFP Otherproteins

CALUX CAFLUX

…

DRE

AhR ligands

AhRTranslocation

CytochromeP450 1A1

EROD

Luciferase EGFP Otherproteins

CALUX CAFLUX

…

Figure 5. Simple schematic diagram of the events in the cell initiated by aryl hydrocarbon receptor (AhR) ligands (such as dioxin molecules). The ligand-Arnt-AhR-complex binds to dioxin-responsive elements (DREs), initiating transcription and translation. Certain proteins can be used to detect organic pollutants in different bioassays (e.g. EROD, CALUX, and CAFLUX). The genes encoding luciferase and enhanced green fluorescent protein (EGFP) are incorporated in cells through genetic modification.


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Dioxins have never been produced intentionally, but are instead formed as by-products during combustion processes, chlorine bleaching of pulp, and the synthesis and use of organochlorine chemicals, such as pentachlorophenol (Bumb et al., 1980; Hagenmaier and Brunner, 1987; Lexén et al., 1993; Olie et al., 1977; Rappe et al., 1987; Rappe et al., 1991). Consequently, dioxin mixtures found in environmental matrices are very complex, complicating risk evaluations. However, to facilitate risk assessment and regulatory control of dioxin exposure, the concept of toxic equivalency factors (TEFs) has been developed. An expert meeting was organized in 1997 by the World Health Organization (WHO) to derive consensus TEFs for PCDDs, PCDFs, and PCBs, the results of which were reported by Van den Berg et al. (1998). These TEFs has recently been re-examined and an up-dated TEF scheme was suggested (Van den Berg et al., 2006). Both TEF schemes can be found in Table 2, together with cross-reactivity (CR) values, reflecting the extent to which compounds are recognised by the antibody in the immunoassay used in the studies presented in Papers IV and V. Due to the common mechanism of action described above, the toxicity of different congeners and similar compounds can be related to that of the most potent congener, TCDD (TEF = 1). Other criteria for including a compound in the TEF concept are its: (i) structural relationships to PCDDs and PCDFs, (ii) persistence, and (iii) accumulation in the food chain. TEFs are used to obtain toxic equivalency (TEQ) values for complex mixtures, assuming additive effects according to the equation below.

TEQ = ∑(Ci×TEFi) Hence, the TEQ value is the sum of the concentrations of each congener multiplied by their respective TEFs and represents the overall TCDD toxic potency of a sample. Some compounds are known to activate the AhR, but have not yet been assigned TEFs, for instance polybrominated dibenzo-p-dioxins and polybrominated dibenzofurans (PBDD/Fs). However, Olsman (2005) has established consensus rank order potencies for 18 PBDD/Fs and mixed halogenated dibenzo-p-dioxins and dibenzofurans based on four different dioxin-specific bioassays, demonstrating a practical use of bioassays.


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Table 2. Toxic equivalency factors (WHO-TEFs) from Van den Berg et al. (1998), and Van den Berg et al. (2006), and cross-reactivity (CR) values from Shan et al. (2001) for 17 PCDDs and PCDFs with chlorines at all lateral positions.

Compound WHO-TEF 1998

WHO-TEF 2005

CR

2,3,7,8-TCDD 1 1 1.29

1,2,3,7,8-PeCDD 1 1 0.73

1,2,3,4,7,8-HxCDD 0.1 0.1 0.01

1,2,3,6,7,8-HxCDD 0.1 0.1 n.a.

1,2,3,7,8,9-HxCDD 0.1 0.1 0.028a

1,2,3,4,6,7,8-HpCDD 0.01 0.01 0.003

OCDD 0.0001 0.0003 <0.0001

2,3,7,8-TCDF 0.1 0.1 0.26

1,2,3,7,8-PeCDF 0.05 0.03 0.001

2,3,4,7,8-PeCDF 0.5 0.3 0.09

1,2,3,4,7,8-HxCDF 0.1 0.1 <0.0001

1,2,3,6,7,8-HxCDF 0.1 0.1 0.054

2,3,4,6,7,8-HxCDF 0.1 0.1 n.a.

1,2,3,7,8,9-HxCDF 0.1 0.1 0.054

1,2,3,4,6,7,8-HpCDF 0.01 0.01 0.0006

1,2,3,4,7,8,9-HpCDF 0.01 0.01 n.a.

OCDF 0.0001 0.0003 <0.0001

a Paper IV (Nording et al., 2006) n.a. = not analysed

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3. THE CONTAMINATED ENVIRONMENT

POPs, even those with low vapour pressures, may be transported long distances from their primary sources since they are highly resistant to degradation and hence have long residence times in the environment. Consequently, some harmful substances may be ubiquitous and can be found at background levels in ecosystems all over the world, posing a global environmental threat. For instance, the presence of organochlorine contaminants has been demonstrated in Artic marine ecosystems by the identification of PCBs in Polar bears, although PCBs have never been either produced or used in their habitat (Norstrom et al., 1988). Since humans also inhabit or visit very delicate (and remote) ecosystems, environmental pollutants may affect the health and well-being of virtually every individual on the planet. Elevated levels of POPs can be found close to sources like waste incineration sites and chemical and cellulose processing plants, posing threats especially to people living in their vicinity. For instance, Olsson et al. (2005) found significant levels of dioxins in fish collected in the Baltic Sea close to Swedish pulp processing plants, indicating ongoing pollution. However, levels of DDT and PCBs in herring and cod collected at various Swedish sites since the 1970s have declined (Bignert et al., 1998), as have levels of DDT, PCBs, dioxins and other POPs in Swedish breast milk (Lundén and Norén, 1998; Norén and Meironyté, 2000; Norén, 1993). These findings illustrate that earlier emissions caused high degrees of environmental contamination, and that measures to eliminate pollution have helped to reduce environmental levels of POPs. Programmes to monitor POP trends play an important role in elucidating relationships between emissions and contaminant levels in environmental matrices. Screening techniques, involving simplified sample preparation and analyte detection procedures, can facilitate this work by allowing larger numbers of samples to be analysed within the framework of the monitoring programs.

