Assessment of environmental pollutants

in humans from four continents

Exposure levels in Slovakia, Guinea-Bissau,

Nicaragua and Bangladesh

Linda Linderholm

Department of Materials and Environmental Chemistry

Stockholm University

Stockholm 2010

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Doctoral Thesis 2010 Department of Materials and Environmental Chemistry Stockholm University SE-106 91 Stockholm Sweden

Abstract Humans are continuously exposed to complex mixtures of anthropogenic chemicals. This thesis focus on human exposure to persistent organic pollutants (POPs). POPs ability to bioaccumulate and biomagnify together with the extensive historical use of POPs in e.g. agriculture and industry have resulted in detection of these compounds in humans and animals from all over the world. Adverse health effects caused by POPs are of particular concern for newborns and young individuals.

The objective of this thesis is to assess human exposure to a selected set of POPs and their metabolites. More specifically, one aim of my thesis is to determine the exposure to polychlorinated biphenyls (PCBs) and in particular their methylsulfonyl and hydroxylated metabolites in humans from a “hot-spot” area of PCB contamination in eastern Slovakia. The maternal transfer of these chemicals is studied. Further, another specific aim is to determine occurrence, levels and, when possible, temporal trends of POPs in children and adults from three developing countries, Nicaragua, Guinea-Bissau and Bangladesh.

High concentrations of PCBs and their metabolites are shown in men and women from Michalovce in eastern Slovakia. Placental transfer of methylsulfonyl-metabolites of PCBs and 4,4’-DDE was observed for the first time. Decreasing temporal trends of the majority of POPs are shown in serum from a cohort of policemen from Guinea-Bissau. In contrast, the levels of polybrominated diphenyl ethers (PBDEs) show an increasing time trend. Within five years, decreasing levels of POPs were also shown in children working and living at a waste disposal site in Nicaragua. Children working and living at waste disposal sites in Bangladesh have considerably lower levels of POPs compared to the children from Nicaragua except for 4,4’-DDT and 4,4’-DDE that are present at very high concentrations, indicating ongoing use of technical DDT.

There are many studies on levels and trends of environmental pollutants from the developed industrial countries in the world, whereas data from developing countries is still scarce. This thesis contributes to partly fill this data gap since it includes assessments of POPs in children and adults from four countries on four continents. © Linda Linderholm ISBN 978-91-7447-136-6 Universitetsservice US-AB, 2010

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Till min pappa

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List of papers This thesis is based on following papers:

I. Hovander L., Linderholm L. , Athanasiadou M., Athanassiadis I., Bignert A., Fängström B., Kocan A., Petrik J., Trnovec T., Bergman Å. (2006). Levels of PCBs and their metabolites in the serum of residents of a highly contaminated area in eastern Slovakia. Environ. Sci. Technol. 40, 3696-3703.

II. Linderholm L. , Park J.S., Kocan A., Trnovec T., Athanasiadou M.,

Bergman Å., Hertz-Picciotto I. (2007). Maternal and cord serum exposure to PCB and DDE methyl sulfone metabolites in eastern Slovakia. Chemosphere. 69, 403-410.

III. Linderholm L. , Biague A., Månsson F., Norrgren H., Bergman Å.,

Jakobsson K. (2010). Human exposure to persistent organic pollutants in West Africa - A temporal trend study from Guinea-Bissau. Environ. Int. 36, 675-682.

IV. Cuadra S.N., Linderholm L. , Athanasiadou M., Jakobsson K. (2006).

Persistent organochlorine pollutants in children working at a waste-disposal site and in young females with high fish consumtion in Managua, Nicaragua. Ambio. 35, 109-116. © Royal Swedish Academy of Sciences.

V. Linderholm L., Cuadra S., Bergman Å., Jakobsson K. (2010).

Brominated and chlorinated persistent organic pollutants (POPs) in children working and living at two waste disposal sites in Nicaragua – a revisit. Manuscript.

VI. Linderholm L., Jakobsson K., Lindh T., Zamir R., Shoeb M., Nahar N.,

Bergman Å. (2010). Environmental exposure to POPs and heavy metals in urban children from Dhaka, Bangladesh. Manuscript.

Permissions to print paper I, II, III and IV were kindly obtained from the publishers.

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The following studies have also contributed to the contents of this thesis:

• Park J.S., Linderholm L. , Charles M.J., Athanasiadou M., Petrik J., Kocan A., Drobna B., Trnovec T., Bergman Å., Hertz-Picciotto I. (2007). Polychlorinated biphenyls and their hydroxylated metabolites (OH-PCBs) in pregnant women from eastern Slovakia. Environ Health Perspect. 115, 20-27.

• Park J.S., Bergman Å., Linderholm L. , Athanasiadou M., Kocan A.,

Petrik J., Drobna B., Trnovec T., Charles M.J., Hertz-Picciotto I. (2008). Placental transfer of polychlorinated biphenyls, their hydroxylated metabolites and pentachlorophenol in pregnant women from eastern Slovakia. Chemosphere. 70, 1676-1684.

• Park H.Y., Park J.S., Sovcikova E., Kocan A., Linderholm L. ,

Bergman Å., Trnovec T., Hertz-Picciotto I. (2009). Exposure to hydroxylated polychlorinated biphenyls (OH-PCBs) in the prenatal period and subsequent neurodevelopment in Eastern Slovakia. Environ. Health Perspect. 117, 1600-1606.

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Cover pictures by:

Kristina Jakobsson, Tomas Trnovec and Hans Norrgren

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Table of contents Abstract .............................................................................................................. ii List of papers..................................................................................................... iv Table of contents.............................................................................................. vii Abbreviations..................................................................................................viii 1 Introduction............................................................................................... 1

1.1 Aim of the thesis ................................................................................ 4 2 Persistent organic pollutants (POPs) -Sources and exposure ............... 5

2.1 PCBs................................................................................................... 5 2.2 DDT ................................................................................................... 8 2.3 HCHs................................................................................................ 10 2.4 PBDEs.............................................................................................. 12 2.5 PCP................................................................................................... 13

3 Analytical methodology.......................................................................... 15 3.1 Sample selection .............................................................................. 15 3.2 Analytical methods........................................................................... 16

3.2.1 Lipid determination ................................................................. 16 3.2.2 Extraction and clean-up........................................................... 17 3.2.3 Identification and quantification.............................................. 20

3.3 Quality control (QC) ........................................................................ 22 4 Toxicokinetics and thyroid disruption.................................................. 24

4.1 ADME..............................................................................................24 4.1.1 Absorption............................................................................... 24 4.1.2 Distribution.............................................................................. 25 4.1.3 Metabolism.............................................................................. 26 4.1.4 Excretion ................................................................................. 28

4.2 Thyroid disruption............................................................................ 29 5 Results and discussion ............................................................................ 32

5.1 “Hot-spot” exposure in Slovakia (Paper I and II) ............................ 32 5.2 Temporal trends in policemen from Guinea-Bissau (Paper III)....... 37 5.3 Children’s exposure to POPs in Nicaragua (Paper IV and V) ......... 38 5.4 Children’s exposure to POPs in Bangladesh (Paper VI).................. 41

6 Concluding remarks and future efforts ................................................ 44 7 Acknowledgement................................................................................... 47 8 References................................................................................................ 49

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Abbreviations ADME Absorption, distribution, metabolism, excretion BCF Bioconcentration factor DDT Dichlorodiphenyltrichloroethane (technical product) 4,4’-DDT 2,2-Bis(4-chlorophenyl)-1,1,1-trichloroethane 4,4’-DDE 2,2-Bis(4-chlorophenyl)-1,1-dichloroethene ECD Electron capture detector ECNI Electron capture negative ionization EDC Endocrine disruptor chemical EI Electron ionization GC Gas chromatography HCHs Hexachlorocyclohexanes HRMS High resolution mass spectrometry IRS Indoor residual spraying LLE Liquid-liquid extraction LOD Limit of detection LOQ Limit of quantification LRMS Low resolution mass spectrometry LVI Large volume injector MAP Mercapturic acid pathway MeO-PBDEs Methoxylated polybrominated diphenyl ethers MeSO2-DDE Methylsulfonyl-DDE or DDE methyl sulfone MeSO2-PCBs Methylsulfonyl-PCB or PCB methyl sulfones MS Mass spectrometry OH-PBDEs Hydroxylated polybrominated diphenyl ethers OH-PCBs Hydroxylated polychlorinated biphenyls PBDEs Polybrominated diphenyl ethers PCBs Polychlorinated biphenyls PCDDs Polychlorinated dibenzo-p-dioxins PCDFs Polychlorinated dibenzofurans PCP Pentachlorophenol POPs Persistent organic pollutants PTV Programmed-temperature vaporizer SIM Selected ion monitoring SPE Solid phase extraction T3 3,3’,5-Triiodothyronine hormone T4 Thyroxine TSH Thyroid stimulating hormone TTR Transthyretin QC Quality control

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1 Introduction

Humans are exposed to a complex mixture of chemicals such as pesticides,

chemicals in materials, articles and products, and by-products from industrial

processes, energy production and combustion. Around 30 000 chemicals have

an annual global use above one ton [1,2]. For many of these chemicals, the

environmental fate or toxicity is not known. Persistent organic pollutants

(POPs) is one group of chemicals whose toxicity, occurrence and fate in the

environment has been well studied. POPs have been in focus for decades due to

their very slow degradation rates and the extensive historical use in agriculture,

public health and industry. Humans are exposed to these chemicals via food, air

and dust. POPs do undergo long distance transport and are therefore detected in

biota, animals and humans from all over the world even in remote areas like the

Arctic where these compounds never have been used [3,4]. The stability and

lipophilic properties of POPs make them bioaccumulate and biomagnify. The

POPs are accumulated in fatty tissues in animals and humans since the

metabolism and excretion is very slow, which leads to a constant internal

exposure to these compounds. Also, the higher up in the food chain, the higher

concentrations of POPs are found. POPs are also considered to be toxic to

humans and wildlife.

