Kelly GUILBOT, 2
ndyear of Engineering School of Chemistry.
Monday, May 7
thto Friday, August 17
th2012.
Under the direction of :
Supervisor : Bert van BAVEL, Director of the Analytical Environmental Chemistry division and
Professor.
Supervisor : Jessika HAGBERG, Lecturer.
Examiner : Anna KÄRRMAN, Lecturer.
Determination of dioxins in Cretaceous strata from the
South of Sweden. Can the environmental anthropogenic
pollutant dioxin be of natural origin ?
Determination of dioxins in Cretaceous strata from the
South of Sweden. Can the environmental anthropogenic
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Abstract
Eleven sediment samples from different geological layers and four fossils from the
South of Sweden were collected and estimated to be 80 million years old (approximately
late of Cretaceous period). The samples were analyzed for polychlorinated dibenzo‐p dioxins
(PCDD/Fs) to investigate whether these samples are likely to contain dioxins from a natural
formation. For over thirty years, the scientific community has discussed the possibility of a
natural formation of dioxins. Several hypothesis have been put forward, but often rejected
by the evidence of a source of anthropogenic pollution in the samples.
In order to answer this issue, two types of analyses have been performed : high
resolution gas chromatography‐high resolution mass spectrometry (HRGC/HRMS) and
elemental analyzer‐isotopic ratio mass spectrometry (EA‐IRMS). HRGC/HRMS provides
information about the source of dioxins comparing the distribution of all PCDD/Fs to
experimental isotopic patterns from past publications. δ
13C of organic carbon gives
information about the nature of carbon present in soils and can be helpful to trace
paleoclimates.
Keywords : old clay layers, octachlorodibenzo‐p‐dioxin, high resolution gas chromatography‐
high resolution mass spectrometry, analyzer‐isotopic ratio mass spectrometry, isotopic
dilution.
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Acknowledgments
I wish to thank first, Bert van Bavel, Professor and Head of the Environmental
Analytical Chemistry team, for receiving me in the laboratory and without whom this project
would not have been possible. I am also grateful to him for his kindness, his educational and
scientific qualities. I learned a lot with this project and I express to him my gratitude.
I extend my warmest thanks to Jessika Hagberg, Senior Lecturer at the Man
Technology and Environment Research Center, Environmental Analytical Chemistry team, for
her availability and without whom I would surely have been lost, her interest in my work and
the relevance of her remarks.
I want to thank Alf Ekblad, Professor of Biology in the Ecosystem Ecology team, Ulrika
Eriksson, a doctoral student in the Environmental Analytical Chemistry team and Anna
Mikunsinska, a doctoral student in the Ecosystem Ecology team, for having taken the time to
explain to me the basis of EA‐IRMS technique and their help about interpretation of the
results obtained.
I thank Elisabeth Einarsson, a doctoral student at the Department of Earth and
Ecosystem Sciences at Lund University, for her nice visit of the excavation site, her
explanations about the description of the different sediment samples and all notions of
geology which were hidden under this project. I would also thank Vivi Vajda, Professor of
geology in Lund University for her help with the stratigraphic diagrams. I thank Mohammed,
Robby and the others (Åsen team) too, to have made me share their passion and thanks to
them these two days will remain unforgettable !
Finally, I thank the whole laboratory for their warm welcome and their cheerfulness.
This immersion in the Research Center allowed me to discover what is "fika" for a Swedish
and to attend a thesis defense.
Table of contents
Abstract ... 4 Acknowledgments ... 5 Abbreviations ... 7 List of tables ... 8 List of figures ... 8 Människa‐Teknik‐Miljö Research Center, Örebro university ... 9 Introduction ... 10 I. Literature review ... 11 I.1. What are dioxins ? ... 11 I.2. Health effects ... 11 I.3. Toxicity calculus ... 12 I.4. Where do dioxins come from ? ... 12 I.5. Bioaccumulation and biomagnification ... 13 II. Materials and Methods ... 15 II.1. Presentation of the samples... 15 II.2. Samples pre‐treatment ... 16 II.2.i. Preparation of the samples ... 16 II.2.ii. Multi‐layers H2SO4 silica column ... 17 II.2.iii. Alumina oxide column ... 17 II.2.iv. Carbon column ... 17 II.3. High Resolution Gas Chromatography/High Resolution Mass Spectrometer (HRGC/HRMS) .... 18 II.4. Isotope dilution principle ... 19 II.5. Elemental Analyzer‐Isotopic Ratio Mass Spectrometry (EA‐IRMS) ... 19 III. Results and Discussion ... 21 III.1. Results of HRGC/HRMS analysis ... 21 III.1.i. Results of the blanks ... 21 III.1.ii. Results of the sediment samples ... 22 III.2. Results of EA‐IRMS analysis ... 24 III. 3. Scenario proposed by Schmitz et al. to explain why dioxins are loaded in clays ... 26 Conclusion, future research and experience ... 27 References ... Error! Bookmark not defined. Appendices ... 327
Abbreviations
CPs : Chlorinated Phenols dw : dry weight EA‐IRMS : Elemental Analyzer‐Isotopic Ratio Mass Spectrometry EPA : Environmental Protection Agency FID :Flame Ionization Detector HAH: Halogenated Aromatic Hydrocarbons HRGC : High Resolution Gas Chromatography HRMS : High Resolution Mass Spectroscopy IAEA : International Atomic Energy Agency IARC : International Agency for Research on Cancer LOD : Limit Of Detection LOQ : Limit Of Quantification Na‐PCP : sodium PentaChloroPhenol OCDD : OctaChloroDibenzo‐p‐Dioxin PBDE : PolyBrominated Diphenyls Ethers PCBs : PolyChlorinated Biphenyls PCDDs : PolyChlorinated Dibenzo‐p‐Dioxins PCDFs : PolyChlorinated Dibenzofurans PCP : PentaChloroPhenol PDB : Pee Dee Belemnite POP : Persistent Organic Pollutant RF : Response Factor RRF : Relative Response Factor SD : Standard Deviation TCD : Thermal Conductivity Detector TCDDs TetraChloro Dibenzo‐p‐Dioxins TEFs : Toxic Equivalent Factors TEQs : Toxic Equivalent WHO : World Health OrganizationList of tables
Table 1. WHO TEFs for human risk assessment based on the conclusions of the World Health Organization meeting in Stockholm, 15‐17 June 1997 (WHO European Centre for Environment and Health, 1998). ... 12 Table 2. Physical chemical properties of some PCCDs (PEREIRA, 2004) (SHLU et al., 1988) (WU et al., 2001). .... 14 Table 3. Identification and brief presentation of the different samples. ... 16 Table 4. Carbon content in the samples and pH... 25 Table 5. Table for the identification of PCDFs. ... 39 Table 6. Table for the identification of PCDDs. ... 39 Table 7. Different formulas used for the quantification of PCDD/Fs. ... 39List of figures