3 . T H E C O N T A M I N A T E D E N V I R O N M E N T

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Dioxins and polycyclic aromatic hydrocarbons in soil

The fate of the PCDDs and PCDFs is determined by their physical and chemical properties, and soil is considered to be their primary sink in the environment (Harrad and Jones, 1992). Therefore, PCDDs and PCDFs can be found in soils and sediments, especially at industrial production sites or user facilities, such as wood treatment sites, where chlorophenol agents contaminated with PCDDs and PCDFs have been used for preservative purposes. The total amount of dioxins in soil originating from such activities in Sweden is estimated by the Swedish Environmental Protection Agency (SEPA) to be 5-50 kg WHO-TEQ, distributed at up to 500 sites (SEPA, 2005). This is of historical origin, since chlorophenol agents were banned as of the 1st of January 1978. Furthermore, chlorine gas and lye were produced at eleven sites in Sweden, by the so-called chloralkali industry, which also contribute to elevated dioxin levels in the environment. Samples from both wood treatment sites and chloralkali production sites in Sweden were investigated in the studies reported in Papers IV and V. In addition, soil samples from sites where uncontrolled combustion has taken place in Uruguay were investigated, and the results show that dioxins also occur in non-industrialized countries (Paper IV). PAHs are formed during the incomplete combustion of organic material. Hence, in the process of deriving combustible gases from coal, historically used as town gas, PAHs were formed and ended up in the coal tar. These by-products were initially left at the production sites, but eventually the coal tar was refined to generate products, such as creosote (by distillation), which mainly consists of PACs with two, three or four fused aromatic rings (Howsam and Jones, 1998; Mueller et al., 1989). In Papers I and II, PAH-contaminated soil samples from old gasworks and wood impregnation sites were investigated. The PAHs originated from both coal gasification processes, and the wood preservative creosote. SEPA has currently identified 41,000 contaminated sites within Sweden alone (SEPA, 2005), and set limits that ideally should not be exceeded for PAHs, PCDDs, PCDFs, and other contaminants in the environment (SEPA, 1997). For soils, these guidance values can be found in Table 3. The preliminary remidation goal set by USEPA for dioxin contaminated residential soil is 1,000 ng TEQ/kg (Fields, 1998). Currently, extensive work is dedicated to the remediation of polluted sites and the level of the pollution is a key determinant of the costs involved, which can vary extensively. For instance, the estimated costs of remediating creosote-contaminated sites can range from 10,000,000 to 70,000,000 SEK (SEPA 2005). Furthermore, the cost of mapping dioxin levels and distributions at a single contaminated site is estimated to be 1,000,000 - 2,000,000 SEK. To streamline this process, fast, accurate and field-adapted screening methods are


1Non-ortho PCBs: 77, 81, 126, and 169; Mono-ortho PCBs: 105, 114, 118, 123, 156, 157, 167, and 189

15

valuable tools. Therefore, efficient sample preparation and bioanalytical techniques for surveying PAH- and dioxin-contaminated soils were investigated, as reported in Papers I, II, IV, and V. Table 3. Guidance levels, per kg dry matter, for dioxins (PCDD/Fs) and polycyclic aromatic hydrocarbons (PAHs) in soils intended for sensitive use (e.g. residential areas), and less sensitive use (e.g. industrial areas) reported by SEPA, 1997.

Compound Sensitive use Less sensitive use

PCDD/Fs 10 ng TEQ/kg 250 ng TEQ/kg

Carcinogenic PAHsa 0.3 mg/kg 7 mg/kg

Other PAHsb 20 mg/kg 40 mg/kg

abenzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, indeno[1,2,3,cd]pyrene, and dibenz[a,h]anthracene bnaphthalene, acenaphthalene, acenaphtene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, and benzo[ghi]perylene

Dioxins in food and feed

The major route for human intake of POPs is through food, especially polluted fish since aquatic food webs have greater contaminant loads and more levels than their terrestrial counterparts (Bernes, 1998). Imposing limits on the levels of PAHs in fish sold for human consumption would have limited value since the parent compounds are metabolized. However, there are guidelines for maximum tolerable dioxin levels (both dioxins and dioxins together with dioxin-like PCBs1) for total weekly intake and also for fish (Table 4). These levels are occasionally exceeded in some fish, for instance herring in the Baltic Sea (Ankarberg et al., 2004). However, fish may be consumed within Sweden and Finland, owing to exceptions from the regulations for a transitional period, since there are extensive surveillance programs and dietary recommendations in these countries. Besides the common intake of dioxins through fish consumption, incidents involving dioxin-contaminated food and feed may also cause elevated human exposure. The first known incidents of poisoning by dioxin-contaminated foodstuffs occurred in Japan and Taiwan in the 1960s and 1970s (as reported, inter alia, by Hsu et al., 1984). Since then, several extensive dioxin-contamination incidents have been reported, for instance PCB and dioxin contamination episodes of animal feed in Belgium in 1999 leading to elevated levels in chicken and eggs intended for human consumption (Ashraf, 1999).


16

Furthermore, elevated levels of dioxins in chicken were traced back to ball clay used as an anti-caking agent in poultry feed (Ferarrio et al., 2000, Ferrario and Byrne, 2000). More recently, elevated levels in pigs were traced to dioxin-contaminated bakery waste used as animal feed (Hoogenboom et al., 2004). The occurrence of dioxins in ecosystems leading to human exposure via both ordinary food intake and food/feed poisoning incidents highlights the need for rapid dioxin screening methods, such as those investigated in Paper III. Table 4. Maximum tolerable weekly intakes and levels in fish of dioxins (PCDD/Fs) and dioxin-like PCBs (DL-PCBs) established by the European Union (EU, 2006).

Compound Weekly intake Fish

PCDD/Fs n.a. 4.0 pg WHO-TEQ/g fresh weight

Sum PCDD/Fs and DL-PCBs

14 pg WHO-TEQ/kg bodyweight

8.0 pg WHO-TEQ/g fresh weight

n.a. = not applicable

17

4. STEPS REQUIRED TO DETECT

POLLUTANTS To protect public health and safeguard the environment, reliable analytical measurements of all kinds of hazardous compounds are needed. Guidelines to ensure the overall quality of environmental analytical measurements were published in 1980 by the American Chemical Society's Committee on Environmental Improvement, and a revision was subsequently published devoted to principles of environmental analysis (Keith et al., 1983). This covers initial planning, sampling, sample pre-treatment and preparation, and finally the measurements of the analyte(s). At every stage, a quality assurance program is essential for a sound analytical protocol to detect and correct problems in the analytical process. The expectations regarding accuracy, sensitivity, precision, reliability, interferences, matrix effects, limitations, cost, and the speed of the analysis have to be realistic. The ultimate user of the data has to consider every aspect of these issues when evaluating, interpreting, and communicating the results. An assignment might, for instance, not necessarily require highly accurate and reproducible quantitative estimates of the target analytes. Qualitative or semi-quantitative analyses often provide sufficient data (in screening approaches, for example). In a qualitative analysis, the target analyte is merely identified, which is often sufficient for a first step in evaluating the presence of a pollutant. Furthermore, through a semi-quantitative analysis it is possible to decide whether a sample is more, or less, contaminated than a certain level, e.g. a guidance value. Since analytes in environmental samples often need to be measured at trace levels (parts-per-billion or even parts-per-trillion), techniques with very low detection limits are essential, placing high demands on the techniques used, for instance, in sample preparation and analyte detection. In the following sections, the key steps in the analytical scheme are discussed, including alternatives to conventional sample preparation methods. Bioanalytical substitutes for the instrumental detection techniques are discussed in Chapter 5. The focus is on solid samples, in many cases with contaminated soils as illustrative examples.