The Stockholm Convention on Persistent Organic Pollutants is a global treaty

that was adopted in Stockholm in 2001 [5] and since then 172 countries have

ratified the convention. The goal of the convention is to protect human health

and the environment from persistent organic chemicals that could cause adverse

health effects such as dysfunctional reproductive systems, birth defects and

cancer [5]. Today, there are twenty-one POPs that should be eliminated or

restricted (use and production) according to the Stockholm Convention [5]. All

twenty-one POPs and three new chemicals proposed to be included as POPs, are

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listed in Table 1.1. Among the POPs listed in Stockholm convention,

dichlorodiphenyltrichloroethane (DDT), polychlorinated biphenyls (PCBs),

hexachlorocyclohexanes (HCHs) and polybrominated diphenylethers (PBDEs)

as well as possible metabolites will be discussed in this thesis.

Table 1.1. A list of the 21 POPs listed in the Stockholm Convention and three proposed chemicals to be included.

Annex A (elimination) Use/Source Aldrin Pesticide Chlordane Pesticide Chlordecone Pesticide Dieldrin Pesticide Endrin Pesticide Heptachlor Pesticide Hexabromobiphenyl Industrial chemical Hexa- and hepta-bromodiphenyl ether Industrial chemical Hexachlorobenzene (HCB) Pesticide/ Industrial chemical Alpha hexachlorocyclohexane (α-HCH) Pesticide/ By-product Beta hexachlorocyclohexane (β-HCH) Pesticide/ By-product Lindane (consists mostly of γ –HCH) Pesticide Mirex Pesticide Pentachlorobenzene Pesticide/ Industrial chemical Polychlorinated biphenyls (PCB) Industrial chemical Tetra- and penta-bromodiphenyl ether Industrial chemical Toxaphene Pesticide Annex B (restriction) DDT Pesticide Perfluorooctane sulfonic acid and salts and perfluorooctane sulfonyl fluoride

Industrial chemical

Annex C (unintentional production) Polychlorinated dibenzo-p-dioxins (PCDD) By-product Polychlorinated dibenzofurans (PCDF) By-product Hexachlorobenzene (HCB) By-product Pentachlorobenzene By-product Polychlorinated biphenyls (PCB) By-product Proposed chemicals Short-chained chlorinated paraffins Industrial chemical Endosulfan Pesticide Hexabromocyclododecane (HBCD) Industrial chemical

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The number of exposure assessment studies focusing on POPs in humans is

almost uncountable when it comes to reports from developed industrialized

countries, whereas information on levels and trends of POPs in developing

countries is scarce as reviewed in e.g. [6,7]. Further, there are only very few

studies on POPs in children. This lack of data is unfortunate since children

cannot be seen as small adults, neither with respect to exposure assessments nor

for health risk estimates. Also, POP exposure to the fetus is an important subject

since the developing fetus might be particularly sensitive to chemicals.

The rich world’s constant demand for the latest technology and the fast and

extensive development of new articles such as electronics, toys, clothes and

home furnishing creates a market for chemicals and enormous amounts of

waste. This waste contains a large number of chemicals that, if they end up in

nature, could cause adverse effects in environment, animals and humans.

Knowledge, functioning infrastructure and financial resources are some of the

requirements for proper waste handling, but developing countries often lack

these requirements. Poor handling of waste may lead to extensive leakage of

toxic chemicals from “hot-spots” and waste disposal sites, leading to

contamination of the air, the surrounding lands, drinking-water and people that

work or live close to, or at, these sites (Paper I-II and IV-VI). In addition,

electric and electronic waste (e-waste) is exported from rich, industrial countries

to developing countries in Asia and Africa, in particular [8-10]. This has lead to

very high concentration of for instance flame retardants in the people that are

working with e-waste [11,12], in soil from dumping sites [13] and in biota [14].

The Basel Convention (www.basel.int) is a global agreement that address

problems and challenges posed by hazardous waste. The key objectives for the

convention are to minimize the generation of hazardous waste and to reduce

transboundary movement of this waste. Paper IV, V and VI in this thesis deal

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with children’s exposure to POPs and heavy metals as a result of their work at

waste disposal sites.

1.1 Aim of the thesis

The main objective of this thesis is to assess human exposure to a selected set of

POPs and their metabolites in four countries from four continents. One specific

aim of the thesis (Paper III-VI) is to determine occurrence, levels and, when

possible, temporal trends of POPs in three developing countries from three

continents; Nicaragua, Guinea-Bissau and Bangladesh. A specific aim of the

thesis is exposure of the selected POPs in children/young humans that were

working and living close to waste disposal sites (Paper IV-VI). Another specific

aim is to study the exposure and also the maternal transfer of POPs and their

metabolites in a PCB contaminated area, a “hot-spot”, in eastern Slovakia

(Papers I-II).

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2 Persistent organic pollutants (POPs) -Sources and exposure

A countless number of studies have dealt with exposure to POPs over the years.

In this thesis, I will only focus on the POPs included in the articles (Papers I-VI)

and focus on the aspects that are of interest for the conclusions that can be

drawn from this thesis.

2.1 PCBs

PCBs have been produced and used since 1929 [15]. There are 209 theoretical

PCB congeners depending on degree of chlorination and differences in chlorine

substitution pattern. The structure of one of the PCB congeners (CB-153) is

shown in Figure 2.1. PCBs are considered stable because of their general

resistance to degradation and low chemical reactivity. Properties such as low

electrical conductance and low flammability made PCB products suitable in

numerous applications; for instance as insulating oil in transformers and

capacitors, as additives in sealants for constructions, and as heat transfer agents

among many other areas [15]. PCBs were detected in marine animals for the

first time in 1966 by Sören Jensen [16,17]. The fact that an industrial chemical

that was not deliberately spread in the nature could be found at high

concentrations in wild animals was alarming. In 1968, rice bran oil was

contaminated with PCB oil in Japan leading to poisoning of over 1800 people

[18]. This incidence gave rise to concerns of possible adverse effects that this

chemical could cause humans and animals, and contributed to the knowledge on

PCB toxicity.

As a consequence of the chemical properties of PCBs (e.g. their persistence),

their widespread use also in non-closed applications, the over time large

volumes being handled, and not the least the ignorance of their environmental

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fate, made PCBs ubiquitous environmental contaminants long before they were

discovered in humans, wildlife and abiotic matrices. PCB was restricted or

banned in most countries in the 1970’s, but the continuous use of PCB

containing products and equipment for years after the ban has contributed to the

relatively high PCB levels still found in biota. Today, decades after the ban,

PCBs are still one of the major POPs found in humans, wildlife and the

environment all over the world even though their concentrations have decreased

in general [19-22].

In addition to the general environmental contamination of PCBs there are also

some “hot-spot” areas with exceptionally high contamination of PCBs, for

instance in eastern Slovakia where 22 000 tons of PCBs were produced between

1959 and 1984. Improper storage and release of untreated contaminated waste

water from the factory polluted the surroundings and the nearby river and lake

with PCBs and high concentrations of PCBs have been found in air, soil, water,

fish, meat and human blood from the area [23-25]. In paper I and II, we

measured PCBs and their metabolites in serum from men and women and in

maternal and cord serum from Michalovce and Svidnik districts in eastern

Slovakia. The former being the “hot-spot” and the latter a control area holding

background levels of PCBs [26].

Although PCB has been banned and the production ceased many years ago,

there are a lot of old equipment containing PCBs still in use, especially in

developing countries. In for instance Bangladesh, there are 400 000

transformers in use today, of which an unknown percentage still contain PCB

oil [27] (Paper VI). It is likely that this is true also for other developing

countries. If old equipment ends up in landfills or poorly controlled waste

disposal sites, there might be an upcoming problem with PCBs in the future in

these countries. It has been reported that oil containing PCBs has been sold,

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mixed with cooking oil, finally ending up in roadside food shops selling deep

fried food in Bangladesh (unconfirmed information in The Daily star, Dhaka,

Bangladesh, 29 Dec. 2006). In some African countries, transformers are

vandalized and emptied of their transformer oil. The oil is either mixed with

diesel and sold as fuels, or mixed with vegetable oil and sold as cooking oil

(unconfirmed information in The Post Newspapers, Zambia, 29 Mar. 2010).

PCBs are metabolized to hydroxylated PCBs (OH-PCBs) and methylsulfonyl-

PCBs (MeSO2-PCBs) as further discussed in section 4.1.3. Hydroxylated

metabolites of PCBs were identified in the environment for the first time in seal

and guillemot droppings from the Baltic sea in the 1970’s [28]. In 1994 it was

shown that certain OH-PCB congeners had a strong selective retention in blood

from PCB dosed rats and in seals from the Baltic Sea and in humans [29]. This

retention is explained by the strong binding affinity of OH-PCBs to

transthyretin (TTR), one of the transport proteins for the hormone thyroxine

(T4) in blood [30,31]. OH-PCBs have been analyzed in several human serum

studies [32-36] (Paper I) and in cord blood [37,38].

MeSO2-PCBs were found in the environment for the first time in 1976, in seals

from the Baltic Sea [39]. Since then have PCB methyl sulfones been detected in

various animals such as fish [40], birds [41] and polar bears [42]. Some MeSO2-

PCBs show a specific retention, for example MeSO2-PCBs with the sulfone

group in para-position bind specifically to uteroglobin, a secretory protein

found e.g. in Clara cells in the lung [43,44]. Specific retention of MeSO2-PCBs

to the kidneys has also been shown [44] and meta-substituted MeSO2-PCBs are

retained in the liver [45,46]. PCB methyl sulfones have also been detected in

human blood and milk as well as in several other tissues [46-50] (Paper I). In

Paper II, MeSO2-PCBs were found in human cord blood for the first time,

proving that these metabolites are able to cross the placenta.

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Cl

Cl

Cl

Cl

Cl

Cl

CB-153

O

Br

Br

Br

Br

BDE-47

CCl3

Cl Cl4,4’-DDT

CCl2

Cl Cl4,4’-DDE

CHCl2

Cl Cl4,4’-DDD

OH

Cl

Cl

Cl

Cl

Cl

PCP

H

Cl

Cl

H

ClCl

HCl

HH

Cl

H

α-HCH

H

H

H

H

ClCl

HCl

ClCl

ClH

H

Cl

Cl

Cl

ClCl

HCl

HH

HH

β -HCH γ-HCH

Figure 2.1. Some selected structures of the chemicals that have been assessed in this thesis.