Figure 1. Halogenated aromatic hydrocarbons' family (BERGQVIST, 1998). ... 11 Figure 2. PCDF, PCDD and PCB skeletal structures, can be chlorinated at any of the suitable positions of the aromatic rings (SROGI, 2008). ... 11 Figure 3. Skeletal structure of the 2,3,7,8‐TCDD (SROGI, 2008). ... 12 Figure 4. Dioxins sources and distribution. Modified from (MOFFAT, 2008). ... 13 Figure 5. U.S. dioxin emissions from made‐man sources have declined over 92% thanks to government regulations and industry efforts (US Environmental Protection Agency, 2006). ... 13 Figure 7. Geologic time scale 2004 : eras are in bold and periods are in italic. Ma means million years ago, symbol "a" from the Latin annus (GRADSTEIN & OGG, 2004). ... 15 Figure 6. Log of the unconsolidated marine Upper Cretaceous unit at Åsen, showing the different layers where the samples have been collected (ERIKSSON et al., 2011). ... 15 Figure 8. Schematic presentation of the various steps of extraction and cleaning of sediment samples. ... 18 Figure 9. Principle of isotope dilution from population analysis in zoology (MEIJA & MESTER, 2008). ... 19 Figure 10. Principle of a EA‐IRMS in a combustion mode (Nature 's fingerprint, a division of Molecular Isotope Technologies LLC). ... 20 Figure 11. Repartition of the concentrations of the different PCDD/Fs congeners in comparison with their LoD, first blank prepared. ... 21 Figure 12. Repartition of the concentrations of the different PCDD/Fs congeners with their LoD, last blank prepared. ... 21 Figure 13. Distribution of the concentrations of PCDD/Fs found in the sediment sample n°6. ... 22 Figure 14. OCDD concentrations in the series of sediment samples. Maybe three of them are slightly different. 22 Figure 15. Distribution of the different PCDD/Fs in a raw ball clay, pattern expected for all clay products (FERRARIO, BYRNE, & CLEVERLY, 2000). ... 23 Figure 16. Delta 13C values of the different sediment and fossil samples for bulk carbon and organic carbon. Error bars represent SD. ... 24 Figure 17. Depth distributions of total carbon, total organic carbon, total inorganic carbon, proportion of organic carbon to the total carbon, pH and δ13Corganic for sand sediments (S1, S2, S4 to S7) and river sediments (S8 to S11). The glacial clay S3 is not represented in this graphic because it does not match with age deposition. ... 25Figure 18. Hypothetical geological and paleoenvironmental model of natural PCDD formation and accumulation in clays. The grey arrows indicate dioxin transfer paths (SCHMITZ et al., 2011). ... 26
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Människa‐Teknik‐Miljö Research Center, Örebro university
The MTM Research Center (Människa‐Teknik‐Miljö in Swedish, Man‐Technology‐ Environment) was created in 1998 by Professor Bert Allard and is established at the University of Örebro. The university has 17,000 students and 1,200 staff. It offers a wide variety of teaching like medicine, sciences, technology, business, law, psychology etc as presented below.
The research center has two principle aims of research, which are biogeosphere dynamics and environmental chemistry & health and ecotoxicology. The different research projects at the center focus on soil remediation, geologic waste deposition, carbon dynamics in soil, persistent organic pollutants in the environment (analysis at ultra‐trace levels, occurrence, stability…), exposure to humans and their evolution. The laboratory is organized in five teams which are the following :
The staff of MTM is composed of six professors, six lecturers and fourteen PhD students and post‐doctors. The research center is financed by several means : Örebro University and external partners from Sweden which work on collaborative projects with the MTM, private companies which grant funds in order to carry out a project, different scholarships from the government, United Nations projects (UNEP) and the European Union.
Introduction
During the past 50 years, several cases involving dioxins have made the headlines.
During the Vietnam war, between 1961 and 1971, the U.S. army used a defoliant
called "Agent Orange", for its herbicide warfare program. The name of the defoliant
originates from the orange‐striped barrels in which it was shipped. The goal was to defoliate
forested and rural land, depriving guerrillas of cover. Later, this chemical product,
manufacturing by Monsanto, was discovered to be contaminated by 2,3,7,8
tetrachlorodibenzo‐p‐dioxin, the most toxic dioxin congener. Exposure to this pollutant
caused many terrible birth defects and several diseases as soft‐tissue sarcoma, non‐
Hodgkin's lymphoma, Hodgkin's lymphoma and chronic lymphocytic leukemia.
In 1976 in Italy, a small chemical manufacturing plant released 2,3,7,8 tetrachloro
dibenzo‐p‐dioxin to the atmosphere due to an overheated reactor. This accident did not
cause deaths but provoked many chloracne cases among children. Eight years after the facts,
breath cancers, diabetes and an altered sex ratio at birth were reported (excess of girls
compared to boys). The city being the most exposed to this contaminant was Seveso, where
17,000 inhabitants at the time of the accident. The city gave its name to the Seveso II
Directive. It is an European Union law aimed at improving the safety of sites containing large
quantities of dangerous substances by considering all possible accident scenarios, by
implementing a prevention plan, a contingency plan, by limiting urbanization around the
sites, informing residents and establishing a competent authority for inspection of hazardous
sites.
During the 2004 Ukrainian presidential campaign which pitted against Viktor
Yanukovych, the government‐supported candidate, and Viktor Yushchenko, this later one
was poisoning with 2,3,7,8 tetrachlorodibenzo‐p‐dioxin by government agents. Yushchenko
finally won the election after three rounds of voting due to frauds in favor of Yanukovych.
During the last thirty years, there has been discussions whether dioxins, these
extremely toxic compounds, could originate from natural formation. Several hypotheses
were advanced for instance forest fires, eruption of volcanoes.
The aim of this project is to quantify the amount of dioxins in very old strata, because
it is likely that these layers would be free of anthropogenic pollutant and thus to obtain a
new evidence of the natural formation of dioxins.