4 . S T E P S R E Q U I R E D T O D E T E C T P O L L U T A N T S

18

Sampling

After the initial planning of a study to assess the occurrence of pollutants, the first practical step is to collect potentially contaminated samples. A summary of environmental sampling has been reported by Keith (1990). He states that "the objective in collecting samples for analysis is to obtain a small and informative portion of the population being investigated". In such work, a number of strategies can be employed, depending on the objectives of the study. When collecting soil samples, for instance, their heterogeneity often causes problems. Consequently, errors associated with the representativeness of the samples frequently exceed the analytical uncertainties. The implementation of sampling plans to minimize erroneous results can be found in Harvey (2000).

Extraction and clean-up

After the sampling, samples have to be prepared for detection of the target compounds. A review of sample preparation techniques for environmental analysis is provided by Lopez-Avila (1999). In general, the target analyte has to be extracted and isolated from its matrix and other anthropogenic substances in order to detect trace amounts of pollutants in the environment. Soxhlet extraction is still one of the widely used extraction techniques, although it was originally invented in 1879, and it was used to extract dioxins from soil samples in the investigations reported in Papers IV and V. In the 1990's, Richter and co-workers introduced an extraction procedure involving the use of elevated temperatures and pressures, named accelerated solvent extraction (ASE), or pressurized liquid/fluid extraction (Richter et al., 1996). ASE has been successfully used for various purposes, including the extraction of PAHs, PCBs, and dioxins from reference materials and contaminated solid samples (Björklund et al., 2000; Richter et al., 1997; Schantz et al., 1997). A schematic representation of an ASE system is shown in Figure 6. Other alternatives to Soxhlet extraction include supercritical fluid extraction and microwave-assisted extraction (Björklund et al., 2002; Sparr Eskilsson and Björklund, 2000; Sporring et al., 2005; Turner et al., 2002). Co-extracted material has to be separated from the target analytes through clean-up of the extract prior to the final detection step. This can be done in various ways, for instance by multi-step column clean-up or integrated extraction and clean-up techniques, which may be faster than conventional techniques and consume less solvents (Björklund et al., 2006). In the study reported in Paper II, ASE was used in accordance with Ong et al. (2003), with sulfuric acid silica in the extraction cell, for the simultaneous extraction and purification of PAHs from soil. This approach has been further refined by Lundstedt et al. (2006) for fractionation of PAHs


19

and their derivatives. Other authors have developed similar methods for analysing PCBs in food and feed, for instance Müller et al. (2001). A further development of the ASE technique, with a carbon trap in the extraction cell, was used for the simultaneous extraction and fractionation of dioxins from food and feed (Paper III), and from soil (Paper IV), referred to as ASE-C (ASE with an integrated Carbon trap). The approach was originally described by Sporring et al. (2003), who used it for recovering [13C12]-labelled PCBs and PCDD/Fs from spiked fish oil. The ASE-C technique has the potential for selective elution of other analytes, co-occurring with the PCDD/Fs in the extracts. Hence, fractionation of PCBs and PCDD/Fs may be possible. This is especially attractive in view of the up-dated guideline values, since both PCDD/F levels together with the total PCDD/F and dioxin-like PCB levels have to be accounted for (EU, 2006), as discussed in Chapter 3.

NitrogenSolvent

Collection vial

Pump

Oven

Valve

Extractioncell

Load cell

Fill cell with solvent

Pressurize and heat cell

Hold at elevated pressureand temperature

Purge solvent from cell with nitrogen

Collect extract

NitrogenSolventSolvent

Collection vial

Pump

Oven

Valve

Extractioncell

Load cell





Collect extract

Load cell





Collect extract

Figure 6. Schematic representation of an accelerated solvent extraction (ASE) system. A flowchart of the extraction procedure is shown on the right. If necessary, several extraction cycles can be run in sequence.


20

Instrumental separation and detection

The last steps in conventional assessments of organic pollutants are separation of the analytes by gas or liquid chromatography (GC or LC) and their detection by a suitable instrument, e.g. a mass spectrometer (MS), flame ionization detector, or electron capture detector (ECD). Patterson (2005) has reviewed developments in POP and PAH measuring systems over the last 30 years. During this time, advances in analytical systems have lowered the detection limit from 100 picograms to 313 attograms of TCDD, by comprehensive GC×GC and high resolution (HR) MS. The basic components of an instrumental PAH or PCDD/F detection system are shown in Figure 7. The quantification of selected PAHs in the work underlying this thesis was accomplished using GC-time of flight (TOF)-MS, which allows the use of isotopically labelled internal standards and provides sufficient sensitivity and selectivity for the levels of PAHs in the samples. Congener-specific analysis of the PCDD/Fs, on the other hand, was accomplished by GC-HRMS in selected ion monitoring mode with a resolution of 8,000-10,000. This high resolution technique also allows isotopically labelled internal standards to be used, and offers unrivalled sensitivity. In both cases, analytes are initially separated in the GC and ionized by electron impact ionization, but the MS analyzers work in different ways. In TOF systems, all ions are initially accelerated by an electric field and ions with differing mass to charge (m/z) ratios are then separated due to differences in their flight time through a field-free region. In HRMS systems, ions are separated by adjusting magnetic and electric fields in such a way that only ions with certain m/z ratios reach the detector.

GC Ionization Mass analyzer Detector

Mass spectrometer

GC Ionization Mass analyzer Detector

Mass spectrometer

Figure 7. A general schematic diagram of instruments used to quantify organic pollutants. In the work underlying this thesis, analytes were initially separated by a gas chromatograph (GC), then ionized through electron impact, and the resulting ions were separated in a magnetic sector (HRMS) or TOF analyzer and detected using instrument-specific detection/amplification systems.


21

PAHs targeted in the studies were identified by matching chromatographic retention times and mass spectra of the analytes detected in the samples with those in a reference standard mixture. The PCDD/Fs were identified by the former, and by inspection of isotope ratios. Quantification was generally based on the isotope dilution technique (USEPA, 1994), in which peak areas for analytes in the samples and reference standards were compared. The quantification was aided by fully deuterated PAHs, or [13C12]-labelled PCDD/Fs added prior to clean-up to compensate for analyte losses. As a quality assurance (QA) and quality control (QC) measure for the analysis, isotopically labelled recovery standards were added prior to injecting samples into the instrument. In all experiments, a method blank was included to detect potential contamination problems. This was especially important for low molecular weight PAHs, which are abundant in the environment and are also present at high background levels in the laboratory, so they were omitted from the evaluation of GC-TOF-MS results in Papers I and II.