2.2 DDT

DDT is an insecticide that has been widely used for malaria control and in

agriculture to control pest insects. It was synthesized for the first time in 1874

but not used in agriculture before its insecticidal properties were discovered in

the 1930’s. During the Second World War, DDT was used by the allied forces

to protect the soldiers from malaria and typhus and in 1945 DDT was released

to the commercial market [51]. The technical DDT mixture contains mainly 2,2-

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bis(4-chlorophenyl)-1,1,1-trichloroethane (4,4’-DDT) but 2,4’-DDT is also

present at about 15-20% [52]. In the environment, 4,4’-DDT is degraded to 2,2-

bis(4-chlorophenyl)-1,1-dichloroethene (4,4’-DDE) and 2,2-bis(4-

chlorophenyl)-1,1-dichloroethane (4,4’-DDD) (Figure 2.1). 4,4’-DDE is

extremely stable and is today the DDT compound found at the highest

concentrations, abiotically and in vivo. A 4,4’-DDT concentration as high or

close to as high as that of 4,4’-DDE is a sign of recent exposure to technical

DDT whereas higher concentration of 4,4’-DDE reveals mainly 4,4’-DDE

exposure, indicating main exposure via the diet.

Most developed countries banned DDT in the 1970’s. The main reason for the

ban was that DDT seemed to have negative effects on wildlife, for instance

were birds of prey and fish-eating birds decreasing dramatically in the 1960’s in

Western Europe and North America. Ratcliff and colleagues observed a

decrease in eggshell weight in certain birds of prey that seemed to occur after

the introduction and use of pesticides [53]. Later this eggshell thinning was

revealed to be caused by DDT exposure, as shown for instance in Swedish

white-tailed sea eagles [54]. DDT is one of the twelve original POPs listed in

the Stockholm convention in Annex B, i.e. it is a compound that should be

restricted. Still, WHO recommends indoor residual spraying (IRS) with DDT

together with use of impregnated bed nets for malaria control [55]. In 2008,

DDT was produced in three countries, India, China and the Democratic

People’s Republic of Korea. The global use was estimated to 4-5000 tons per

year with India as the main consumer [56]. Even though DDT is banned in e.g.

Nicaragua and Bangladesh, it is still possible to buy it on the black market in

Nicaragua and it is used in Bangladesh e.g. in the preparation of dried fish [57].

In Paper III-VI, we have measured high levels of DDT compounds in

Nicaragua, Bangladesh and Guinea-Bissau.

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4,4’-DDT is metabolized to DDE methyl sulfones (MeSO2-DDE) (see section

4.1.3). The main sulfones metabolite, 3-MeSO2-DDE, accumulates in fat tissue

but it also binds irreversibly to the adrenal cortex in both mice and human after

metabolic activation, causing cell death [58,59]. Irreversible binding of 3-

MeSO2-DDE to the adrenal cortex has also been observed in mice fetus [60]. 3-

MeSO2-DDE has been detected in several human tissues [46,48] (Paper I) and

in cord blood (Paper II).

The use of DDT is controversial, health risks and benefits of DDT have been

reviewed by for instance Rogan and Chen [61]. Their conclusion is that more

research about the human health risks of DDT is needed, and that the risks for

children might outweigh the benefits of DDT spraying. Turosov and colleagues

conclude that even though the actual and potential risk of DDT should not be

diminished, a total ban would be devastating for poor countries [62]. Very high

concentrations of DDTs were found in malaria vector-control workers from

South Africa [63] and in non-occupationally exposed men living in an endemic

malaria area in South Africa [64]. The DDT levels were correlated with low

sperm counts and sperm defects [63,64]. Reduced seminal parameters have also

been observed in non-occupational DDT exposed men from Mexico [65]. High

concentrations of DDTs were detected in blood from delivering women from

villages in South Africa were IRS with DDT takes place [66]. Human health

effects that have been connected with DDT exposure are, except for impaired

semen quality, for instance preterm births, reduced lactation length,

neurodevelopmental effects, and cancers [61,67].

2.3 HCHs

There are four major isomeric forms of hexachlorocyclohexanes; α-HCH, β-

HCH, γ-HCH and δ-HCH. HCHs have been used as insecticides in for instance

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agriculture, for fruit, seed and soil treatment and for lumber protection. They

have also been used medically on humans for treatment of lice. HCHs have

been used either as a technical mixture that consists mainly of α-HCH (65-70%)

followed by γ-HCH (14-15%) and β-HCH (7-10%), or as Lindane that consists

of the active isomer γ-HCH (99%) [68,69]. The HCH isomers have different

physical properties depending on the number of axial or equatorial chlorines.

The structures of α-HCH, β-HCH and γ-HCH are illustrated in Figure 2.1. The

gamma isomer has the highest vapor pressure, the lowest bioconcentration

factor (BCF) and the shortest half-life due to its three axial chlorines that

facilitates degradation [70,71]. β-HCH has all the chlorines in equatorial

positions and has therefore by far the highest BCF and is the most persistent and

metabolically inactive isomer resulting in a much longer half-life compared to

γ-HCH [70]. The use of Lindane instead of the technical HCH mixture is

therefore preferred but the other HCH isomers are formed as by-products in the

production of Lindane. For each ton of Lindane that is produced, 6-10 tons of

the other isomers are formed as by-products, so good waste management is of

great importance at these production sites to prevent the surrounding areas from

being contaminated with a HCH mixture [72]. Lindane, α-HCH and β-HCH

were added to the list of POPs in the Stockholm convention in 2009 [5]. Ratios

between HCH isomers can be used to find out whether the technical mixture or

Lindane has been used and how recent the HCH input is. Relatively high

concentration of γ-HCH indicates recent use of Lindane due to the relatively

short half-life of that isomer and low content in the technical HCH mixture.

Both α-HCH and γ-HCH are relatively volatile and are therefore more easily

evaporated and undergo long range transport to a larger extent than β-HCH,

hence leading to higher concentration of α -HCH in the Arctic. Global use and

contamination consequences of HCHs have been summarized by e.g. Li [73].

Levels of γ-HCH and β-HCH in Nicaragua and Bangladesh were determined in

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children’s serum in Paper IV and VI and changes of HCHs in human serum

over time in Guinea-Bissau are presented in Paper III.

2.4 PBDEs

Polybrominated diphenyl ethers (PBDEs) have been used as additive flame

retardants since the early 1970’s. The major uses have been in high-impact

polystyrene, acrylonitrile butadiene styrene (ABS) plastic, polyurethane foam,

textile coatings, wire and cable insulation and in electronics [74]. Three

commercial PBDE mixtures have been used; PentaBDE, OctaBDE and

DecaBDE. The names correspond to the number of bromines in the major

components of the technical mixtures. One of the major PBDE congeners,

BDE-47, is illustrated in Figure 2.1. PBDEs were among the nine new POPs

listed by the Stockholm convention in 2009 [5]. The PentaBDE and OctaBDE

mixtures are banned by the European Union since 2004 [75] and DecaBDE has

been eliminated for use in electronic equipment in the European Union since

2008 [76]. The production of PentaBDE and OctaBDE has ceased in the United

States [77] and major producers of DecaBDE in the U.S. have decided to phase-

out this chemical product [78].

The use of PBDEs in a wide range of materials and articles such as in furniture,

textiles and in home electronics has led to high levels of these compounds in

household dust [79]. This exposure pathway is different compared to other

POPs such as the PCBs (that mainly derives from the diet) and has led to higher

levels in small children than in adults (e.g. due to their “hand to mouth

activity”) [80-83]. Among the main toxicological effects related to PBDEs are

adverse effects on neurobehavioral development, the thyroid hormone system

and reproductive systems as reviewed by e.g. Darnerud [84] and Talsness [85].

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PBDEs can be metabolized to hydroxylated metabolites (OH-PBDEs). This has

been shown in rats dosed with single PBDE congeners or with PBDE mixtures

[86-90]. Metabolism of PBDEs to OH-PBDEs and brominated phenols has also

been shown in human hepatocytes in vitro by Stapleton and colleagues [91]. But

OH-PBDEs, and also methoxylated PBDEs (MeO-PBDEs), also occur

naturally, produced by sponges and algae [92-94]. In naturally produced OH-

PBDEs, the hydroxy group is often in ortho-position whereas the hydroxylated

PBDE metabolites seem to be substituted preferably in meta- or para-position

[95]. However, 6-OH-BDE-47 can be both a metabolite of BDE-47 and a

natural product. High concentrations of OH-PBDEs and PBDEs were reported

in children from a waste disposal site in Nicaragua that were investigated for

chlorinated POPs in paper IV, by Athanasiadou and colleagues [96]. A few

years later, new samples were collected from Nicaraguan children. These

samples revealed a marked decrease in PBDE concentration over time (Paper

V). Relatively low concentrations of PBDEs were detected in human serum

from Guinea-Bissau and Bangladesh (Paper III and VI).

2.5 PCP

Pentachlorophenol (PCP) is a pesticide that has been used mainly as a wood

preservative but also as an insecticide and as an herbicide [97]. It is more

volatile and water soluble than the other compounds discussed in this thesis and

rather than being accumulated and stored in body fat, PCP is bound to proteins

and the highest concentrations are found in liver and kidney [98]. During

combustion, PCP is known to form polychlorinated dibenzo-p-dioxins

(PCDDs). This is of great interest since Paper IV and V in this thesis deals with

children that are working and living at waste disposal sites in Nicaragua where

the waste is burned daily. PCP is not on the list of twenty-one POPs listed in the

Stockholm Convention but production and use has been prohibited in the

14

European Union (with some exemptions) since 1992. Pentachlorophenol is one

of the major halogenated phenolic compound found in human serum [34,37,99]

Paper IV. PCP inhibits the oxidative phosphorylation in the cells and is also

known to bind to TTR, one of the transport proteins for thyroxine in human

blood [97,100].