This report contains a brief presentation of dioxins and the state of knowledge. A part,
Materials and Methods, explains the procedure to follow in order to analyze these
compounds, from the sampling of the samples, extraction and the clean up, and finally the
analytical equipment and settings to analyze the dioxins. The last part, Results and
Discussion, deals with the results and suggests a possible way to continue this project.
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I. Literature review
I.1. What are dioxins ?
Dioxin is the generic name for a large family of environmental contaminants that share a similar chemical structure and a common mechanism of toxic action. Dioxins belongs to the group of compounds known as Persistent Organic Pollutants (POPs) because they have high melting points and are stable to acids and bases. Thus, these characteristics make them very persistent in the environment. The term "dioxin" is used to designate polychlorinated dibenzo‐p‐dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs). This term can also make reference to some of the polychlorinated biphenyls (PCBs), which are commonly called dioxin‐like compounds, because they share a structural similarity and toxic mode of action with dioxin (BERGQVIST, 1998).
Figure 1. Halogenated aromatic hydrocarbons' family (BERGQVIST, 1998).
The basic structure of PCDD/Fs comprises of two benzene rings joined by either a single (furan) or a double oxygen bridge (dioxin), whereas in the PCBs' structure the two benzene rings are bound by a single bond C‐C (SROGI, 2008). (See appendix 1 for nomenclature of PCDFs, PCDDs and PCBs). Figure 2. PCDF, PCDD and PCB skeletal structures, can be chlorinated at any of the suitable positions of the aromatic rings (SROGI, 2008). Considering all the possible positions for the chlorine atoms, we can identify 75 PCDDs, 135 PCDFs and 209 PCBs. Each different form is called a "congener." They are known to bio‐accumulate due to their lipophilic nature and, therefore, have health implications. As a result, their emission into the environment and food chain is strictly controlled. Only 7 of the 75 dioxins, 10 of the 135 furans, and 12 of the 209 PCBs have 2,3,7,8‐TCDD's toxicity. Limits are published by the World Health Organization (WHO) and local authorities.
I.2. Health effects
Various health effects associated with exposure to PCDD (primarily 2,3,7,8‐TCDD) have been reported. Chloracne, characterized by comedones occurring with or without cysts and pustules and patchy darkening of the skin, were classical manifestations caused by chronic exposure. Long‐term exposure is linked to impairment of the immune system, the developing nervous system, the endocrine system and reproductive functions. Chronic exposure of animals to dioxins has resulted in several types of cancer (WATANABE et al., 1999). 2,3,7,8‐TCDD is considered by the International Agency for Research on Cancer (IARC) as a human carcinogen. The WHO recommended in 1998 that the human ingestion should stay within the limits of 1‐4 pg kg‐1 of body weight per day (KOGEVINAS, 2001).
1 2 3 4 6 7 8 9 1 2 3 4 6 7 8 9
I.3. Toxicity calculus
The complex nature of PCDD/Fs and biphenyl mixtures complicates the risk evaluation for humans. For this purpose, the concept of toxic equivalency factors (TEFs) has been developed and introduced to facilitate risk assessment and regulatory control of exposure to these mixtures.
The “Toxic Equivalent” (TEQ) scheme weighs the toxicity of the less toxic compounds in relation to the most toxic 2,3,7,8‐TCDD. Each compound is attributed a specific “Toxic Equivalency Factor” (TEF). This factor indicates the degree of toxicity compared to 2,3,7,8‐TCDD, which is given a reference value of 1.
Figure 3. Skeletal structure of the 2,3,7,8‐TCDD (SROGI, 2008).
To calculate the total TCDD toxic equivalent (TEQ) of a dioxin mixture, the amounts of each toxic compound are multiplied with their TEF values and then added together (WHO European Centre for Environment and Health, 1998).
Table 1. WHO TEFs for human risk assessment based on the conclusions of the World Health Organization meeting in Stockholm, 15‐17 June 1997 (WHO European Centre for Environment and Health, 1998).
I.4. Where do dioxins come from ?
Current ambient levels of dioxins are the result of human activities. PCDDs and PCDFs are inadvertently produced through a number of human activities, as well as by natural processes. Dioxins are released into the air from combustion processes such as commercial, municipal or medical waste incineration, from burning fuels (i.e., wood, coal, oil) and burning of household waste. Chlorine bleaching of pulp and paper, certain types of chemical manufacturing and industrial processing can create low quantities of dioxin as undesired by‐products. Dioxins have also been detected at low concentrations in exhaust from cars. Burning of materials that contain chlorine, such
13
as plastics, wood treated with pentachlorophenol (PCP), and pesticide‐treated waste produce dioxins. Dioxins can also be formed during forest fires and volcanic eruptions (LEMIEUX et al., 2004).
Figure 4. Dioxins sources and distribution. Modified from (MOFFAT, 2008).
The amounts of dioxins that have been released from various sources have changed significantly over time. Historically, the majority of dioxins released were from commercial, municipal and medical waste incineration, the manufacture and use of certain herbicides and chlorine bleaching of pulp and paper. U.S. dioxin emissions from man‐made sources have declined over 92 percent since 1987 due to a combination of effective government regulations and voluntary industry efforts. Industrial man‐made sources have decreased so significantly that household trash burning is currently the largest man‐made source of dioxin emissions to the environment (US Environmental Protection Agency, 2006).
Figure 5. U.S. dioxin emissions from made‐man sources have declined over 92% thanks to government regulations and industry efforts (US Environmental Protection Agency, 2006).
I.5. Bioaccumulation and biomagnification
When released into the air, dioxins disperse and travel long distances because they are extremely persistent compounds. They can deposit themselves onto soils, water and vegetation. There were also discovered in the Arctic and Antarctic regions, which are far away from industrial areas (RAPPE et al., 1997).