22

23

5. BIOANALYTICAL TECHNIQUES Bioanalytical techniques have gained popularity in environmental analysis since they provide ways to eliminate uncontaminated samples and rank the potency of suspected contaminated samples before more costly instrumental analyses are applied. They involve the use of biologically derived components to obtain analyte-specific responses. The intensity of the signals elicited is related to the amounts of inductive chemicals in a sample or extract. There are two types of bioanalytical techniques, immunoassays and bioassays, both of which have been used within the framework of this thesis. Behnisch et al. (2001a) have reviewed current state-of-the-art bioanalyses of dioxin-like chemicals. Furthermore, attempts have been made to compare the performance of immunoassays and bioassays for such compounds, both in previous studies (Li et al.,1999; Roy et al., 2002) and in Paper V.

Immunoassays

Immunochemical techniques were initially used for clinical purposes, following the first application of immunoassays by Yalow and Berson (1959) to detect insulin in human blood. Since then, extensive research has widened the scope of their applications as well as the range of formats and labels used in the assays. The potential utility of immunoassays for monitoring environmental pollutants was suggested in the early 1970s, but it was not until 1980 that they began to be more widely applied for analyzing hazardous compounds like pesticides (Hammock and Mumma, 1980). There are now several immunoassays for detecting organic pollutants in the environment (reviewed inter alia by Plaza et al., 2000; and Van Emon, 2001). A few examples of immunoassays for detecting PAHs are provided by McDonald et al. (1994), Knopp et al. (2000), and Chuang et al. (2003). Furthermore, the immunochemical analysis of dioxins has been reviewed by Harrison and Eduljee (1999). The successful application of an enzyme-linked immunosorbent assay (ELISA) to estimate dioxin contents in contaminated soil in a country with limited resources (Uruguay) demonstrated its suitability as a low-cost alternative to conventional dioxin analysis (Trindade et al., 2006).

5 . B I O A N A L Y T I C A L T E C H N I Q U E S

24

The antibody is the key component in any immunoassay, and the interaction between the antibody and antigen must be unfettered in order for the assay to give reliable results. Specific structures (epitopes) in the antigen are recognized by the antibody and the interaction (which is reversible) involves electrostatic, hydrophobic and van der Waals forces and hydrogen bonding. Enzymes are among the most popular labels for recognising the binding of the antibody to the antigen and ELISA has proven to be very useful for environmental applications. Two common ELISA formats are shown in Figure 8.

Coating antigen

Analyte

Primary Ab

Secondary Ab

Substrate

Product

Indirect competitive ELISA Direct competitive ELISA

Enzyme tracer

Coating antigen

Analyte

Primary Ab

Secondary Ab

Substrate

Product

Indirect competitive ELISA Direct competitive ELISA

Enzyme tracer

Figure 8. Schemaric diagram of two common heterogenous formats for detecting low molecular weight analytes by the enzyme-linked immunosorbent assay (ELISA): the indirect competitive ELISA used in 96-well microtiter plates as reported in Papers IV and V (left), and the direct competitive ELISA in tubes described in Papers I and II (right). Pictures by Malin L. Nording, 2003 and 2004. A direct competitive ELISA for PAHs was used in the studies presented in Papers I and II. In this format, the antibodies are immobilized on a surface, and the analyte and enzyme tracer compete for interactions with the antibody. If the target analyte is present in the sample being analysed it will inhibit the binding of the analyte-enzyme complex to the antibody, thereby reducing the level of bound enzyme and, hence, the catalytic conversion of the substrate to the coloured product in a concentration-dependent manner. The analyte concentration is therefore inversely proportional to the optical density of the


25

coloured product, and measurements of the resulting colour provide indications of the concentration of the analyte in the sample. In the investigations reported in Papers IV and V, an indirect competitive ELISA for dioxins was used, in which the antibody is in solution and a coating antigen and analyte compete to bind to the antibody. Similarly, a high amount of analyte will result in low optical density. However, in order to detect the interaction between the antibody and coating antigen, a secondary antibody labelled with an enzyme is required. Whenever immunoassays are used for measuring specific analytes in complex matrices, interferences may be present and affect the immunochemical reaction. A matrix effect is indicated by deviations between the parameters defining the sample extract curve and the standard curve. Examples are provided by the crude sample extracts analysed in Paper IV, see Figure 9.

Abs

orba

nce

Concentration (pg/ml)

TMDD 0.461 0.997 115 0.036 0.997

Crude extract I 0.455 0.711 59.0 0.05 0.999

Crude extract II 0.454 1.368 1.74 0.335 0.992

Crude extract III 0.446 1.108 7.50 0.233 0.996

AbsMax Slope IC50 AbsMin R2

0.01 0.1 1 10 100 1000 10000 1000000

0.1

0.2

0.3

0.4

0.5

Abs

orba

nce

Concentration (pg/ml)

TMDD 0.461 0.997 115 0.036 0.997

Crude extract I 0.455 0.711 59.0 0.05 0.999

Crude extract II 0.454 1.368 1.74 0.335 0.992

Crude extract III 0.446 1.108 7.50 0.233 0.996

AbsMax Slope IC50 AbsMin R2

0.01 0.1 1 10 100 1000 10000 1000000

0.1

0.2

0.3

0.4

0.5

Figure 9. Illustration of deviations from the shape of the standard curve (TMDD) of curves obtained from three uncleaned soil extracts. The curves are defined by their maximum absorbance (AbsMax), slope at the inflection point (Slope), inflection point (IC50), minimum absorbance (AbsMin), and regression coefficient (R2). The samples were collected from wood treatment sites in Sweden.


26

The interferences may be non-specific (e.g. pH, ionic strength or organic molecules with no structural resemblance to the analyte) or specific, i.e. compounds that are recognized by the antibody (cross-reactants). The epitopes on the cross-reactant may be the same, or structurally related to, the epitope in the analyte, as described by Van Regenmortel (1998). The antibody usually binds most strongly to epitopes that are homologous to those used to raise it, but it is not uncommon for antibodies to bind with higher affinity to other, related epitopes. Such antibodies are called heteroclitic or heterospecific (Mäkelä, 1965; Underwood, 1985). For example, in Paper I, the cross-reactivity of anthracene was shown to be 140% as compared to phenanthrene, which the antibodies were designed to detect. However, most of the tested compounds display lower cross-reactivities. In some cases, cross-reactivity is advantageous, for instance when the levels of a class of compounds can be estimated using antibodies raised against a certain molecule that can be regarded as representative for the whole class. On the other hand, when cross-reactants interfere with the immunochemical reaction, results may be difficult to interpret, as investigated in Paper I. Therefore, it is important to minimize interferences by unwanted cross-reactants. This is achieved by isolating the analytes before the immunoassay (clean-up and fractionation of extracts), optimizing assay conditions and/or developing more specific antisera (Miller and Valdes, 1991). Polyclonal antiserum (used in studies described in Papers IV and V) contain antibodies that recognize several different epitopes in the antigen since it is produced by immunizing an animal in which several B-cells, constituting part of the immune system, are responsible for the production of the antibodies. Monoclonal antibodies (used in studies described in Papers I and II), on the other hand, are produced by clones of a single cell in vitro that generate a unique type of immunoglobulin G molecule, i.e. antibody. Hence, greater specificity can be obtained with monoclonal antibodies than with polyclonal antibodies. However, monoclonal antibodies are generally more expensive to produce and have lower affinities for small molecules (like PAHs and dioxins) than polyclonal antibodies. Therefore, many immunoassays for environmental applications are based on polyclonal antibodies. In general, matrix effects can be suppressed by dilution of the sample extract, at the expense of sensitivity, and changes in assay performance, for instance by adding additives to the reaction buffer, thereby mimicking the matrix (Nichkova, 2003). However, it is recommended that interferences be monitored by analyses of appropriate blanks and controls, and analyses of contaminated sample extracts by confirmatory methods since matrix effects may vary with reagent batches and matrix sources.