15

3 Analytical methodology

3.1 Sample selection

Sample selection is an important first step for assessment of anthropogenic

chemical exposure. Preferably we want to have a good participation rate and as

much information about the subjects as possible. Also, and most importantly,

we want to have large enough sample volumes to enable us to find and quantify

as many contaminants, and their metabolites, as possible. In the present thesis

this means that low concentrations of the analytes should be able to be

quantified. It may be difficult to get sufficient volumes of human blood (plasma

or serum) for analysis, especially when working with samples from children. It

is often desirable or even crucial to have volumes enough of samples to make

individual analysis when cause effect relations are to be established. Individual

analysis of samples adds valuable information on the distribution and variation

of the contaminants in a cohort or population. Such work makes it possible to

identify highly exposed or sensitive individuals.

If the available sample volumes are too small, individual analysis of the samples

is unfeasible, but pooling of the samples may then be a solution to the problem.

A pooled sample equals the mean concentration of all samples in the pool, but

there is no information on the distribution among the individuals included in the

pool, one or a couple of the individuals in the pooled sample might have

extreme values which will influence the value for the whole pool. As an

example, pooled human milk samples have been used to study time-trends of

POPs in mother’s milk [19,101,102]. When pooling samples, it is important to

consider which samples to pool i.e. determine which the important descriptors

are. In paper IV, we based the pooling on possible work at the waste disposal

site, living area and fish intake among children in the study. In paper III, we

16

based it on age and availability. A strength with pooled samples is that a larger

number of subjects can be analyzed compared to individual analysis when time

and costs for analyses are limiting factors.

3.2 Analytical methods

3.2.1 Lipid determination

Prior to extraction and clean up it is necessary to decide how the results are to

be reported for the work. Due to the lipophilic character of POPs they are to a

large degree associated with the lipids in the body and should therefore

preferably be presented on a lipid weight basis. Also, if the concentration is

given per gram of fat it is possible to compare POP concentrations between

different matrices such as serum, blood, milk or any other tissue. Total lipids in

serum are the sum of triglycerides, cholesterol and phospholipids. The lipid

weight (i.e. total lipids) in serum is either determined through gravimetric lipid

determination or via enzymatic lipid determination. The latter method is

preferable since gravimetric lipid determination is time consuming and requires

an experienced laboratory worker and a good extraction method that will extract

all the lipids from the matrix. The gravimetric method becomes particularly

uncertain when only small serum volumes are available.

The enzymatic method for estimation of lipid content in serum was established

by Akins and colleagues based on summation of cholesterol, free cholesterol,

triglycerides and phospholipids for estimation of total lipids in the sample [103].

However, whereas enzymatic measurements of triglycerides and cholesterol are

a routine procedure at most hospital laboratories, analysis of phospholipids is

usually not. Several publications have therefore calculated total lipids from

triglycerides and cholesterol only [104-106]. The formulas in these publications

17

are built on blood lipid content in adult men and women. Hence, it is not known

how well these equations work for estimation of total lipids in children’s serum.

Gravimetric lipid determination were used in Paper I, III and IV whereas

enzymatic lipid determination and total lipids calculation according to Covaci et

al. [104] have been used in Paper V and VI and Akins in Paper II [103].

Enzymatic and gravimetric lipid determination has been shown to correlate well

[107]. Laboratory quality control samples that are included in each batch of

samples in all projects serve as a control of the accuracy of the two lipid

determination procedures. To avoid errors in the lipid content independent of

method for assessing the lipid content, blood samples should preferably be

taken after over-night fasting to ensure equilibration of POPs and lipids in the

body. This may, however, be very difficult to accomplish in field studies.

3.2.2 Extraction and clean-up

Analytical methods used for analysis of POPs are based on extraction of the

substances of interest and separation of substance groups depending on

chemical properties of the analytes. Liquid-liquid extraction (LLE) is a common

extraction method where two liquid phases are used to extract and separate the

analytes from the matrix. Advantages with LLE methods are that they are

usually robust and it is possible to analyze a broad range of substances and their

metabolites in a large number of matrixes. For instance are LLE used for both

solid samples such as different types of tissues, dust and sludge and liquid

matrixes such as water, serum and milk e.g. [24,102,108-110]. Drawbacks with

LLE are that the methods are in general tedious and require large volumes of

organic solvents.

18

A modified version of the extraction and clean-up method for neutral and

phenolic organohalogen compounds in serum developed at the department has

been used in all studies included in this thesis [110]. A flow chart of the method

is shown in Figure 3.1. Neutral substances like the PCBs, HCHs and DDTs can

be analyzed from the same sample as phenolic compounds such as hydroxylated

metabolites of PCBs and PBDEs and chlorinated and brominated phenols. Also,

methylsulfonyl metabolites of PCBs and 4,4’-DDE can be separated from other

neutral compounds by partitioning with conc. sulfuric acid [39] or dimethyl

sulfoxide partitioning [111]. Further, the PBDEs can be separated from the

chlorinated compounds on a silica gel column and be analyzed separately. A

fast semi-automated extraction method based on the method designed by

Hovander et al. has been developed by Jones and colleagues [112].

Other extraction methods used for POPs in serum are based on solid phase

extraction (SPE) as described by e.g. Thomsen, Covaci and Sandau [113-115].

SPE methods are fast and usually require less volume of solvents. Even though

good results and recoveries has been reported in the literature, when I tested

SPE for analysis of POPs and their metabolites, it was difficult to obtain

consistent and high recoveries of all substance groups of interests (unpublished

work).

To separate phenolic substances from neutrals, a 0.5 M potassium hydroxide

solution in 50% ethanol has been used. The phenolic compounds are ionized,

partitioned into the water phase, and recovered by addition of acid [110]. Before

analysis by gas chromatography (GC), the phenols should preferably be

derivatized to improve their stability and to improve their chromatographic

behavior by making them less polar. One way to accomplish this is to methylate

the hydroxyl group with ethereal diazomethane. This method is used in all

papers included in this thesis. Diazomethane is very effective and the products

19

are stable methoxylated compounds. A disadvantage is that diazomethane is

carcinogenic and explosive, hence cautiousness is required. A special permit

from the Swedish work environment authority (Arbetsmiljöverket) is mandatory

for use of diazomethane.

Separation of neutral and phenolic compounds

Phenols Neutrals

Derivatization

H2SO4 treatment Neutrals

H2SO4:Silica gel column

GC-ECD

H2SO4:Silica gel column

GC-ECD

GC-MS (ECNI)

DMSO

MeSO2-metabolites

H2SO4 treatment

Denaturation and Extraction of Plasma

Silica gel column

GC-MS (ECNI)

PBDEs

Silica gel column

GC-MS (ECNI)

OH-PBDEs

90% H2SO4 + KOH Silica gel column

OH-PCBs, PCP

PCBs, DDTs, HCHs

Figure 3.1. General scheme of the extraction and clean-up procedure used in paper I-VI in this thesis.

There are other methods for derivatization of halogenated phenols as well, e.g.

iodopropane [116], acetonitrile/methanol/water/pyridine [117] and

pentafluorobenzoylchloride [118,119]. Underivatized phenols has also been

20

analyzed with liquid chromatography coupled to a mass spectrometer (LC/MS)

[117,120]. A drawback with analysis with LC/MS is that it is difficult to get as

good sensitivity and separation as you obtain by gas chromatography (GC).

Sulfuric acid partitioning and sulfuric acid treated silica gel has been used as

clean-up steps in Paper I-VI. Sulfuric acid is a destructive lipid removal

procedure to eliminate lipids. All compounds of interest in this thesis have been

shown to be stable in concentrated sulfuric acid. The hexachlorocyclohexanes,

4,4’-DDT and 2,4’-DDT are on the other hand susceptible to potassium

hydroxide, but good recoveries of these compounds were obtained with the

KOH concentration used (0.5 M) in the separation of the phenolic and neutral

compounds [110]. Activated silica gel has been used to separate PCBs from the

PBDEs after analysis of PCBs by GC coupled to an electron capture detector

(GC/ECD), but prior to PBDE analysis by GC coupled to a mass spectrometer

(GC/MS), since PCBs might co-elute and interfere with PBDE congeners [121].

3.2.3 Identification and quantification

Gas chromatography coupled to an electron capture detector (ECD) or a mass

spectrometer (MS) has been used for detection, identification and quantification

of the analytes in this thesis. GC/ECD is very sensitive for halogenated

compounds and it is inexpensive, robust, and user-friendly. On the other hand,

the identification is only based on retention time and this may cause some

problems with co-elutions. A selective and complete clean-up is essential. In

this thesis, GC/ECD has been used for analysis of chlorinated POPs such as the

DDTs, PCBs and HCHs as well as the OH-PCBs and PCP.

Gas chromatography coupled to mass spectrometry was used for the analysis of

brominated compounds (PBDEs) in paper III, V and VI and the methylsulfonyl

21

metabolite of PCB and DDE in paper I-II. We used electron capture negative

ionization (ECNI) and selected ion monitoring (SIM) scanning for the ions 79

and 81 (the bromine ions) for the PBDEs, which is a very sensitive method

[122]. However, using this detection method may still cause some problems

with co-elutions since this technique is not as selective as electron ionization

(EI) where the major ion is the molecular ion [M]+ and [M-2Br]+ [121,123].

Since the molecular ion is more stable for PCB and DDE methyl sulfones

compared to PBDEs, GC/MS (ECNI) scanning for [M]- and [M+2]- was used

for these compounds.

Large volume injection (LVI) is a valuable tool when concentrations of the

analytes may be low. This is also good when only very small sample volumes

are available. Instead of injecting about one microliter onto the GC column, up

to a hundred microliters can be injected on a programmed-temperature

vaporizer (PTV) injector. Extracts are injected into a cold injector with the split

exit open. The solvent is evaporated and as the temperature in the injector rises,

the split exit is closed. Large volume injections using PTV injectors have been

used for GC/MS analysis of brominated and chlorinated compounds such as

PBDEs and PCBs by for instance Covaci [124], Tollbäck [125] and Zhao [126].