Table 2 shows that PCDDs have high boiling points. They don't easily dissolve in water because their solubility in water is extremely low. This involves that air is their preferential means of transport. Higher is the number of chlorine atoms (molecular weight of PCDD/Fs increases) and lower
is the solubility in water because the (apolar) aromatic ring has a significant hydrophobic effect, enhanced by the presence of chlorine atoms. These substances are found preferentially bound to particulate material and to organic matter (log Koc < log Kow), in aquatic medium and in soil
(PEREIRA, 2004). Table 2. Physical chemical properties of some PCCDs (PEREIRA, 2004) (SHLU et al., 1988) (WU et al., 2001). PCDD congener Molecular weight (g mol‐1) Fusion point (°C) Boiling point (°C) Solubility in water (µg L‐1) at 25 °C Log Kow Log Koc 2,3,7,8‐TCDD 322.0 305 446 0.2 6.80 6.64 1,2,3,7,8‐PeCDD 356.5 240 464 ‐ 7.40 6.92 1,2,3,4,7,8‐HxCDD 391.0 273 487 0.004 7.80 7.11 1,2,3,4,6,7,8‐HpCDD 425.2 264 507 0.002 8.00 7.20 OCDD 460.0 330 510 0.0004 8.20 7.30
Terrestrial and aquatic animals consume dioxins on plants and in the air, water, sediment, and soil. It is, in that way, that dioxins enter in the food chain. This is called bioaccumulation. This corresponds to an increase of a pollutant from the environment to the first organism in a food chain. Dioxins in plants are concentrated in the bodies of plant eating animals, and are then further, concentrated when meat eating animals eat plant eating animals. This second phenomenon is called biomagnification. Another way to explain it, it is an increase in concentration of a pollutant from one link in a food chain to another.
A lot of publications mention the fact that dioxins have been found in different types of soil. PCDDs have been found in marine sediments, irrigation drain sediments and topsoil from Queensland, Australia (GAUS et al., 2001) (GAUS et al.). High concentration of OCDD has been found in ball clay from the USA, Japan and Germany (HORII et al., 2008). Ball clay is sedimentary in origin, and it is usually composed of kaolin (20‐80 %), mica (10‐25 %) and quartz (6‐65 %). "Ball clay" is a term of art or industry rather than a purely mineralogical term. The name derives from the original practice of mining such clay in cubes that would become rounded into balls during handling and storage, and hence was referred to as "ball clay" (The ball clay heritage society, 2000). (Photographs of ball clay in appendix 2).
Examples of biomagnification of dioxins contamination from ball clay has been found in various animal products, including chicken and catfish, due to the use of ball clay as an anti‐caking additive in feed (FERRARIO et al., 2000).
Given that the congener profiles analyzed do not correspond to normal profiles of anthropogenic sources, like fly ash for example, most of the discussions are based on the fact to know if dioxins could be of a natural origin. These unusual profiles are characterized by a dominance of octachlorodibenzo‐p‐dioxin (OCDD) and a low concentration of PCDFs (GAUS et al., 2002). Most of the hypothesis going to the direction of a natural origin, like an abiotic natural formation due to surface‐promoted reactions associated with clay minerals (HORII, et al., 2008), are often contradicted by the evidence of the presence of dioxin precursors like pentachlorophenol (PCP), which is a fungicide and whose the use was considerable in timber industry and in agriculture, since the late 1930s. PCP contaminated soil is also typically characterized by elevated OCDD levels (GAUS et al.). Consequently, even samples which can be considered as "pristine", "undisturbed" could have been polluted by an anthropogenic source, as it was the case in Queensland, Australia (GAUS et al.).
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II. Materials and Methods
II.1. Presentation of the samples
Kristianstad is located at 69 km northeast of Lund, in the southern part of Sweden. This basin, where the samples come from, is limited by two faults in the southwest, which have formed two horsts : Nävlingeåsen and Linderödsåsen (see map in appendix 3).
A warm and humid climate resulted in extensive kaolinization of the principally Precambrian crystalline basement. Tectonic block movements in the Early Cretaceous to early Campanian in the Late Cretaceous, formed a basin by tilting the basement towards Nävlingeåsen and Linderödsåsen horsts. The area was flooded repeatedly during the Cretaceous and an archipelago with small islands and rocky shorelines was developed in the northern part (LINDGREN & SIVERSON, 2004). Figure 7. Geologic time scale 2004 : eras are in bold and periods are in italic. Ma means million years ago, symbol "a" from the Latin annus (GRADSTEIN & OGG, 2004). The Åsen locality is an old kaolin quarry now serving as a refuse dump.
Thanks to the index fossils contained in the different layers, it is possible to estimate the age of settlement layers. The excavation site is divided into two zones : Belemnellocamax Balsvikensis (1.2 to 2.0 m) and Belemnellocamax Mammilatus (0 to 1.2 m) (See figure 5 next). These two species of belemnites lived 80 million years ago, late Cretaceous. This type of information enables paleologists to date sediments precisely. These two zones are separated by a glacial clay (erosional surface) from quaternary cover. This clay is younger than layers from B. Mammilatus and Balsvikensis zones, but it is in between the two belemnite zones probably due to tectonic movements. (Photographs of Åsen site are available in appendix 4) (ERIKSSON et al., 2011). Cambrian (542-488 Ma) Ordivician (488-443 Ma) Silurian (443-416 Ma) Devonian (416-359 Ma) Carboniferous (359-299 Ma) Permian (299-251 Ma) Triassic (251-199 Ma) Jurassic (199-145 Ma) Cretaceous (145-65 Ma) Paleogene (65-23 Ma) Neogene (23-0 Ma) S1 S2 S3 S5/S6 S4 S7 Figure 6. Log of the unconsolidated marine Upper Cretaceous unit at Åsen, showing the different layers where the samples have been collected (ERIKSSON et al., 2011). S11 S9 S10 S8
Table 3. Identification and brief presentation of the different samples.
Identification
number Description Localities Color Depth(m) pHa)
S1 Upper part B. Balsvikensis zone Åsen yellow 1.8 9.09
S2 Lower part B. Balsvikensis zone Åsen green 1.4 9.18
S3 Balsvikensis‐Mammilatus zone : glacial clay Åsen brown 1.2 9.07
S5 B.Mammilatus zone : oyster Åsen yellow 1.1 9.13
S6 B.Mammilatus zone : oyster bed Åsen green 1.1 8.75
S4 Upper part of B.Mammilatus zone : sand Åsen green 0.8 8.63
S7 B.Mammilatus zone : crushing layer Åsen green 0.6 8.76
S11 River plane Åsen black 0 3.23
S9 River plane Åsen grey ‐1 7.17
S10 River plane Åsen grey ‐20 6.98
S8 River plane Åsen grey ‐25 5.76
F1 Large clams Ullstorp ‐ F2 Shark teeth Ullstorp ‐ F3 Belemnite Åsen ‐ F4 Oyster Åsen ‐ a) The pH was measured according to NF X 31‐103, i.e., sediments are dissolved in de‐ionized water in a ratio 1/2.5. The suspension is then stirred for 60 min and the pH was measured in the supernatant (the upper part of a PPT tube) after two hours of settling.