27

Cell-based bioassays

In the 1960s, it was found that the AHH enzyme system is induced by PAHs (inter alia) in several cell lines (Nebert and Gelboin, 1968). Furthermore, the same effect was found to be induced by TCDD in chick embryo liver (Poland and Glover, 1973). Consequently, a bioassay, utilizing rat hepatoma cells (H4IIE), derived by Pitot et al. (1964), was suggested by Niwa et al. (1975). Furthermore, as an alternative to AHH measurements, a fluorimetric assay to measure the enzymatic activity of EROD was developed by Pohl and Fouts (1980). For details of the enzymatic reactions involved, see Figure 10. Among others, Safe and colleagues, performed profound work in the 1980s to establish the relationship between in vivo toxicity and induction of AHH and EROD in H4IIE cells (e.g. Bandiera et al., 1984; Safe et al.,1987).

OH

N

OC2H5O O

N

OOH O

Benzo[a]pyrene 3-Hydroxybenzo[a]pyrene396 nm/522 nm

Resorufin7-Ethoxyresorufin550 nm/585 nm

AHH

EROD

Figure 10. Cytochrome P4501A1 catalyses inter alia the transformation of benzo[a]pyrene to 3-hydroxybenzo[a]pyrene and 7-ethoxyresorufin to resorufin (cf. Figure 3). For the former reaction to take place, aryl hydrocarbon hydroxylase (AHH) activity is essential, as is 7-ethoxyresorufin-O-deethylase (EROD) activity for the latter reaction. The levels of the products can be easily determined and provide indications of the expression of CYP1A1, which is equivalent to the level of the inducing chemicals, such as dioxins.


28

Since then, H4IIE cell bioassays have been used extensively as indicators of dioxin-like chemicals in both wildlife and the environment (reviewed by Whyte et al., 2004). However, a severe limitation of the assay is that reductions in the signal strength are often observed when the concentrations of the contaminants exceed a certain level, causing bell-shaped response curves (e.g. Verhallen et al., 1997), prompting the development of alternative approaches involving the use of recombinant cell lines. In the beginning of the 1990s, stably transfected recombinant mouse and human cell lines that could be used as cell bioassay systems were described by Aarts et al. (1993), Denison et al. (1993) and Postlind et al. (1993). The cell lines contained reporter genes expressing firefly luciferase for detecting AhR ligands. Since then various recombinant cell-based bioassays, based on measuring AhR-dependent gene expression resulting from dioxin-like chemical burdens, have been developed and an overview of the work in this research field is provided by Denison et al. (2004). Mammalian cell lines based on transfected and/or endogenous reporter genes as well as recombinant yeast cell bioassays are discussed therein. As an extension of the early work in which recombinant cell lines were introduced as bioassay systems, Garrison et al. (1996) reported a variety of cell lines, including rat (H4IIE), mouse (Hepa1c1c7), guinea pig, hamster, and human lines transfected with expression vectors for detecting dioxin-like chemicals. The cited workers constructed pGudLuc1.1 (Figure 11), a reporter plasmid that responds to dioxin-like chemicals in an AhR- dose-, and time-dependent manner, eventually causing induction of the firefly luciferase. Several advantages over the EROD assay, including the stability of the luciferase, low background activity, and lack of substrate inhibition, make this bioassay attractive for identifying bioactive compounds, although its results do not necessarily reflect toxicity. This was demonstrated by Aarts et al. (1995), in an examination of the species-specificity of AhR agonists' and antagonists' actions, and by Murk et al. (1996), who showed its utility for analysing sediments and pore water. Murk and co-workers named the assay chemically activated luciferase gene expression (CALUX). Later, the assays based on the recombinant H4IIE and Hepa1c1c7 cell lines became known as the dioxin-responsive, DR-CALUX bioassay, and the CALUX bioassay, respectively. In a refinement of the CALUX bioassay, Nagy et al. (2002a), developed a recombinant cell line in which enhanced green fluorescent protein (EGFP) expression is induced in response to dioxin-like chemicals. This chemically activated fluorescent gene expression (CAFLUX) bioassay is more rapid, more convenient, and cheaper than the CALUX bioassay. In addition, CAFLUX provides a means to measure reporter gene activity in "real-time" since the fluorescence is measured in intact cells.


29

Wild-type cell Plasmid with DRE

TRANSFECTION

Recombinant cell

DREs

Luciferase gene

pGudLuc1.1 (9225 bp)

Wild-type cellWild-type cell Plasmid with DRE

TRANSFECTION

Recombinant cell

DREs

Luciferase gene

pGudLuc1.1 (9225 bp)

Figure 11. Overview of the procedure for obtaining CALUX recombinant cells. Dioxin-responsive elements (DREs) are incorporated in the expression vector pGudLuc1.1 (adapted from Garrison et al., 1996). The CAFLUX assay has been used in the identification of novel AhR agonists (Nagy et al., 2002b) and compared to other CALUX bioassays (Han et al., 2004). However, the utility of the CAFLUX bioassay, especially for authentic dioxin contaminated samples, has not yet been comprehensively evaluated, although Papers III and V provide valuable information related to this issue. The CA(F)LUX bioassays are sufficiently sensitive to detect dioxins at levels found in both food and feedstuffs (Paper III), and soil (Paper V), using the dose-response curve typically obtained for TCDD (Figure 12), which was used as a standard in every CA(F)LUX assay presented in the work this thesis is based upon. The limit of quantification, defined as the blank value plus ten times the standard deviation (SD), was in this case 0.06 pg/well, theoretically corresponding to 3 pg/g sample with a sample amount of 1 g and a final extract volume of 100 µl.


30

Figure 12. A typical standard dose-response curve from a CAFLUX assay, with TCDD-concentrations in the wells ranging from 0.3 to 1900 pM. The EGFP induction of DMSO (blank)-treated cells (19000) has been subtracted from all points. The lower left and upper right pictures show non-fluorescent and fluorescent cells, respectively. Pictures by Erik Spinnel, 2004. The applications of recombinant and other cell-based bioassays have been reviewed by Behnisch et al., (2001b). Successful applications in assessments of dioxin-like potency in various matrices are provided inter alia by Engwall et al. (1999), Murk et al. (1997), Scippo et al. (2004), Spinnel et al., 2005, and Ziccardi et al. (2002). The usefulness of cell-based bioassays in dioxin analysis has been further confirmed by intra- and inter-laboratory comparative studies (Behnisch et al., 2002; Besselink et al., 2004), and the CALUX bioassay has proven very useful in dioxin food incidents and identification of novel dioxin sources (Hoogenboom, 2002; Hoogenboom et al., 2004; Hoogenboom et al., 2006).