A PTV injector coupled to a GC/ECD was successfully used for the analysis of

PCBs and pesticides in Paper III, V and VI.

In the present thesis, all samples were quantified by single point calibration

versus an external standard. The linearity of the response was checked by

injection of external standards at different concentrations. Surrogate standards

were used to make up for the losses during extraction and clean-up and injection

standards were added prior to instrument analysis to calculate the recovery of

the surrogate standards. The surrogate standard should be as closely related to

the analytes as possible. Accordingly, congeners of PCBs and PBDEs that are

22

not present in the samples may be used as surrogate standards. 13C-labelled

internal standards are preferable, but then high resolution mass spectrometry

(HRMS) is required [121,123]. Thomsen and colleagues found GC-LRMS

(ECNI) and GC-HRMS (EI) equally well suited for determination of PBDEs in

serum, plasma and milk samples [127].

3.3 Quality control (QC)

As a quality control, blank solvent samples were always included in each batch

of samples in this thesis. If there were any contaminations of the blanks, the

amount found in the blanks was subtracted from the amount in the samples.

Contamination is usually not a problem with the chlorinated POPs analysed for

in this thesis, but it can be problematic in PBDE analysis since PBDEs have

been used more recently in a variety of applications and are therefore present in

for instance electrical equipments and dust in the laboratory. This is especially a

problem when background levels are measured. To reduce PBDE contamination

of the samples, all sample preparations, extractions and clean-up have been

performed in a special laboratory built for trace analysis of chemicals. The

laboratory is cleaned every morning to reduce collection of dust and an airlock

must be passed and shoes changed before entering the clean room. All solvents

are of the highest quality available and all glass equipment is baked at 300°C

before use. Laboratory reference serum samples have also been included in each

batch of samples. These samples work as a quality control of the measured

concentrations of POPs.

The limit of detection (LOD) and the limit of quantification (LOQ) are

important factors in trace analysis. LOD is generally estimated as three times

the signal-to-noise ratio (S/N) in the chromatogram and LOQ as ten times the

S/N. LOD and LOQ can either be determined in low concentrations of

23

analytical standards or in spiked serum samples. This can be used when there is

no background contamination, which is when the compound of interest is not

present in the solvent blanks. This is usually the case in the analysis of

chlorinated POPs such as PCBs and DDTs. Some PBDE congeners on the other

hand are normally found in the blank solvent samples. I have used three times

the amount found in the blank solvent samples as LOQ. This way of determine

LOQ has also been used by others [128,129]. Sjödin et al. use three times the

standard deviation of the amount detected in the blanks as their LOD whereas

the LOQ is not defined [130]. There are many ways of dealing with LOD and

LOQ, but when reading articles on PBDE analysis it is often impossible to find

information on what the limits are, how they have calculated them and how

blank contaminations have been dealt with. That is indeed unfortunate.

24

4 Toxicokinetics and thyroid disruption

Toxicokinetics, i.e. absorption, distribution, metabolism and excretion (ADME)

of potentially toxic chemicals [131], is a key for how exogenous chemicals

behave in biota. In this thesis, the ADME in humans are of particular interest.

Exposure routes, distribution, metabolism excretion as well as thyroid

disruption of the POPs of interest for this thesis, will be discussed in this

chapter.

4.1 ADME

4.1.1 Absorption

Exposure pathway is the course a chemical takes from the source to the target

whereas absorption, or the exposure route, is the way a chemical enters the

target after contact. Inhalation, ingestion, dermal contact and placental transfer

are possible exposure routes. Factors that affect absorption are both

physiological like age, gender, genetics and health status as well as factors

related to human behavior such as life-style, socio-economic situation and diet.

In many industrial countries where several POPs were banned decades ago, the

major exposure route for POPs is through ingestion, i.e. food. PCBs and 4,4’-

DDE are accumulated in fat making ingestion of fatty fish a major source of

these chemicals [32,34,132,133]. In contrast, exposure to presently used

pesticides occurs via direct contact, inhalation or dermal contact as well as via

ingestion of contaminated water and food. For PBDEs, dust has proven to be an

important pathway of exposure and inhalation of dust particles, ingestion and

dermal uptake are main exposure routes [79,134]. This is especially true for

children due to their hand to mouth activity [135]. Very recently, a correlation

between the PBDEs in house dust and PBDE levels found on children’s hands,

25

using hand wipes, was presented for U.S. children [136]. Furthermore, hand

wipe residues (of PBDEs) as well as the number of times that the hands were

washed per day have been correlated with serum levels of PBDEs in adults

[137]. For people working and living at waste disposal sites where un-controlled

fires are common, the exposure to smoke and inhalation of particulate matters is

probably a major exposure route to POPs. Actual handling of dangerous waste

such as cleaning, disassembling and storing may also lead to dermal absorption

of at least some anthropogenic chemicals.

The fetus is exposed to chemicals via the umbilical cord and right after birth via

breast milk. POPs are known to both pass the placenta and to accumulate in

breast milk [138-141]. The lipid content in cord blood is low, which results in

lower total levels of lipophilic compounds that are incorporated in the blood

lipids. POP exposure from breastfeeding is higher than the exposure in utero,

but the intrauterine exposure seems to be more critical. Children’s and babies’

exposure are also different from adults’ exposure. For example, infants have a

very monotonous diet of breast milk and/or infant formula which affects their

exposure to chemicals. Children also have an increased food and water

consumption and inhalation volume per kilogram body weight as well as a

higher water content compared to adults. The fact that small children live close

to the ground and frequently put things in their mouth also influence their

exposure and make it different from a typical adult’s exposure.

4.1.2 Distribution

When POPs such as PCBs and DDTs are transported in blood they are

associated with lipoproteins and proteins such as albumin [142]. The

distribution of POPs depends mainly on their lipophilic character and due to

their stability and generally slow metabolism, they are to a high degree

26

accumulated in fatty tissues. The distribution may also depend on specific

retention to certain tissues caused by e.g. binding to macro molecules. This

binding could either be covalent and irreversible as in the case with MeSO2-

DDE in the adrenal cortex [58,59] or, reversible as exemplified by the binding

of para-substituted methylsulfonyl-PCBs to uteroglobin in lung [43,44] or the

binding of OH-PCBs to transthyretin in blood [30].

4.1.3 Metabolism

The metabolism of xenobiotics, such as PCBs, generally leads to formation of

more polar metabolites that are more readily excreted than the parent

compounds. However, sometimes the metabolism give rise to reactive

intermediates that may bind to biomacromolecules like DNA and proteins [143]

and also to lipids [90,144,145]. Metabolites may however be more persistent,

bioaccumulative and/or toxic than the original molecule as in the case of 4,4’-

DDE. Factors that influence the metabolism of chemicals include for instance

age, sex, diseases, genetic factors and diet. Hence, fetuses, neonates and also

elderly have a metabolism that is not comparable to the adults’.

The first step in PCB and PBDE metabolism is the formation of an arene oxide

mediated by cytochrome P-450 oxidation is [146]. The arene oxide will be

transformed to hydroxylated metabolites (OH-PCBs/OH-PBDEs) spontaneously

or to a dihydrodiol metabolite. The latter metabolite is formed via epoxide

hydrolase catalyzed reactions. An example of OH-PCB formation is illustrated

by the metabolism of CB-138 in Figure 4.1. OH-PCBs or OH-PBDEs are in

general further metabolized through conjugation with glucoronic acid or sulfate,

which significantly improves the water solubility and thereby facilitates the

excretion.

27

Cl

Cl

Cl

Cl

ClOH

Cl

Cl

Cl

ClCl

Cl

Cl

Cl

Cl

Cl

Cl

ClClO

O

P-450

1,2-shift

CB-138

4-OH-CB146

Cl

Cl

Cl

Cl

Cl

Cl

HO

Cl

Cl

Cl

Cl

Cl

ClOH

3’-OH-CB138 4’-OH-CB130

1,2-shift

Figure 4.1. Formation of hydroxylated metabolites of 2,2’,3,4,4’,5’-hexachlorobiphenyl

(CB-138).

Methylsulfonyl-metabolites of PCBs and DDE are formed when the arene oxide

formed by cytochrome P-450 react with the tripeptide glutathione (GSH)

forming a conjugate via glutathione-S-transferase. After the loss of water the

conjugate undergoes peptidase hydrolysis via the mercapturic acid pathway

(MAP) [147]. The sulfur-cysteine conjugate is excreted with the bile to the

intestine. The intestinal micro flora converts the sulfur-cysteine conjugate to a

thiol which can be excreted or undergo methylation in the intestinal mucosa.

The methylthio-PCB is then oxidized to a MeSO2-PCB by cytochrome P-450 in

the intestinal mucosa, or after transportation back to the liver. An example of

the formation of a PCB methyl sulfone is illustrated in Figure 4.2.To enable

reactions with glutathione, the arene oxide needs to be rather stable. All PCB

methyl sulfones found in the environment has a 2,5 or a 2,5,6 chlorine

28

substitution pattern since chlorines on both sides of the epoxide group has a

stabilizing effect.

CB-101 -glu

ScysCl

Cl

ClCl

Cl

Cl

Cl

ClCl

Cl

SHCl

Cl

ClCl

Cl

SCH3

ox ox

P-450

Mercapturic Acid PathwayMAP

C-S β-lyaseSAM

Intestinalmicroflora

Intestinalmucosa

Bile

IntestinalMucosaor liver

-gly

Cl

Cl

ClCl

Cl

SO2CH3

Cl

Cl

Cl

Cl

Cl

O ClSG

OH

Cl

Cl

Cl

Cl

SGCl

Cl

Cl

Cl

Cl

Cl

Cl

ClCl

Cl

P-450

Glutathione-S-transferase

-H2OGlutathione=GSH

CB-101 -glu

ScysCl

Cl

ClCl

Cl

Cl

Cl

ClCl

Cl

SHCl

Cl

ClCl

Cl

SCH3

ox ox

P-450

Mercapturic Acid PathwayMAP

C-S β-lyaseSAM

Intestinalmicroflora

Intestinalmucosa

Bile

IntestinalMucosaor liver

-gly

Cl

Cl

ClCl

Cl

SO2CH3

Cl

Cl

Cl

Cl

Cl

O ClSG

OH

Cl

Cl

Cl

Cl

SGCl

Cl

Cl

Cl

Cl

Cl

Cl

ClCl

Cl

P-450

Glutathione-S-transferase

-H2OGlutathione=GSH

Figure 4.2. Metabolic pathway for the formation of one of the two methylsulfonyl-isomers

formed as metabolites of 2,2’,4,5,5’-pentachlorobiphenyl (CB-101).

