1. science
2. ecology
3. pollution

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
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
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
i
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
ii
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.
iii
Abbreviations
AHH
AhR
Arnt
ASE
ASE-C
CAFLUX
CALUX
CR
DDE
DDT
DR-CALUX
DRE
ECD
EGFP
ELISA
EROD
EU
GC
HR
IARC
Kow
LC
MFO
MS
PAC
PAH
PCB
PCDD
PCDF
POP
QA
QC
SD
SEPA
TCDD
TEF
TEQ
TOF
USEPA
WHO
Aryl hydrocarbon hydroxylase
Aryl hydrocarbon receptor
AhR nuclear translocator
Accelerated solvent extraction
ASE with an integrated carbon trap
Chemically activated fluorescent gene expression
Chemically activated luciferase gene expression
Cross reactivity
1,1-dichloro-2,2-bis(4-dichlorodiphenyl)ethylene
1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane
Dioxin responsive chemically activated luciferase gene
expression
Dioxin-responsive element
Electron capture detector
Enhanced green fluorescent protein
Enzyme-linked immunosorbent assay
Ethoxyresorufin-O-deethylase
European Union
Gas chromatography
High resolution
International Agency for Research on Cancer
Octanol-water partitioning coefficient
Liquid chromatography
Mixed function oxidase
Mass spectrometry
Polycyclic aromatic compound
Polycyclic aromatic hydrocarbon
Polychlorinated biphenyl
Polychlorinated dibenzo-p-dioxin
Polychlorinated dibenzofuran
Persistent organic pollutant
Quality assurance
Quality control
Standard deviation
Swedish Environmental Protection Agency
2,3,7,8-tetrachlorodibenzo-p-dioxin
Toxic equivalency factor
Toxic equivalency
Time of flight
United States Environmental Protection Agency
World Health Organization
iv
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
1.
I N T R O D U C T I O N
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 indepth 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.
2
1.
Sampling
Pre-treatment
Extraction
Clean-up
A vast
amount of
samples
None
Agitation with
solvent
Filtration
A selection
of samples
Drying and
sieving
I N T R O D U C T I O N
Detection
Bioanalytical
techniques
Immunoassays
Bioassays
Multi-step
columns
Soxhlet
Instrumental
techniques
GC-MS
Semiquantitative
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).
3
1.
I N T R O D U C T I O N
4
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 (1,1dichloro-2,2-bis(4-dichlorodiphenyl)ethylene) is toxic. DDT and DDE are
both persistent and currently DDE levels in Sweden generally exceed those of
5
2.
P O L L U T A N T S
C O N S I D E R E D
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
2×10
-7
-5
0.001
0.02
8.4×10
Vapor pressure (Pa)
2×10
-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).
6
2.
P O L L U T A N T S
C O N S I D E R E D
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 PAHintermediates might form, possibly inducing cancer (IARC, 1983). As an
7
2.
P O L L U T A N T S
C O N S I D E R E D
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).
1.
cyt P450
O
Benzo[a]pyrene
OH
Benzo[a]pyrene-7,8-oxide
7-hydroxybenzo[a]pyrene
2.
Glucuronide
Sulphate ester
Gluthathione conjugate
HO
OH
trans-7,8-dihydroxy-7,8dihydrobenzo[a]pyrene
cyt P450
O
HO
OH
trans-7,8-dihydroxy-7,8dihydrobenzo[a]pyrene-9,10-oxide
BINDING TO DNA
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).
8
2.
P O L L U T A N T S
C O N S I D E R E D
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).
Clx
9
8
7
6
O
O
1
Clx
Cly
8
2
Cly
1
9
2
3
7
3
6
4
O
4
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 coworkers 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
9
2.
P O L L U T A N T S
C O N S I D E R E D
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 speciesand 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).
AhR ligands
AhR
Translocation
DRE
Cytochrome
P450 1A1
Luciferase
EGFP
EROD
CALUX
CAFLUX
…
Other
proteins
Figure 5. Simple schematic diagram of the events in the cell initiated by aryl
hydrocarbon receptor (AhR) ligands (such as dioxin molecules). The ligand-ArntAhR-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.
10
2.
P O L L U T A N T S
C O N S I D E R E D
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.
11
2.
P O L L U T A N T S
C O N S I D E R E D
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.
WHO-TEF
WHO-TEF
1998
2005
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
Compound
a
Paper IV (Nording et al., 2006)
n.a. = not analysed
12
CR
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.