0

5000

10000

15000

20000

25000

30000

35000

40000

0,01 0,10 1,00 10,00 100,00 1000,00

TCDD (pg/well)

EGFP

indu

ctio

n

0.01 0.1 1.0 10 100 10000

5000

10000

15000

20000

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30000

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0,01 0,10 1,00 10,00 100,00 1000,00

TCDD (pg/well)

EGFP

indu

ctio

n

0.01 0.1 1.0 10 100 1000

31

6. PRACTICAL USE OF THE

SCREENING CONCEPT There have been continuous improvements in PCDD/F and PAH analyses. Initially, only TCDD and benzo[a]pyrene were measured. However, with current technology, the majority of dioxin congeners and many PAHs and PAH metabolites can be measured in a vast number of matrices. The sensitivity has also improved along with the development of better sample preparation techniques, new types of chromatographic columns, and more powerful detection instruments. The ultimate aims are to lower the detection limits and further increase the efficiency of the analysis. An avenue of possibilities is provided by novel and integrated extraction and clean-up techniques in addition to bioanalytical detection techniques, some of which have been mentioned in Chapters 4 and 5. Instrumental approaches that can be used instead of bioanalytical systems for detecting analytes include hyphenated techniques such as LC-LC-GC-ECD, for analysis of marker congeners, and comprehensive GC×GC-ECD, for congener-specific analysis (Haglund et al., 2004). Preparing samples to detect trace amounts of analytes like dioxins is not as straightforward as preparing samples to detect PAHs, which can be found at parts-per-millions and more in contaminated soil samples. For the latter, agitation with an appropriate solvent without further clean-up prior to immunoanalysis might be sufficient, as demonstrated in Paper II. This approach is not recommended for immunoassays or CA(F)LUX analyses of dioxins in soil, for which elaborate sample preparation is necessary (Papers IV and V). Other authors have also reported the importance of clean-up prior to CALUX analysis (Schroijen et al., 2004; Van Wouwe et al., 2004). Sample preparation with traditional use of Soxhlet and multi-step clean-up is tedious and requires relatively large amounts of resources. Developments in this area would speed up analyses considerably (Björklund et al., 2002). In 2004, Focant et al. reviewed current state-of-the-art techniques for preparing samples containing dioxins, including an automated extraction and clean-up system based on ASE coupled to Power-PrepTM, a commercially available clean-up system (Abad et al., 2000; Eljarrat et al., 2001). With this approach they

6 . P R A C T I C A L U S E O F T H E S C R E E N I N G C O N C E P T

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reduced the total sample preparation time from several days to less than half a day. The enhanced sample preparation techniques can be utilized prior to conventional instrumental analysis, thereby facilitating rapid analytical processing while maintaining the quality of the data. A further step in high-throughput methodology is to couple fast sample preparation techniques to parallel processing at the detection stage, provided by bioanalytical techniques. This screening approach is illustrated in Figure 13, a concept that may be useful for many practical purposes, for instance in large-scale food and feed monitoring programs, mapping of pollutants, or for screening samples before and after certain treatments. It can be used to eliminate negligibly contaminated samples and to identify samples contaminated above a certain limit to be further analysed by confirmatory techniques. It can also be used to rank samples for priority of analysis.

SamplingSoil, biota,…

Confirmatorytechnique

Bioanalyticaltechnique

Rapid sample preparation

Above guidancevalue

Belowguidance

value

Data baseMapping, monitoring…

Confirmation

SamplingSoil, biota,…

Confirmatorytechnique

Bioanalyticaltechnique

Rapid sample preparation

Above guidancevalue

Belowguidance

value

Data baseMapping, monitoring…

Confirmation

Figure 13. Overview of the screening approach. Large numbers of samples are initially processed by a rapid preparation technique and then analysed by a bioanalytical technique. Some of the extracts, including samples with analyte levels exceeding guidance values and e.g. one tenth of the samples with analyte levels below these values undergo analysis with a confirmatory technique (e.g. GC-HRMS). The results are incorporated in a database, used for instance in pollutant mapping and monitoring programs.


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For cell-based bioassay screening of food and feedstuffs, several criteria must be met to assure the quality of the analyses (EU, 2002). These include stipulations that the SD of triplicate determinations and three independent experiments must not exceed 15% and 30%, respectively, of the mean values. Since the presence of isotopically labelled internal standards may bias bioassay responses, quality criterias should be recorded by other means, e.g. by the inclusion of reference samples. Furthermore, a blank should be analysed in parallel with each unknown sample. QA/QC methods for cell-based bioassays in food and feed control may convincingly be extended to other bioanalytical techniques and matrices.

Summary of Papers I-V

In the work underlying this thesis a number of rapid sample preparation and bioanalytical techniques were applied to the analysis of PAHs and PCDD/Fs in authentic contaminated environmental matrices, including soil, food and feed. The feasibility and utility of several approaches, in both currently available and refined formats were explored. The techniques used and analytes detected are listed in Figure 14. A brief summary of each study follows.


Creosote & gasworks

soilsNone Agitation with

solventFiltration Immunoassay

Creosote & gasworks

soilsNone Agitation with

solventFiltration Immunoassay PAHs in

Papers I and II

Fish oil and fish meal

Homogenization and mixing with

Na2SO4

Integrated extraction and clean-up with ASE

CAFLUX PCDD/Fs in Paper III

Chlor alkali soils...

Homogenization and sieving

or integrated extraction and clean-up with ASE

Immunoassay


PCDD/Fs in Paper IV

Homogenization and sieving

CALUX CAFLUX

Immunoassay

Chlor alkali soils...

Soxhlet Multi-stepcolumns PCDD/Fs in

Paper V

Figure 14. Overview of sample preparation and bioanalytical techniques used in the studies underlying the thesis. Each arrow is accompanied by a reference method based on appropriate protocols for instrumental analysis.