The B. Mammillatus zone comprises sandstones resting upon either residual kaolin clay, reworked kaolin and quartz sand or directly upon the crystalline basement. This zone is rich in belemnites, oysters, coprolites, vertebrate teeth, bones and fossil coquinas.
The B. Balsvikensis zone is characterized by a sandier facies, which is exceptionally poor in reptile remains contrary to the Mammillatus zone and manifests only small fishes and sharks.
The marine extinction observed between these two zones corresponds to an environmental shift, but it also correlates with a recognized trans‐Atlantic mid‐Campanian extinction event and might have been part of a broader global phenomenon.
II.2. Samples pre‐treatment
Due to the high lipophilicity of dioxins, the analysis requires many purification steps. A multi‐ step procedure consisting of sample extraction, adsorption chromatography columns clean‐up, and finally, analysis using high‐resolution gas chromatography coupled to high‐resolution mass spectrometry (HRGC/HRMS) the isotopic dilution mode to isolate and quantify these analytes. The aim of the extraction step is to transfer the compounds of interest into a non‐polar solvent.
The following steps are in accordance to EPA method 1613b, 1994 : Tetra‐through Octa‐Chlorinated Dioxins and Furans by Isotope Dilution HRGC/HRMS.
II.2.i. Preparation of the samples
An average of 10 g of the sample was ground in a mortar and homogenized with sodium sulfate Na2SO4 (≈25 g) in order to remove water from the sample. This mixture was ground until obtaining a fine powder and put in an extraction thimble. The internal standard 13C‐PCCD/Fs in toluene at 20‐40 pg µL‐1 (see appendix 5 to know all the compounds present in this internal standard) was spiked into the sample, 25 µL with a Hamilton syringe, prior to extraction in a Soxhlet device (see appendix 6 for scheme). During the extraction the sample was refluxed in toluene during 24 hours. This extraction solvent was removed using a rotary evaporator. Youngest Oldest
17
II.2.ii. Multi‐layers H2SO4 silica column
First, a glass‐wool plug was placed at the bottom in a 15 mm ID Chromatography column, in order to keep the different sorbents. This column was prewashed with ethanol, n‐hexane and dichloromethane. Secondly, this column was packed with : 3 cm of potassium hydroxide‐silica gel (KOH), 0.5 cm of neutral silica gel 60, 3 cm of 40 % of acid (H2SO4) silica gel, 1.5 cm of 20 % of acid (H2SO4) silica gel, 1 cm of neutral silica gel 60 and 1 cm of anhydrous sodium sulfate Na2SO4 (Fluka). Thirdly, the column was washed with n‐hexane with a volume of 2 times the column height. Last, the sample was added on the top of the column and extracted with n‐hexane with a volume of 4 times the column height (appendix 7). The extraction solvent was evaporated in order to obtain a final volume between 1 and 5 mL. This column is used to remove non‐polar, polar interferences including organic matter.
II.2.iii. Alumina oxide column
First, a glass‐wool plug was placed at the bottom in a 15 mm ID Chromatography column, in order to keep the different sorbents. This column was prewashed with ethanol, n‐hexane and dichloromethane. Secondly, this column was packed with : 9 g of Basic Alumina B‐Super grade I and 3 g of anhydrous sodium sulfate Na2SO4. Thirdly, the column was washed with n‐hexane with a volume of 2 times the column height. Last, the sample was added on the top of the column and the elution was performed in two steps. The first fraction was for the "bulk" PCBs and the second, for the planar PCBs, PCDDs and PCDFs. The first elution was done with 70 mL hexane/dichloromethane (49:1) and the second elution with 100 mL hexane/dichloromethane (1:1) (appendix 7). The second fraction was evaporated in order to obtain a final volume between 1 and 5 mL. II.2.iv. Carbon column First, a glass‐wool plug was placed at the bottom in a 14 mm ID Chromatography column, in order to keep the different sorbents. This column was prewashed with ethanol, n‐hexane and dichloromethane. Secondly, this column was packed as follows : 1.5 g of a carbon‐celite mixture realized with 3.6 g of Carbopack C 80‐100 mesh and 16.4 g of celite 545 Fluka (kept in a desiccator at room temperature) and approximately 1 cm of anhydrous sodium sulfate Na2SO4. Prior to use it, this column was rinsed by two times the column height, with n‐hexane. The elution was also performed in two steps : the first fraction, containing planar polychlorinated biphenyls (PCBs) and polybrominated diphenyls ethers (PBDEs), was eluted with 7 mL of n‐hexane and the second fraction, which gather the planar PCDDs and PCDFs, was eluted with 80 mL of toluene. This second fraction was evaporated in order to obtain a final volume between 1 and 5 mL (appendix 7).
Jensen and Sundström discovered the capability of carbon to separate the PCBs on the number of ortho chlorine atoms. This can be explained by the fact that the number of ortho chlorine atoms is related to the planarity of the different PCBs. PCBs with no or one chlorine atom in the ortho position can adapt a planar or semi‐planar configuration. Given that PCDDs and PCDFs are also planar, but at a lower concentration that PCBs, it is important to separate them before GC/MS analysis (VAN BAVEL, 1995).
Figure 8. Schematic presentation of the various steps of extraction and cleaning of sediment samples.
Before being analyzed on the HRGC/HRMS, the samples were poured in vials containing 25 µL of a recovery standard : 13C‐PCCD/Fs in toluene at 20 pg µL‐1 (see appendix 8). The total volume of vials was reduced under nitrogen stream until approximately 50 µL. If the GC/MS analysis was not performed the same day, the vials were stored in the dark at <‐16°C.