13
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
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 nonindustrialized 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 byproducts 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 creosotecontaminated 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
14
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
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
a
benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene,
indeno[1,2,3,cd]pyrene, and dibenz[a,h]anthracene
b
naphthalene, 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 dioxincontamination 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).
1
Non-ortho PCBs: 77, 81, 126, and 169; Mono-ortho PCBs: 105, 114, 118, 123, 156,
157, 167, and 189
15
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
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 dioxincontaminated 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
16
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 semiquantitative 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.
17
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
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
18
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
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.
Pump
Load cell
Fill cell with solvent
Pressurize and heat cell
Extraction
cell
Oven
Solvent
Nitrogen
Hold at elevated pressure
and temperature
Valve
Purge solvent from cell with
nitrogen
Collect extract
Collection vial
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.
19
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
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
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.
20
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
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.
21
4.
S T E P S
R E Q U I R E D
T O
D E T E C T
22
P O L L U T A N T S
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).
23
5.
B I O A N A L Y T I C A L
T E C H N I Q U E S
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
Enzyme tracer
Primary Ab
Secondary Ab
Substrate
Product
Indirect competitive ELISA
Direct competitive ELISA
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
24
5.
B I O A N A L Y T I C A L
T E C H N I Q U E S
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.
0.5
Absorbance
0.4
0.3
0.2
0.1
0
0.01
0.1
1
10
100
1000
10000
100000
Concentration (pg/ml)
AbsMax
Slope
IC50
AbsMin
R2
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
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.
25
5.
B I O A N A L Y T I C A L
T E C H N I Q U E S
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 crossreactivity 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 crossreactants. 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.
26
5.
B I O A N A L Y T I C A L
T E C H N I Q U E S
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).
AHH
OH
Benzo[a]pyrene
3-Hydroxybenzo[a]pyrene
396 nm/522 nm
C2H5O
O
O
EROD
N
HO
O
O
N
7-Ethoxyresorufin
Resorufin
550 nm/585 nm
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.
27
5.
B I O A N A L Y T I C A L
T E C H N I Q U E S
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 timedependent 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 dioxinresponsive, 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.
28
5.
B I O A N A L Y T I C A L
T E C H N I Q U E S
DREs
pGudLuc1.1
(9225 bp)
Luciferase
gene
Wild-type cell
Plasmid with DRE
TRANSFECTION
Recombinant cell
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.
29
5.
B I O A N A L Y T I C A L
T E C H N I Q U E S
40000
35000
EGFP induction
30000
25000
20000
15000
10000
5000
0
0,01
0.01
0,10
0.1
1,00
1.0
10,00
10
100,00
100
1000,00
1000
TCDD (pg/well)
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).
30
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 cleanup system (Abad et al., 2000; Eljarrat et al., 2001). With this approach they
31
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
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 highthroughput 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.
Sampling
Soil, biota,…
Rapid sample preparation
Bioanalytical
technique
Below
guidance
value
Above guidance
value
Confirmatory
technique
Confirmation
Data base
Mapping, monitoring…
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.
32
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
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.
Sampling
Pre-treatment
Extraction
Clean-up
Detection
Creosote &
gasworks
soils
None
Agitation with
solvent
Filtration
Immunoassay
Fish oil and
fish meal
Homogenization
and mixing with
Na2SO4
Chlor alkali
soils...
Homogenization
and sieving
Chlor alkali
soils...
Homogenization
and sieving
Integrated extraction and
clean-up with ASE
Soxhlet
Multi-step
columns
CAFLUX
PCDD/Fs in
Paper III
Immunoassay
PCDD/Fs in
Paper IV
or integrated extraction and
clean-up with ASE
Soxhlet
Multi-step
columns
PAHs in
Papers I and II
CALUX
CAFLUX
Immunoassay
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.
33
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
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 prioritypollutant 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 crossreactivity 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 crossreactivities 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
34
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
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.
35
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
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.
36
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;
c®
• the GC-MS and PAH RIS results agree well, considering the less
efficient extraction prior to ELISA analysis and the differences in crossreactivities 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.
37
7.
C O N C L U D I N G
R E M A R K S
Table 5. Some advantages and limitations of immunoassays, CALUX, and
CAFLUX. The features of CALUX also apply to CAFLUX.
Advantages
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.
Easy to field-adapt.
A biologically relevant sum of
AhR ligands is reported.
Cheaper than CALUX.
User friendly.
Limitations
Potentially infinite production of cells. Measurements in real-time.
Cross-reactivity with non-target
compounds.
Restrictions for use due to
genetic modification.
Cumulative signal.
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 userfriendly 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.
38
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 Wallenbergstiftelsen.
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 …
39
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.
40
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