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An immunoassay for analysing PAHs in contaminated soil In the studies reported in Papers I and II, the PAH RISc® soil test was used. This is a field-adapted and commercially available test kit for screening samples with PAHs above or below certain levels in contaminated soil. The kit contains antibody-coated tubes and all the reagents needed to detect PAHs by ELISA. In addition, kits for efficient extraction, based on agitation in methanol, can be obtained from the same manufacturer. Initially, the structure/cross-reactivity relationships of three priority-pollutant PAHs and 13 methyl-, phenyl-, and carbonyl-PAHs, together with NSO-heterocyclic PACs, were evaluated to obtain detailed information about the specificity of the antibody. The inhibition of the absorbance caused by test compounds was compared to that of phenanthrene, which the antibodies were raised against. It was found that the structural features of the chemicals strongly influence the degree of inhibition they display. For instance, methylated and carbonyl-substituted PAHs generally have lower cross-reactivity than compounds with structural similarities to phenanthrene. Therefore, it is important to monitor changes in the PAC composition of contaminated soil samples, for instance during biological treatment, in order to interpret the ELISA inhibition correctly. However, since the degradation products are smaller, often hydroxy-, or carbonyl-substituted molecules, than the target analytes, and the antibodies are likely to have limited sensitivity to them, the PAH RISc® soil test should still give a reasonable estimate of the abundance of large, recalcitrant PAHs. With knowledge of the antibody's sensitivity to PACs beyond those already reported, it was possible to evaluate the site-specific performance of the kit using field samples. Eleven PAH-contaminated soil samples collected from coal gasification and wood impregnation sites in Sweden were used to examine the contributing factors to the ELISA measurement uncertainties. It was found that: (i) non-target compounds co-extracted with the analytes do not seem to inhibit the ELISA, (ii) agitation of the samples in methanol prior to the ELISA does not provide comprehensive extraction of the analytes, and (iii) ELISA and GC-MS results were in good agreement provided that individual cross-reactivities and insufficient extraction were taken into account. Integrated extraction/purification of contaminated food and feedstuffs for detecting dioxins by CAFLUX A high-throughput method for monitoring dioxins in food and feedstuffs was developed and its usefulness for analysing contaminated samples was demonstrated in Paper III. The ASE 300 instrument was equipped with specifically designed extraction cells to allow extraction and fractionation in a single step. The only other treatment required was passage through a miniaturized multilayer silica column as a polishing step to remove fat residues from the extracts prior to CAFLUX analysis. There was no significant


35

difference between the mean CAFLUX, DR-CALUX, and GC-HRMS values of the dioxin contents in fish oil (t-test, p = 0.05). The results of CAFLUX and GC-HRMS analyses of the dioxin content in fish meal were also very close. Furthermore, the lipid recoveries for both food and feed samples were close to 100%, allowing simultaneous gravimetric lipid weight determination. In all, it was concluded that the developed method is a promising screening method for more rapid and cheaper dioxin control of food and feedstuffs than currently used methods. However, further optimisation to eliminate the blank signal, improvement of the repeatability and reproducibility, and further tests on a larger set of samples for validation purposes are required before the integrated extraction/purification and CAFLUX method can be used on a larger scale. Integrated extraction/purification of contaminated soil samples for detecting dioxins by ELISA In the studies described in Paper IV, soil samples were extracted and purified according to conventional Soxhlet and multistep column clean-up and also by a modified version of the integrated extraction/purification ASE method presented in Paper III. The extracts were analysed by GC-HRMS and an ELISA developed by Bruce D. Hammock and co-workers (Sanborn et al., 1998; Sugawara et al., 1998). The optimal concentrations of coating antigen (III-BSA) and primary antibody (7598) in the ELISA were determined before the method was applied to soil extracts. The correlation coefficients after the Soxhlet and enhanced ASE procedure were 0.78 for the GC-HRMS and 0.99 for the ELISA results, respectively. Furthermore, the correlation coefficient between the GC-HRMS and ELISA results after the enhanced ASE procedure was 0.90. Hence, the combination of integrated extraction/purification and ELISA was concluded to be a more promising high-throughput screening approach for dioxin contaminated soil than conventional methods. However, further validation on a larger set of contaminated soil samples is necessary to establish the robustness and reliability of the method. The relationship between the ELISA and GC-HRMS results was linear, although dioxin contents were underestimated by the ELISA. Therefore, the PCDD/F profile of the investigated samples was evaluated by principal component analysis of data obtained by instrumental analyses of Soxhlet extracts, and the results showed that the relative amounts of various congeners affected the ELISA. In addition, important congeners in specific contaminated samples were identified and correlated to the pollution source of the respective sites (chlor-alkali, wood treatment, and uncontrolled combustion). Hence, a site-specific correction factor for accurate interpretation of the ELISA results was recommended.


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Comparison of ELISA, CALUX, and CAFLUX for analysing dioxins in contaminated soil samples In the investigations reported in Paper V, Soxhlet extracts derived from soil samples obtained from wood treatment and chlor-alkali sites, and samples of the artificial soil used in the studies reported in Paper IV, were also analysed by two bioassays; CAFLUX and CALUX. The results obtained with the different bioanalytical techniques for detecting dioxins were compared to GC-HRMS results and against each other. It was concluded that the bioanalytical techniques provide satisfactory alternatives to GC-HRMS analyses. They were found to be selective and accurate, and may be used to screen soil samples for compliance with the soil quality criteria recommended by the USEPA, i.e. <1000 ng TEQ/kg.

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7. CONCLUDING REMARKS The major conclusions from the studies underlying this thesis can be summarized as follows: • the specificity of the antibodies supplied in a commercial kit for an

immunoassay for detecting PAHs in soil, the PAH RISc® soil test kit, depend on the structural features of the analytes. Structural similarities to phenanthrene are associated with high cross-reactivity;

• the GC-MS and PAH RISc® results agree well, considering the less efficient extraction prior to ELISA analysis and the differences in cross-reactivities of specific PAHs;

• ASE-C and CAFLUX have potential utility for efficient screening of dioxins in food and feed;

• ASE-C in combination with ELISA is a promising approach for efficient screening of dioxin-contaminated soil; and,

• ELISA, CALUX, and CAFLUX can all be used to prioritize potential dioxin-contaminated soil samples before congener-specific GC-HRMS analysis.

A range of accessible paths for increasing the analytical capacity of organic compounds has been illustrated, which also was the main goal of the studies. With the aid of efficient sample preparation and bioanalytical detection techniques, large numbers of samples could be screened. The benefits of such approaches are that they would allow high sample throughputs and rapid, convenient determinations of total contaminant levels, i.e. the sum contents of a group of compounds. The cost-efficiency of such analysis also makes otherwise expensive analytical procedures accessible to countries and laboratories with limited resources. Among the drawbacks are the lack of acceptance due to limited validation studies in the research community, the costly development, and the fact that the data are semi-quantitative rather than quantitative. Furthermore, complete dose-response curves can be difficult to generate and interpret, and confirmatory analysis are necessary. Specific pros and cons of the bioanalytical techniques examined are summarized in Table 5. Finally, the screening methodology presented in this thesis might extend the analytical framework, expanding the database for decision-making. However, the results need to be treated with caution and always complemented with confirmatory techniques.

7 . C O N C L U D I N G R E M A R K S

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Table 5. Some advantages and limitations of immunoassays, CALUX, and CAFLUX. The features of CALUX also apply to CAFLUX.

Immunoassay CALUX CAFLUX

Kits for a broad range of pollutants are commercially available.