II.3. High Resolution Gas Chromatography/High Resolution Mass
Spectrometer (HRGC/HRMS)
A GC/MS instrument is composed of a GC instrument connected to a MS instrument. The GC instrument is used to separate a mixture on boiling points and interactions with the stationary phase of the GC column. By passing the mixture through a long capillary column, the different components of the mixture emerge and elute, one by one, and are recorded like peaks with a specific retention time, enabling further identification and quantification. The MS instrument, then, ionizes each of the components isolated from the mixture by electron impact into several fragments and separates them. This leads to a characteristic mass spectra (m/z ratio). The connection between the two instruments is performed with two pumps to produce a high vacuum > 10‐7 Pa range (Photograph in appendix 10). The "high" vacuum conditions are important to avoid : Ion scattering : if the ions collide with any residual gas molecules their trajectory will be modified resulting in peak broadening. Contamination : residual gases in the ionization chamber are also ionized together with the sample material giving rise to an instrument background. Mass resolution can be defined as follows : R=m/∆m where m is the mass and ∆m the mass difference calculated at 10 % of the valley (see appendix 11). Usually, dioxin analysis is performed at R=10 000. This allows distinction between two fragments whose mass varies only 0.01. This results in higher sensitivity, selectivity and signal‐to‐noise (S/N) ratio and lowers the limit of detection (LOD) and limit of quantification (LOQ).
OCDD concentrations were determined by using a HP 6890 GC with a micromass AutoSpec mass spectrometer operated in the electron impact selected ion monitoring (SIM) mode at a resolution R > 10 000 MU (10 % valley). The GC was equipped with a BP5MS capillary column (0.25
300 mL toluene
sample in 3x1 mL hexane elution with 10 mL hexane
elution with 80 mL hexane/MeCl2 (49:1) elution with 100 mL hexane/MeCl2 (1:1)
elution with 7 mL hexane elution with 80 mL toluene
sample in 3x1 mL hexane
19 µm film thickness; 30 m length x 0.25 mm i.d., SGE Analytical Science, Australia). The column oven temperature was programmed from 90 (1.5 min) to 260 °C at a rate of 9 °C min‐1, and to 300 °C at 6.5 °C min‐1 which was held for 4 min, then finally to 310 °C at 30°C min‐1.
II.4. Isotope dilution principle
Isotope dilution principle is not the fruit of analytical chemistry. Indeed, zoologists were used to use the catch and release methods for fish population estimation in ponds, before the discovery of isotopes. The principle is quiet simple : a known amount of fish were caught, labeled and released in the pond with the other fish. Once the labeled fish were intermingled with all the others, a second capture was made. This step enables zoologists to know the ratio of labeled and unlabeled fish. Knowing this ratio and the total amount of labeled fish, zoologists could easily estimate the total population of fish in the pond. For example, if we have caught 5 fish in order to labeled them and after the second capture, we have obtained a ratio of 1/3, the population of fish in the pond N is equal to : 1/3=5/(N‐5), so N= 15/1+5=20 fish. This paradigm demonstrates the exact working principle of isotope dilution (MEIJA & MESTER, 2008). The ratio 13C/12C is average 1.1%. The skeletal structure of dioxins includes 12 carbon atoms. A 13C labeled standard dioxin will have a 12 mass units greater than the dioxin molecules originally present in the sample. Thanks to a high resolution mass spectrometer, it is easy to separate the signal of initially present dioxin molecules from one of the standards added. Consequently, it is possible to determine the concentration of dioxins in the sample.The internal standard shares the same chemical and physical properties as the compounds of interest and is used to control the extraction and cleanup efficiency, to adjust variations in analytical response due to instrumental and/or matrix effects and variations in the amount of sample provided for analyses due to variable injection volumes. The recovery standard allows the measurement of the rate of recovery of internal standard added just before the extraction step. The calibration standard works as a single point calibration standard (see appendix 9).
Isotope dilution improves the precision and the accuracy of analytical results. This method can be used to identify native analytes in samples, it also improves the accuracy of analytes at ultra‐ trace concentrations and it can correct for loss of analytes during preparation. (See appendix 14 for the identification and quantification of dioxin congeners on MassLynx).
II.5. Elemental Analyzer‐Isotopic Ratio Mass Spectrometry (EA‐IRMS)
Isotope Ratio Mass Spectrometry (IRMS) is a specialized technique used to provide information about the geographic, chemical and biological origins of substances. This powerful tool had been used to study the origin of PAHs in sediments of Lake Erie, lower Great Lakes, USA (SMIRNOV et al., 1998). This technique permits the establishment of δ13C, which was introduced in 1950 by McKinney to easily establish the isotopic ratios (CALDERON, 2005) :
δ
13C(‰)= (
‐1)*1000
Delta values are either heavier (enriched) or lighter (depleted) than the standard. For example, if a sample has a delta value of +5 ‰ then it is 5 parts in 1000 enriched in 13C compared with the
Figure 9. Principle of isotope dilution from population analysis in zoology (MEIJA & MESTER, 2008).
standard. If it has a delta value of ‐5 ‰, then it is 5 parts in 1000 depleted in 13C. The standard used for carbon was the Pee Dee Belemnite (PDB), based on a Cretaceous marine fossil, Belemnitella americana, which was from the Pee Dee Formation in South Carolina, USA (Photographs in appendix 12). It was replaced by a artificial Vienna Pee Dee Belemnite in 1987, because of the exhaustion of this resource (VERKOUTEREN, 1999).
13
C/
12C
PDB=0.11237
The standard used for nitrogen is the atmosphere. The International Atomic Energy Agency (IAEA) now controls the creation and distribution of all isotopic standard materials.
Figure 10. Principle of a EA‐IRMS in a combustion mode (Nature 's fingerprint, a division of Molecular Isotope Technologies LLC).
The sample is weighed and placed in a tin or silver capsule, tightly crimped to avoid any trapping of air that would perturb the combustion. Then this capsule, containing the sample, is placed in an autosampler carousel, which leads it to a combustion furnace at 1050°C. When O2 is introduced, the tin capsule ignites. The Sn oxidation creates an exothermic "flash combustion" at 1800°C (Cr2O3 and Co3O4 as oxidant agents), ensuring the complete combustion and oxidation of the sample. Carbon, hydrogen and nitrogen compounds from the sample are respectively converted in CO2, H2O and N2. Any gas formed which contains halogens or sulfur is chemically removed and the remaining combustion products are sent into a 650 °C Cu+ reducing reactor, where incomplete combustion products from the oxidation tube (NOx, CO, etc.) are reduced and excess O2 is removed. Water is then chemically scrubbed from the helium. The final product gases (N2, CO2) are separated on a gas chromatography column and detected by a Thermal Conductivity Detector (TCD) also called katharometer, before entering the MS. TCD is a universal detector but its sensitivity is lower compared to this of a Flame Ionization Detector (FID) (See appendix 13 for more explanations about the functioning of a TCD). TCD generates an electrical signal proportional to the concentration of the gases presents. The gases effluents are then sent to the MS. (MUCCIO & JACKSON, 2009) (GRASSINEAU, 2006).