4.1.4 Excretion

POPs are excreted via urine or feces, either as the parent compound or after

metabolism. Feces were shown to be the major route of excretion for CB-101

and its metabolites in both mouse and mink by Klasson Wehler et al. [145]. The

feces contained except for CB-101, hydroxylated and methylsulfonyl-

metabolites, also lipid bound, non-extractable (i.e. covalently bound to macro

molecules) and water soluble metabolites. Örn and Klasson Wehler have

demonstrated that feces was also the major route of excretion for BDE-47 in rat,

while urine was the main excretion route in mouse [90].

29

4.2 Thyroid disruption

POPs are well known for their toxicity. A large number of cause – effect

relationships have been demonstrated including effects leading to cancers,

immunotoxicity, and impaired reproduction. Endocrine disruption is one known

POPs related effect that could lead to numerous endpoints [148]. In this chapter,

only effects on the thyroid hormone system and neurodevelopment will be

discussed.

Chemicals that alter the functions of the endocrine system and thereby cause

adverse health effects are known as endocrine disrupting compounds (EDCs).

Such chemicals may cause developmental, reproductive and neurological

effects at trace level concentrations in both humans and wildlife, and mixtures

of EDCs could have synergistic or antagonistic effects [148]. EDCs may either

act directly through binding to a receptor and activate or block its effect, or

indirectly by changing synthesis, secretion, transport, metabolism or elimination

of the endogenous hormones. The timing of exposure is very critical when

assessing possible effects of EDCs. The developing fetus and young children

are of particular concern when considering EDC exposure.

Both PCBs and PBDEs are suggested to affect the thyroid hormones as

reviewed by for instance Boas and colleagues [149], Brouwer et al. [150] and

Legler [151,152]. The hydroxylated metabolites of both PCBs and PBDEs bind

to transthyretin (TTR) which is one of the transport proteins for T4 in human

blood [30]. TTR is important for transport of T4 to the fetus and across the

blood-brain barrier. OH-PCBs can also inhibit the deiodinase activity,

preventing the transformation of T4 to the active 3,3’,5-triiodothyronine (T3)

hormone [150]. Further, OH-PCBs and PCP inhibits sulfatation which is an

important regulation pathway of the levels of the free hormone in fetuses [150].

The effect of PCB and PBDE exposure in several animal and human studies has

30

been decreased T4 and T3 levels and increased thyroid stimulating hormone

(TSH) level in blood [149]. The consequence of these changes is probably very

subtle in adults whereas small changes in thyroid hormone levels can be crucial

for the growing fetus and for children [149].

Several studies have scrutinized the relationship between organohalogen

compounds and thyroid disruption in adults e.g. [153-156] and neonates e.g.

[154,157-162]. But there are only a few studies on children (except for

newborns) focusing on thyroid hormones and POPs. A significant positive

association between CB-118 and TSH and a negative correlation between PCBs

and free T3 were for instance observed in German children whereas no

correlations where seen for PCB levels and FT4 [163]. In adolescents from the

Akwesasne Mohawk Nation, PCB (but not 4,4’-DDE) was negatively related to

FT4 and T4 whereas the TSH levels increased with higher PCB concentration

[164,165]. Interestingly, this was true only for non-breastfed children even

though they had lower levels of POPs compared to breastfed children.

According to the authors, this could be due to that it is the prenatal exposure

that is critical and the correlations disappears for the breastfed children since the

large intake of POPs through breastfeeding after birth disguise the prenatal

exposure [165].

One health effect that has been correlated to exposure to POPs is

neurodevelopmental effects in children as reviewed by for instance Damstra

[166], Boucher [167], Ribas-Fitó [168] and Schantz [169]. In a recent study

from U.S., neurodevelopmental effects were also significantly correlated to

prenatal concentrations of PBDEs [170]. The neurodevelopmental effects

correlated to POP exposure might be caused by alteration of thyroid hormones

in utero [168,171]. Effects on thyroid function were seen in children from

Menorca, with background exposure to DDTs, PCBs and β-HCH [172] and

31

neurodevelopmental effects were correlated with prenatal exposure to 4,4’-DDT

and 4,4’-DDE in that cohort [173,174]. Despite the higher concentrations of

DDTs in among breastfed children in this cohort, and the negative effects on

cognitive performance correlated to DDTs, the breastfed children had a better

cognitive performance regardless their in utero exposure to DDTs [175]. This

result evinces the beneficial effects of breast feeding and the fetus’ sensitivity

even to background exposures of POPs. The importance of using cord blood in

exposure assessments is also demonstrated. Both cord blood and maternal

concentration of 4-OH-CB107 was significantly associated with reduced mental

development index in children from eastern Slovakia [176]. Thyroid hormones

were measured in Paper V, but no associations between FT4 and TSH with

PCBs, PBDEs and DDTs were determined, probably due to the small sample

size.

32

5 Results and discussion

Since the results from the Papers I-VI are presented and discussed in the

individual publications or manuscripts it is my intention to extract the most

prominent results from each study, discuss them and do some additional

comparisons in this chapter.

5.1 “Hot-spot” exposure in Slovakia (Paper I and II)

In Paper I, PCB metabolites were measured in 319 adults sampled in 2001 from

the PCB contaminated Michalovce district in Slovakia and from Svidnik, a less

PCB contaminated district situated 70 km north-west of Michalovce. Eastern

Slovakia is a known “hot-spot” for PCB contamination in the world and in

Paper I, we found that the levels of PCBs, OH-PCBs and MeSO2-PCBs are still

very high in this population. A comparison of the major PCB, OH-PCB and

MeSO2-PCB congeners in serum is shown in Figure 5.1. The sum of the three

major OH-PCBs was about 15% of the sum of PCBs and the OH-PCB/PCB

ratio was significantly higher in women than in men. A similar difference in

OH-PCB/PCB ratio between the sexes was also observed in victims from the

Yusho accident in Japan [177]. The concentration of MeSO2-PCBs was low,

less than one percent of the PCB concentration Figure 5.1. A comparison of the

levels and patterns of OH-PCBs in Slovakia compared to Sweden, Faroe Islands

and Canadian Inuit reveals that the PCB levels found in Slovakia are

exceptionally high (Figure 5.2) [32,36,99,178]. The sex difference in retention

of OH-PCBs, women having higher concentrations, is worth to stress. It may be

of interest to look further into the retention also of other halogenated phenolic

compounds in males and females. The question is if higher concentrations of

OH-PCBs and possibly other phenols have an influence on thyroid dysfunction,

known to occur more frequently in women than in men.

33

100

200300400500

600700

Background Exposed

1234567

Background Exposed

CB-1534-OH-CB1874-MeSO2-CB149

ng/g

fat

ng/g

fat

Figure 5.1. A comparison of the major PCB, OH-PCB and MeSO2-PCB congeners (ng/g fat) in serum from men and women living in eastern Slovakia (Paper I).

Background Exposed low high low highSlovak Republic Sweden Faroe Island Canada

200

400

600

800

1000

1200

1400

1600

ng/g

fat

Background Exposed low high low highSlovak Republic Sweden Faroe Island Canada

200

400

600

800

1000

1200

1400

1600

ng/g

fat

Figure 5.2. A comparison of 4-OH-CB187 in women from Slovakia [36], in high and low fish consumers from Sweden and Faroe Islands [32,178] and from Canadian Inuit [99] (ng/g fat).

34

In addition to PCBs, people in eastern Slovakia also have a relatively high

exposure to 4,4’-DDE. The 4,4’-DDE exposure is also higher in the PCB

contaminated Michalovce district compared to Svidnik, but the difference is less

pronounced than for PCBs. Michalovce is more of an agricultural area than

Svidnik, which may have lead to a more extensive use of DDT in the

Michalovce area than in Svidnik in the past. DDT was also intensively used and

produced at several plants in the Former Soviet Union, not far from the

Slovakian border, something that was ongoing until late 1980’s [179]. However,

the low 4,4’-DDT/4,4’-DDE ratio found in the cohort (approximately 0.03)

suggests that the present exposure to the technical DDT mixture is very low.

Paper II is an exposure assessment of PCBs, DDTs and their metabolites in

serum from pregnant women and in cord blood from Michalovce and Svidnik.

The samples were collected between 2002 and 2004. The aim included studies

of the placental transfer of PCBs, DDTs and their metabolites and possible

health effects in the children, caused by the exposure. The focus in Paper II was

on methylsulfonyl-metabolites of PCB and DDE and the placental transfer of

those metabolites. The results show, for the first time ever, that MeSO2-PCBs

and MeSO2-DDE are present in cord blood, proving that these metabolites do

cross the placenta. Due to the complex formation of sulfones metabolites, with

critical contribution by intestinal microflora metabolism, it is not likely that

MeSO2-PCBs and –DDE are formed in the fetus itself. OH-PCBs and PCP was

also measured in the cord blood but published separately [37]. In Figure 5.3, the

concentration of PCBs, OH-PCBs, PCP, MeSO2-PCBs and MeSO2-DDE in

maternal and cord serum is compared on a fresh weight basis. The PCB

concentration is markedly higher in the maternal serum compared to the cord

serum since the PCBs are associated with the lipid fraction of the blood and the

lipid amount in cord serum is much lower than the lipid amount in maternal

serum (0.2% versus 1.1%). When presented on lipid weight basis, the

35

concentration is almost the same in maternal and cord serum which is

confirming their behavior as lipid accumulating chemicals. The concentration of

the OH-PCB metabolites are about the same in maternal and cord serum and are

most likely the result of placental transfer of OH-PCBs from the mother. The

PCB concentration is so much lower in the fetus compared to the mother that it

is unlikely that the fetus could produce all the OH-PCBs internally. It is

unlikely, since the metabolism in the fetus would have to be not just as good as,

but far better than in the mother. Just as the OH-PCBs, PCP is associated with

the proteins in the blood rather than the lipids and the concentration is therefore

similar in maternal and cord serum on a fresh weight basis. Strikingly, the PCP

concentration in cord serum is high, even higher than the PCB concentration

[37].