Biological effects are coped with (membrane passage etc.). Easier to handle than CALUX.

Advantages Easy to field-adapt. A biologically relevant sum of AhR ligands is reported. Cheaper than CALUX.

User friendly. Potentially infinite production of cells. Measurements in real-time.

Cross-reactivity with non-target compounds.

Restrictions for use due to genetic modification. Cumulative signal.

Limitations Sensitive to assay conditions. Access to tissue-culture room is necessary. High background signal.

Each assay is unique with respect to antibody specificity etc. Hence, there is

no common ground for an overall immunoassay characterisation.

Interpretation of results is difficult with no prior knowledge

about the nature of contamination.

Limited validation data.

A bright perspective for the future

The development of rapid, inexpensive sample preparation and detection techniques for organic pollutants reflects advances in the scientific field of environmental research driven by the environmental concern of the general public. The next step would be to further refine the techniques and make them more automated, a likely development in view of increasing demands to reduce costs while increasing the effectiveness of the analyses. Furthermore, validation using a large number of contaminated field samples is necessary for sound statistical evaluation and a better understanding of the usefulness of the methods. The ultimate goal has to be a rapid, portable, cheap, and user-friendly instrument that responds to a broad range of pollutants simultaneously, facilitating high throughput screening. With this, and other innovations, in combination with further awareness of environmental issues, our knowledge about the occurrence of harmful substances will expand, and we can pass on a safer environment to today's children and future generations.

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Acknowledgements Ni är många som på ett eller annat sätt hjälpt mig att nå målet på resan som började för fem år sedan. Tack Eva Knekta för att du visade vägen som skulle bli en så stor glädje och utmaning i mitt liv. Tack till guiden Peter Haglund som med beundransvärt tålamod väglett mig i både uppförs- och utförsbackar. En bättre handledare får man leta efter! Tack även till biträdande guide Mats Forsman, min fasta punkt i labyrinterna på FOI, och till min mentor Kerstin Forsén Öberg. Mina medresenärer på Miljökemi förtjänar alla ett stort tack för den fantastiska gemenskap som lägger grunden för tankens vandring mot nya upptäckter. Ett speciellt tack till min kupékompis Anneli Marklund för din aldrig sinande reslust! Tack Ylva Persson för att du så generöst delar med dig av din kunskap inom för mig okända regioner. Tack Lena Burström för din entusiasm som kartläsare och tack till Richard Lindberg för visualiseringen av resans mål. Tack också till resesällskapet på FOI, som så öppenhjärtigt tagit hand om en vilsen doktorand. Till mina vapendragare Erik Spinnel, Erland Björklund, Karin Wiberg, Kristina Frech Irgum och Sune Sporring vill jag rikta ett särskilt stort tack för er ovärderliga medverkan på färden. Jag uppskattar våra samarbeten och är glad för alla konstruktiva diskussioner, bränsle till farkosten som berett nya vägar. Thank you my friends at University of California, Davis: Bruce Hammock, Shirley Gee, Mikaela Nichkova, Mike Denison, and Dave Baston. I am forever grateful for your hospitality. I have learned so much from collaborating with you, especially the importance of being open-minded and not to be afraid of conquering new territories. Thank you Beatriz Brena, University of Uruguay, for giving me a piece of your land to investigate. I am happy to collaborate with you! Jag är också glad över delaktigheten i Marksaneringscentrum Norr (MCN) och de landvinningar som åstadkommits i nätverkets regi. Tack till alla medlemmar för intressanta möten som tvingat mig att lyfta blicken från kartan och skåda verkligheten för en stund! För att ha bekostat biljetten vill jag tacka Forskningsrådet för miljö, areella näringar och samhällsbyggande (FORMAS), Centrum för miljövetenskaplig forskning (CMF), Civilingenjörsförbundets miljöfond och Wallenberg-stiftelsen. Slutligen finns det en handfull personer som delat så mycket mer med mig än bara de senaste årens expedition. Ni har funnits med mig under alla resor som jag företagit mig och det har alltid gått lika bra med er vid min sida. Mamma Berit, pappa Lars-Åke, syster Sofia och bror Jonas, tack för allt ert stöd! Älskade Anton och Alvar, tack för att ni delar era liv med mig! Nu ska vi tillsammans resa mot nya, spännande mål …

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Summary in Swedish

Snabb provupparbetning samt bioanalytisk detektion för effektiv analys av organiska miljögifter

Vid kartläggning av utbredning och förekomst av organiska miljögifter måste ofta stora mängder prover prepareras och analyseras. Vanligtvis används metoder som ger detaljerad information, men som också är dyra och tidskrävande. Därför finns ett stort behov av enklare tekniker. Arbetet som ligger bakom denna avhandling syftade till att undersöka användbarheten hos smidigare tekniker än de konventionella för provupparbetning samt för detektion med hjälp av bioanalytiska tekniker. Till att börja med nyttjades en förenklad extraktionsteknik tillsammans med en antikroppsbaserad detektionsteknik (ELISA) för att analysera polycykliska aromatiska kolväten (PAHer) i förorenade jordprover. Det visade sig att det finns ett samband mellan hur PAHerna ser ut och graden av korsreaktivitet (antikroppsigenkänning) hos de ämnen som förekommer tillsammans med de prioriterade PAHerna. Deras bidrag till skillnaderna mellan resultaten från ELISA-analys och instrumentell analys med en gaskromatograf kopplad till en masspektrometer (GC-MS) är dock liten. Istället berodde dessa skillnader på de prioriterade PAHernas olika korsreaktivitet samt på den begränsade extraktionseffektiviteten som noterades inför ELISA analysen. Förutom PAHer, så undersöktes också dioxiner. Både dioxinförorenad fiskolja, fiskmjöl och jord analyserades med hjälp av ELISA-teknik samt med två cellbaserade tekniker, nämligen CAFLUX och CALUX. Dessutom utvecklades förbättrade provupparbetningstekniker baserade på accelererad lösningmedelsextraktion (ASE). Då ASE användes med en integrerad kolfälla (ASE-C) tillsammans med CAFLUX för att bestämma dioxininnehållet i fiskolja och fiskmjöl stämde resultaten överens med de från konventionella metoder. Även upparbetning av dioxinförorenade jordprover med hjälp av ASE-C och efterföljande ELISA-analys gav goda resultat. Slutligen rapporteras det i avhandlingen om den första jämförelsen av dioxinanalys med hjälp av CAFLUX, CALUX, ELISA och GC-MS på samma upparbetade extrakt från förorenade jordprover. De bioanalytiska teknikerna var tillräckligt bra för att användas i arbetet med att sålla ut jordprov med högre dioxinhalt än 1000 pg/g (ett gränsvärde som amerikanska naturvårdsverket rekommenderar). Sammanfattningsvis är de undersökta teknikerna lovande komplement till den gängse analysen, men mer forskning behövs innan de tillämpas i stor skala.

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