Samples are analyzed using an EuroEA3024 elemental analyzer (Eurovector, Milan, Italy) coupled to a Isoprime isotope‐ratio mass spectrometer (GV‐Instruments, Manchester, UK). The standard deviation of ten replicated samples of wheat was 0.09 ‰. The reactor furnace was set to 1050 °C and the combustion reactor tube packed with quartz wool, chromium oxide and silvered cobaltous cobaltic oxide. Reduction furnace was set to 650 °C and the reduction reactor tube was packed with quartz wool and copper. Oven temperature was 120 °C and He flow 100 mL min‐1. Samples were initially dried in an oven at 80°C overnight and then ground to a homogeneous powder in a shatterbox. Isotope ratios of carbon are reported in parts per mil (‰) and all values reported are relative to the international standard PDB by conventional delta notation (δ13C). Accuracy of the isotope ratio analysis for carbon was verified using wheat (bulk carbon : δ13C = ‐27.19 ‰, SD = 0.14 ‰, n=14), (organic carbon : δ13C = ‐26.23 ‰, SD = 0.075 ‰, n=7).
21
III. Results and Discussion
III.1. Results of HRGC/HRMS analysis
III.1.i. Results of the blanks
One blank containing ≈ 25 g of anhydrous sodium sulfate Na2SO4 was analyzed with every three samples. These blanks reflect the contamination of the laboratory (fume hood and Soxhlet extractors, flasks…). The calculus of the mean of these four blanks and the standard deviation (SD) allow to establish the limit of detection (LoD) for each congener as follows : LoD pg g‐1 3 x SD pg g‐1 Figure 11. Repartition of the concentrations of the different PCDD/Fs congeners in comparison with their LoD, first blank prepared. Figure 12. Repartition of the concentrations of the different PCDD/Fs congeners with their LoD, last blank prepared. The first blank shows relatively high concentrations in dioxins and particularly in OCDF and OCDD congeners, which one is a congener of interest. These concentrations decrease in the order of blank n°1 to 4 as we can see above. Before each extraction, the Soxhlet apparatus was washed with toluene during 24 hours. Given that the samples have very low concentrations of dioxins, the Soxhlet had become more and more clean after four series of extraction. (Blank n°2 and 3 in appendices 15 and 16.)
III.1.ii. Results of the sediment samples
The results for the eleven sediment samples are not supporting our hypothesis and only very small amounts of dioxins were found. All the concentrations found in picograms per grams of dry weight samples fall below the LoD. Moreover, in the publications where OCDD congener was detected in different types of ball clay products, the concentrations were in the range of several 100 ng g‐1 dw (HORII, et al., 2008). There is, consequently, a 105 factor of difference. All the results compared with the LoD are available in appendices 18 to 28. Figure 13. Distribution of the concentrations of PCDD/Fs found in the sediment sample n°6. However, the concentration of OCDD in sample n°3, n°9 and n°11 seems to be slightly higher than in other samples. This might give an indication in which layers to sample in future investigations. Figure 14. OCDD concentrations in the series of sediment samples. Maybe three of them are slightly different. The fact that OCDD concentration seems higher in sediment n°3 is not unexpected given that this sediment is a glacial clay, and thus, contains a lot of kaolin. Sediment n°8 to 11 come from a watercourse. OCDD concentrations in sediment n°9 and 11 are slightly higher than in sediment n°8 and 10 because they come from the upper part of the watercourse (top and one meter below), which zone is less agitated than the river bed, and consequently, fine clay is more gathered. Sediment n°1, 2, and 4 to 7 contain principally sands.
23
To further study the origin/source of dioxins, it is also interesting to have a look on the congener profiles. Indeed, congener profile of mined clay products (not only ball clay) is well‐defined where the OCDD congener dominates the others PCDDs congeners and the concentrations of these congeners decrease with corresponding degree of chlorination. 1,2,3,7,8,9‐HxCDD predominates relative to the two other 2,3,7,8‐ substituted HxCDD isomers (5/17/78). Concentrations of PCDFs are reported to be very low or non‐detectable. This pattern is characteristic and differs from the profile found for anthropogenic sources of contamination (HORII et al., 2008) (FERRARIO et al., 2000). Figure 15. Distribution of the different PCDD/Fs in a raw ball clay, pattern expected for all clay products (FERRARIO, BYRNE, & CLEVERLY, 2000). In stack emissions, PCDF congeners can be measured at the same concentrations as PCDDs. In combustion emission, the most prevalent PCDD congeners are OCDD and 1,2,3,4,6,7,8‐HpCDD, like in mined clay products, but 1,2,3,4,6,7,8‐HpCDF, OCDF, 1,2,3,4,7,8‐HxCDF, 2,3,7,8‐TCDF and 2,3,4,6,7,8‐HxDCF congeners also dominate. In iron ore sintering, the dominant congener in emission is 2,3,7,8‐TCDF (FERRARIO et al., 2000). The typical pattern of waste incineration processes can be describe rapidly as follows :
[PCDFs]>[PCDDs]
PCDFs : [Cl4] ≈ [Cl5] ≈[Cl6] > [Cl7] > [Cl8]
PCDDs : [Cl4] < [Cl5] > [Cl6] < [Cl7] ≈ [Cl8] (MOHR et al., 1999).
In Sweden, chlorinated phenols (CPs) and particularly PCP and sodium pentachlorophenol (Na‐PCP) were used like wood preservative, until its legislation in 1977. Production of these compounds results in the formation of impurities like dioxins and these compounds are also known to produce OCDD under certain conditions (heat, photolysis in environment and in biota). CPs have the potential to migrate through sediment cores, even 50‐60 years after their use. PCP profiles are characterized by a dominance of OCDD, but also marked by relatively high concentrations of PCDFs (dominance of OCDF, followed by 1,2,3,4,6,8,9‐HpCDF and 1,2,4,6,8,9‐HxCDF) and the ratio of the ∑PCDDs to ∑PCDFs concentrations (D/F ratio) ranges from 1 to 10. Besides, 1,2,3,6,7,8‐HxCDD is usually present as the dominant isomer among the toxic HxCDDs (GAUS et al., 2001) (GAUS et al.). Gaus et al. assume that the characteristic pattern of clay materials could be explained by OCDD formation from diffused PCP in sediment cores. This could be a way of explanation of so high concentrations of OCDD in clay samples and the lower PCDFs concentrations. They also suggested that dechlorination processes, via the lateral (2, 3, 7, 8) or peri (1, 4, 6, 9) positions, have altered the original PCDD/Fs signature and it has lead to this specific pattern. Two graphics showing the distribution of dioxins in a Japanese PCP formulation (13.4 % active ingredient, expired in 1971) and
in sediments from Haihe River, in China exposed to a PCP and Na‐PCP contamination are available in appendices 29 and 30.