0.5

1.0

1.5

2.0

CB-118

CB-153

CB-138

CB-180

CB-170

ng/g

ser

um

MaternalCord

PCP

0.2

0.4

0.6

0.8

Maternal

Cord

3'-O

H-CB13

84'-

OH-C

B172

0.05

0.10

0.15

0.20

4-O

H-CB10

7

3-O

H-CB15

3

4-O

H-CB14

64-

OH-C

B187

Maternal

Cord

0.05

0.10

0.15

ΣMeS

O 2-P

CB

3-M

eSO 2

-DDE

Maternal

Cord

Figure 5.3. A comparison of median concentrations of PCBs, OH-PCBs, MeSO2-PCBs and PCP in maternal and cord serum from eastern Slovakia (ng/g serum). Paper II and [37,180].

36

The Slovakian children were followed-up 16 months after birth and the Bailey

Scales of Infant Development II was administered. Mental and psychomotor

developmental status was measured and a significant negative correlation

between the concentration of 4-OH-CB107 in both cord blood and their mental

and psychomotor development scores were determined [176]. However, none of

the other OH-PCB congeners were correlated to how well the children

performed in the test. If this effect is caused by endocrine disruption, altered

neurotransmitter function or reduced thyroid hormones in the central nervous

system is not yet known [176].

The PCB contamination in eastern Slovakia can be compared to Anniston in the

U.S. where a PCB manufacturing plant produced 400 000 tons of PCB between

1929 and 1970. PCB containing waste was disposed in landfill sites that leaked

PCB into the surrounding area [181-183]. The PCB concentration found in

Anniston is not as high as in Michalovce even though there are some

individuals with very high PCB levels [184]. Even though Anniston is a known

“hot-spot” for PCBs, very little information and only a few studies on exposure

studies and possible health effects thereof have been conducted and these

studies started decades after the PCB production had ceased. Accordingly the

studies from eastern Slovakia are particularly interesting and important.

Fish and seafood were not a major component of the diet in Slovakia which is

usually the case for populations with a high PCB exposure [32,34,99]. Instead

locally produced fat rich foods were shown to cause high PCB concentrations in

human serum [185]. The high levels of PCBs in people from eastern Slovakia

have made it possible to look at health effects caused by PCBs and several

adverse health effects have been correlated to PCBs such as

neurodevelopmental effects [176], diabetes [186], dental effects [187], hearing

impairment [188,189] and decreased thymus size [190].

37

5.2 Temporal trends in policemen from Guinea-Bissau (Paper III)

Thanks to a HIV study with an open cohort of police officers in Guinea-Bissau

initiated more than 20 years ago, we were able to get access to serum samples

collected from 1990 to 2007 [191]. Through repeated sampling of the same

policemen at four or five time-points over this time period we got a unique

possibility to study the temporal trend and half-lives/doubling times of some

selected POPs in adults from a country on the west coast of Africa. This study

demonstrates the importance of collaborations across disciplines and

establishments of biobanks. The most important finding was that just as in

industrialized countries, all chlorinated POPs determined in this study were

decreasing over time. These results show that the action taken in the more

developed, industrial countries to reduce human levels of these compounds has

also had an effect in a poor country in Africa. On the other hand, the human

PBDE concentrations were increasing over time, although the levels are still

very low. In a recent study on breast milk from Accra, Ghana, a statistically

significant increase was seen for both PCBs and PBDEs between 2004 and 2009

[192], something that might be influenced by the e-waste handling there [8].

Most time trends studies on POPs and half-life calculations found in literature

are performed on different samples from each time-point. These half-lives can

be seen as population half-lives. In contrast, the study reported in Paper III is

based on measurements in the same individuals over the full time span since

samples were available from each one of them at each time point. However, due

to small sample volumes, the samples had to be analyzed as pools but the same

individuals were pooled from each time point. Although the half-lives in Paper

III might give a better indication of the biological half-life of a compound in

humans compared to a population half-life, they cannot be seen as the biological

38

half-lives since the ongoing exposure to the different POPs are unknown. Only

a few other studies have used repeated sampling (2-3 time-points) of the same

individuals for calculation of half-lives of POPs [193-195]. Hagmar and

colleagues reported a 34% and 55% decrease of CB-153 and 4,4’-DDE between

1991 and 2001 in Swedish men [193] and Tee et al. observed a 49% decrease of

PCBs in men from Michigan between 1980 and 1994 [194]. The decreases over

time in both studies are comparable to the half-lives of PCBs and 4,4’-DDE

seen in Guinea-Bissau (14 and 13 years respectively). In a study on POPs and

breast cancer risk in Denmark, repeated measurements of PCBs and DDTs

changed a non-significant association between these compounds and breast

cancer risk after the first POP measurement to a significant association between

both 4,4’-DDT and CB-138 and an increased risk of breast cancer when the

second POP measurement was included [196]. To conclude, the information

that we receive from temporal studies are valuable, especially when the samples

are from areas with little or no previous data, as e.g. Guinea-Bissau.

5.3 Children’s exposure to POPs in Nicaragua (Paper IV and V)

When we received blood samples from young women and children working and

living at a waste disposal site in Managua, Nicaragua in 2002 we did not know

what to expect. There were very few studies on human exposure to

environmental pollutants from this part of the world and no studies on children.

We expected that their main exposure would be to pesticides, and it was known

to us that there had been a factory producing toxaphene in the outskirts of

Managua, at the shore of Lake Managua, between 1974 and 1991 [197]. But

even though we tried to look for pesticides such as toxaphene and chlordanes,

these compounds were not detected in the analyses. Instead, the historical POPs,

DDTs, PCBs, γ-HCH and PCP were assessed and concentrations determined

39

(Paper IV). Since only limited volumes of serum were available for analysis of

organohalogen compounds the samples had to be pooled.

The children at the waste disposal site in Managua had higher levels of all

compounds analyzed for compared to children living close to the site and

referents. Relatively high levels of 4,4’-DDE were found whereas the 4,4’-DDT

levels were low, indicating very low or no use of technical DDT. The PCB

concentrations were quite high, especially in the children living at or next to the

waste disposal site, indicating that the waste disposal site is a source of local

contamination to PCBs. The waste disposal site also seemed to be the source of

PCP, since the children both working and living at the waste disposal site had

higher levels of PCP than other children and young women in the study.

After this first study, Athanasiadou and co-workers measured the PBDE levels

in the same children and found unexpectedly extremely high levels of PBDEs,

among the highest concentration found at that time [96]. Due to the high PBDE

concentrations in these children, it was possible to determine also the

hydroxylated PBDE metabolites in the blood [96]. To follow up on this study,

new serum samples were collected in 2007-2008 from the same waste disposal

site in Managua and from a second waste disposal site in León, a smaller city 80

km north-west of Managua (Paper V). This time, the aim was to analyze

individual samples to enable determination of inter-individual variation of the

contaminants. We also wanted to investigate any potential changes of serum

concentrations of the POPs over time and to see if a similar exposure situation

was to be found at other waste disposal sites in Nicaragua.

In Paper V, a marked decrease between the levels of especially the PBDEs was

observed between 2002 and 2007-2008. When comparing the mean levels from

2007-2008 with the levels determined in the pooled samples from 2002, the

40

BDE-47 has decreased about fifty times and BDE-153 twenty-five times, and

that in just five years. This decrease was striking since the half-life of these

congeners are expected to be several years in humans [198] and many of the

children that were participating in the second study was working and living at

the waste disposal site already in 2002. One explanation for this large decrease

might be that there were only a couple of children in 2002 with extremely high

PBDE concentrations, individuals that effected the concentration found in the

whole pool. A possible evidence for this theory is the different relations

between the pools that were seen for the chlorinated POPs and the PBDEs. The

difference between Pool 1 and Pool 2, that is the difference between children

both working and living at the waste disposal site and children just working

there, were small with just slightly higher concentrations of PCBs and DDTs in

Pool 1 (see Paper IV, Table 2). If the same comparison is made for PBDEs, the

difference is very large between Pool 1 and Pool 2 (see Paper V, Figure 1),

indicating that there are some children that probably have a specific exposure to

just PBDEs. Also, Athanasiadou analyzed two pools from each distinct group of

children (pools 1-5) collected in 2002 [96]. One pool included exactly the same

children as in Paper IV whereas the other pool had the same set of children

except for a couple of individuals. The large differences seen in POP

concentrations between the two pools, especially in the highly exposed group,

reveal large individual differences between the children [96]. Results like these

show the value of individual analysis.

A peak in the late 1990’s and then decreasing time-trends for PBDEs have been

seen in other countries as well, such as in breast milk from e.g. Sweden

[102,199,200] and Japan [201]. But the decrease is not as fast as the one we saw

in the Nicaraguan children and the BDE-153 congener in the mothers’ milk

studies is not decreasing. One explanation to the differences in POP

concentrations between the two studies might be differences in the kind of

41

waste that is disposed at the waste disposal site or changes in how the waste is

handled. The chlorinated organohalogens also decreased over time but more

moderately, i.e. about three times for the DDTs and ten times for the PCBs.

Another difference between the studies presented in Paper IV and V is that the

concentration of the POPs in the latter study is equal between study groups of

children, i.e. not dependent on where they live and work. The reason for this is

not clear and in fact difficult to explain. Regarding the PBDEs it might be that

the children that did not work or live at or close to any dumping sites were both

younger and sampled a couple of years earlier and that they are exposed to

PBDEs in their home environment. But why they have the same concentration

of PCBs as the waste disposal site is difficult to explain. The marked decrease

of PCB concentration seen in the waste disposal site children might just be due

to that they do not receive as much PCB containing waste anymore. However,

the population half-life for PCBs in humans is several years [19,199].