The congener profiles have been treated as follows : only the concentrations which were twice the level of the blank have been reported after background subtraction. These profiles are available in appendices 31 to 35. Unfortunately, it is impossible to make a correlation between the profiles obtained and those described above due to the low concentrations of dioxins in our samples.
III.2. Results of EA‐IRMS analysis
Measurements of total carbon content (bulk carbon) and organic carbon (Corg) in sediments have been performed to estimate what could be the possible contribution of organic matter in sediment on the isotope ratios. The content in organic carbon is based on the effectiveness of remineralization of organic matter taking place in the sediment. A method based on the acidification of the samples has been tested (See appendix 36 to read the protocole) in order to remove carbonates. Figure 16. Delta 13C values of the different sediment and fossil samples for bulk carbon and organic carbon. Error bars represent SD. The bulk isotope ratio analysis for carbon shows two groups : the first one, from S1 to S7 have δ13C values close to zero. The second group, from S8 to S11, shows more depleted value ranging from ‐7 ‰ to ‐25 ‰. This is relevant because the two groups correspond to geological groups : S1 to S7 sand layers and S8 to S11 watercourse sediments. The δ13Cbulk values of sand layers close to zero can be interpreted by the large amount of carbonates from calcareous shell. The δ13Cbulk values of river sediments close to ‐26 ‰ can be interpreted by remains of pure carbon from prehistoric plants. After the acidification treatment, δ13Corganic values of sand layers have completely changed and are now very depleted (≈‐29 ‰). δ13Corganic values of river sediments are less affected by the treatment, because they contain less inorganic carbon. δ13Corganic values of river sediments are around ‐26 ‰.
25 Table 4. Carbon content in the samples and pH. Identification of the samples Depth (m) Total Carbon (%) Total Organic Carbon (%) Total Inorganic Carbon (%) a) Proportion of organic C to the total C pH S1 1.8 6.74 0.045 6.70 0.007 9.09 S2 1.35 5.65 0.046 5.60 0.008 9.18 S5 1.2 4.21 0.071 4.14 0.017 9.13 S6 1 2.31 0.083 2.23 0.036 8.75 S4 0.8 2.07 0.130 1.94 0.063 8.63 S7 0.65 4.11 0.107 4.00 0.026 8.76 S11 0 1.3 1.606 ‐0.31 1.236 3.23 S9 ‐1 1.61 0.853 0.76 0.530 7.17 S10 ‐20 1.62 0.891 0.73 0.550 6.98 S8 ‐25 0.51 0.312 0.20 0.611 5.76 a) The negative value of TIC is due to errors related to the acidification treatment of the samples. Figure 17. Depth distributions of total carbon, total organic carbon, total inorganic carbon, proportion of organic carbon to the total carbon, pH and δ13Corganic for sand sediments (S1, S2, S4 to S7) and river sediments (S8 to S11). The glacial clay
S3 is not represented in this graphic because it does not match with age deposition.
The youngest samples contain more carbon material (S1 and S2). S1 and S2 had a terrestrial environment with conifers and very few flowers and leaves (indeed very few fossils have been discovered in the B. Balsvikensis zone) whereas S5 to S8 had a marine environment. The samples from the watercourse have a dominant organic part contrary to sand sediments, which are much more inorganic. The sudden change in pH values for the river sediments shows also dramatic changes in the paleoenvironment. For the samples S4 and S7, it is possible to observe a slight increase in the proportion of organic carbon, which can lead to think that something was going on. Some residue of charcoal found in the river sediments testify of the past forest fire activities in this region.
TOC does not correlate with PCDD/F content. Indeed, S9 has the higher OCDD concentration whereas its TOC value is 0.853 %. Therefore, the comparison of the PCDD/F content and the TOC indicates that the percentage of organic carbon is not a reliable proxy of the PCDD/F concentration in clays. It is assumed that not the content of organic carbon, but the type of organic matter could be a very important factor of dioxin enrichment in kaolinitic clays. Sand sediments Watercourse sediments TC (%) TOC (%) TIC (%) Proportion of organic C to the total C pH δ 13C organic (‰)
III. 3. Scenario proposed by Schmitz et al. to explain why dioxins are loaded
in clays
Schmitz et al. have made a distinction between "primary kaolin" and "secondary kaolin". Primary kaolin or in situ kaolin contains many minerals and pieces of rocks because it is formed by weathering of acid‐rocks like granite. On the opposite, secondary kaolin is much more pure clay because it is reworked by geological processes like erosion, transportation and sedimentation of the rocks contained in primary kaolin. This distinction is relevant because it has shown that primary kaolin does not contain dioxins contrary to secondary kaolin which exhibits the natural formation pattern. Based on these findings, Schmitz et al. have proposed a model gathering several hypothesis advanced in previous studies to explain the accumulation of dioxins in secondary clays. Figure 18. Hypothetical geological and paleoenvironmental model of natural PCDD formation and accumulation in clays. The grey arrows indicate dioxin transfer paths (SCHMITZ et al., 2011). The source of dioxins is still unknown and can be released to the atmosphere by forest fires, for instance, and then by aerial deposition, soils act like a tank for dioxins. In that way, dioxins are loaded in soils. Photosynthesis phenomenon would be responsible of the decomposition of furans and the lower chlorinated dioxins. It is the abiotic formation. Other studies have proved that it is possible to create OCDD by catalytic way mixing Fe(III)‐montmorillonite with pentachlorophenol. The biotic formation envisages that microorganisms, bacteria can create precursors of dioxins.
Once loaded into the soils, it is likely that PCDD/Fs undergo other transformations by evaporation and biotransformation which promote PCDDs and particularly the most chlorinated forms because they are more refractory and more lipophilic. This could explain the so typical natural formation pattern observed.
Due to their hydrophobic nature, dioxins accumulate in organic matter and clay minerals and are transported by erosion in secondary kaolin. Secondary kaolin would have preserved dioxins through geological times (SCHMITZ et al., 2011).
27
Conclusion.