5.4 Children’s exposure to POPs in Bangladesh (Paper VI)

In a preliminary study on human exposure to chlorinated POPs in Bangladesh

some time ago, the concentrations of these POPs were surprisingly as high, or

even higher in children and teenagers compared to adults [202]. To further

investigate Bangladeshi children’s exposure to POPs and also to metals, new

serum samples were collected from children from Dhaka in 2008. Both

chlorinated and brominated organohalogens were determined in individual

samples from the children working and living at or next to two waste disposal

sites in Dhaka and in referents as presented in Paper VI.

The most remarkable result from Paper VI is the high 4,4’-DDT concentrations.

This undoubtedly reveals recent use of technical DDT. The concentrations of

DDTs in Bangladeshi children were higher compared to Nicaraguan children

42

(Paper IV) and in the same range as in Mexican children except from an area

with endemic malaria [203]. The other measured chlorinated POPs were found

at very low concentrations and no correlation was observed between serum

concentration and possible work or vicinity to the waste disposal sites (Figure

5.4). The levels of PBDEs on the other hand, were higher in the children

working and living at the waste disposal site in Savar and low levels were found

in the children with less contact with the dump sites indicating that the waste

handling is a source to PBDEs (Figure 5.4).

Savar

Switch G

ate

Matuail

Tejkuni

400

800

1200

10002000300040005000

ng/g

fat

ng/g

fat

4,4’-DDT

4,4’-DDE

102030405060 CB-153

ng/g

fat

20406080

100120

1020304050

β-HCH

γ-HCH

0.40.81.21.62.0

12345

<LOQ

BDE-153

BDE-209

Savar

Switch Gate

Matuail

Tejkuni

Figure 5.4. Box-plots of the levels of DDTs, HCHs, CB-153 and PBDEs in serum from four groups of children from Bangladesh (ng/g fat).

43

The PBDE concentrations in the Bangladeshi children were significantly lower

than the concentrations found in children from Nicaragua. This could either be

due to that there are less PBDE containing waste at the waste disposal sites in

Dhaka or different circumstances at the two waste disposal sites. The waste in

Nicaragua is burned deliberately but spontaneous fires also occur. Independent

of the cause of the fires, the air is filled with smoke and particles. This is not the

case in Bangladesh where the waste is used as landfill. The concentrations of

PCBs were relatively low and in the same range in the children from

Bangladesh, Nicaragua (Paper V) and China [204]. On the other hand, some

very high levels of PCBs (mean ΣPCB= 1700 ng/g fat) were observed in breast

milk from women living close to a waste disposal site in Kolkata, India in 2009

[205]. India is a country with rapid population and industrial growth and a fast

urbanization and the high PCB concentration found in breast milk from women

living close to the dump site in Kolkata indicates that the waste disposal site is a

source of PCBs, probably originating from old transformers and condensers that

have been dumped there [206]. There are still an unknown number of

transformers that contain PCB oil in Bangladesh that could cause severe PCB

contamination in the future if they end up at open waste disposal sites. A

situation similar to that described in Kolkata may potentially emerge at sites in

Bangladesh.

44

6 Concluding remarks and future efforts

The main conclusion that can be drawn from this thesis is that there are

populations and groups that are, for various reasons, highly exposed to different

environmental pollutants despite the fact that many of these pollutants were

banned decades ago. This is for instance the case in eastern Slovakia, where

high levels of PCBs and their metabolites are still found at high concentrations

in humans due to former PCB production, making this a “hot-spot” area for

PCB contamination. Placental transfer of PCB and PCB metabolites were

studied in this population and the placental transfer of PCB and 4,4’-DDE

methyl sulfones were observed for the first time.

Even though there are numerous of studies on exposure, levels and trends of

environmental pollutants from the developed industrial countries in the world,

data from developing countries is still scarce. The exposure assessments of

POPs in humans from three developing countries, as presented in this thesis, are

therefore adding valuable data on POP exposure in humans (including children)

to narrow this data gap. For example, little is known about human exposure to

POPs in Africa. The temporal trends of POPs in the study on police officers

from Guinea-Bissau, indicated decreasing concentrations of DDTs, PCBs and

HCHs over time, whereas the PBDEs were increasing, although still detected at

low concentrations. This is telling us that brominated flame retardants like the

PBDEs are not yet under control.

Poor handling of waste, landfills and un-controlled burning at waste disposal

sites may lead to extensive leakage of toxic chemicals leading to contamination

of air, the surrounding lands, drinking-water and people that work or live close

to, or at, these sites. Children working and sometimes also living at a waste

45

disposal site in Nicaragua in 2002 had higher concentrations of both chlorinated

and brominated POPs compared to reference children. Five years later, the

concentrations had decreased markedly and there were no evident difference

between the children from the waste disposal site and other children. High

levels of 4,4’-DDT and 4,4’-DDE were observed in children living in Dhaka,

Bangladesh. The relatively high level of 4,4’-DDT indicate recent use of

technical DDT. No differences in serum concentration of DDTs, PCBs or HCHs

were seen between children living at, or close to, waste disposal sites and

children living far away from these sites. However, PBDE concentrations were

higher in children from the waste disposal sites than in other children, which

might imply that the waste disposal sites are a possible source of these

compounds. The concentrations of PBDEs were relatively low, pronouncedly

lower in the Bangladeshi children compared to Nicaraguan children.

A great deal of production of articles such as electronics, toys, clothes and home

furnishing is taken place in developing countries. This production involves

many different chemicals, some of them toxic and persistent, and if they leak

into the environment they may cause adverse effects in humans. Also, use,

recycling and re-use, and disposal of these products may pose a hazard.

Knowledge, functioning infrastructure and financial resources are some of the

requirements for proper management of these risks. Developing countries often

lack these requirements.

Future efforts should support biomonitoring to determine levels and trends of

known POPs as well as novel pollutants. Waste disposal sites are a good place

for exposure studies since this is the final destination for many products and

articles containing a large quantity of different chemicals. To understand and

study the entire lifecycle of materials and products that contain hazardous

compounds is also necessary. Moreover, the fact that we are exposed to a

46

mixture of compounds that could possess additive, synergistic or counteracting

effects and therefore act even at low levels must be considered both when

assessing the exposure and when performing risk assessments. It is also

important to identify contamination “hot-spots” and extra sensitive groups of

individuals and populations. Finally, to assess the exposure of developing

fetuses and children are of greatest importance since such exposure might be the

cause of adverse health effects later in life. A global biomonitoring program of

children is required to improve the understanding of the POP problem and to

improve health of children.

47

7 Acknowledgement

Som ni vet så hatar jag verkligen mornar, men trots det så stiger jag faktiskt upp varje morgon och åker till jobbet, kanske inte pigg och glad, men jag gör det. Det som driver mig är att jag tycker att mitt jobb är så himla kul. Dels så brinner jag verkligen för mina små soptippsbarn, men så har jag ju också världens bästa arbetskamrater på Miljökemi. Ni är så härligt roliga, hjälpsamma, busiga, snälla, knasiga, inspirerande, och flamsiga. Först och främst så vill jag tacka mina handledare. Åke för att du är så entusiastisk och inspirerande och för att du alltid tar dig tid när man behöver dig. Maria för att du har lärt mig allt jag kan och för att du tvingar ut mig på djupt vatten och säger att jag kan simma själv. Men jag vet att du har koll och kommer till min undsättning om det skulle behövas. Kristina för att du får mig att se saker ur ett annat perpektiv och för att du alltid kommer med så smarta idéer och lösningar. I would also like to thank all my co-authors for invaluable contributions. Vad vore Piff utan Puff? Inget! Jag menar det verkligen Anna, det här hade inte gått utan dig. Att dela rum, glädje och frustration med dig har varit helt sanslöst kul. Du är fantastisk, glöm aldrig det! Jag vill också tacka: Karin, för att du är en så himla bra vän och för att du aldrig säger nej. Johon, för att du är precis lika knasig som jag är och för att du alltid kan få mig att skratta. Jessica, för att du finns när man behöver fnittra en stund och Ioannis för att du är som du är, alltid. Hans, för att man alltid kan komma in till dig och prata bort en stund. Emelie, för att du alltid är så glad, det smittar. Vickan, för att du säger som det är och för att du alltid bjuder på dig själv och ett skratt. Lisa, du får mig att se saker på ett nytt sätt. Andreas, jag kommer aldrig att glömma vår tid med grisbollen! Maria S, för en oförglömlig Kinaresa och goda bakverk. Hitesh, Good luck with your thesis. Anna-Karin och Cecilia lycka till med doktorerandet och glöm inte att ha kul under tiden!

48

Göran, för att du är så härligt bitter och Per för skojiga diskussioner vid fikabordet. Lillemor , tack för hjälpen med analysbiten. Maggan, Johan E, Sören, Lotta, Birgit och Anita för att ni alltid hjälper till när man behöver det som bäst. Ett stort tack till mina före detta kollegor också, för att ni var var så himla snälla mot mig när jag var ett blåbär: Jana, Anna M, Britta, Karin N, Hrönn, Kaj och Tati m.fl. Det finns en värld utanför Miljökemi också, familj, släkt och vänner: Jag vill speciellt tacka mina kära vänner från kemistlinjen Anna B, Annika, Anna C, Henrik, Chrisse, Maria, Jenny och Malin som gjorde att det var så kul att läsa kemi och för att ni fortfarande är lika fikasugna. Jag vet inte hur jag skulle klara mig utan Hanna, för att du aldrig säger nej till en kopp te och Marianne, för att du känner mig så bra och för att du ser till att jag behåller båda fötterna på jorden. Ett alldeles särskillt tack till min familj: Mina föräldrar för för att ni alltid har stöttat men aldrig pushat. Mamma för att du alltid ställer upp och min allra käraste syster för att du alltid tror på mig. This thesis was financially supported by grants from: • The European Commission’s 5th framework Program

(PCBRISK, QLK4-2000-00488) • US National Institutes of Health, National Cancer Institute

(R01-CA96525) • Swedish International Development Cooperation Agency (SIDA)

(SWE-2006-376)

49

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