Total or biomimetic extracts or direct contact exposure? [Elektronische Ressource] : comparative research towards a realistic ecotoxicological characterisation of sediments / presented by Thomas-Benjamin Seiler

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Dissertationsubmitted to the Combined Faculties for the Natural Sciencesand for Mathematics of the Ruperto-Carola University of Heidelberg, Germany,for the degree of Doctor of Natural SciencesPresented by: Diploma biologist Thomas-Benjamin SeilerBorn in: ErlangenDate of oral examination: …...................................Total or biomimetic extracts or direct contact exposure?Comparative research towards a realisticecotoxicological characterisation of sedimentsReferees: Prof Dr Thomas BraunbeckHeidelberg Institute of Zoology, University of HeidelbergProf Dr Heinz KarraschGeographical Institute, University of HeidelbergAcknowledgements"Each of us has cause to think with deep gratitude of those who have lighted the flame within us."Albert SchweitzerIf a PhD thesis represents more than just the product of laboratory work and scientific writing, this is surely due to the persons who were directly and indirectly involved. Hence, my deep gratitudes go to all those colleagues and friends accompanying me throughout these very enjoyable years of scientific research.First of all, I would like to thank Prof Dr Thomas Braunbeck, Aquatic Ecology & Toxicology Section at the Heidelberg Institute of Zoology, for giving me the opportunity to prepare my thesis under his official supervision and, thus, being my first referee.

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Dissertation
submitted to the Combined Faculties for the Natural Sciences
and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany,
for the degree of Doctor of Natural Sciences
Presented by: Diploma biologist Thomas-Benjamin Seiler
Born in: Erlangen
Date of oral examination: …...................................Total or biomimetic extracts or direct contact exposure?
Comparative research towards a realistic
ecotoxicological characterisation of sediments
Referees: Prof Dr Thomas Braunbeck
Heidelberg Institute of Zoology, University of Heidelberg
Prof Dr Heinz Karrasch
Geographical Institute, University of HeidelbergAcknowledgements
"Each of us has cause to think with deep gratitude of those who have lighted the flame within us."
Albert Schweitzer
If a PhD thesis represents more than just the product of laboratory work and scientific writing, this is
surely due to the persons who were directly and indirectly involved. Hence, my deep gratitudes go to
all those colleagues and friends accompanying me throughout these very enjoyable years of scientific
research.
First of all, I would like to thank Prof Dr Thomas Braunbeck, Aquatic Ecology & Toxicology Section
at the Heidelberg Institute of Zoology, for giving me the opportunity to prepare my thesis under his
official supervision and, thus, being my first referee.
I am no less thankful to Prof Dr Henner Hollert, Department of Ecosystem Analysis at RWTH Aachen
University, my supervisor, who let me experience as much freedom as required to evolve my academic
as well as creative spirits. Besides, I am truly grateful to Prof Hollert and Dr Jan Wölz for the pleasant
collaboration during the 2007 establishment of the Department of Ecosystem Analysis at RWTH
Aachen University.
My expressed appreciation also go to Prof Dr Heinz Karrasch for being my second referee and by this
showing his interest in research on sediment toxicity.
What made the years of my thesis so special was a very friendly working atmosphere created by the
various colleagues in Heidelberg and Aachen, in particular Nina Best, Kerstin Bluhm, Markus
Brinkmann, Marit Ernst, Tilman Floehr, Andrea Gerstner, Prof Dr Arnold V Hallare, Sebastian
Hudjetz, Dr Steffen Keiter, Dr Thomas Kosmehl, Dr Eva Lammer, Erik Leist, Sibylle Maletz, Sabine
Niebergall, Sabrina Peddinghaus, Dr Paula Suares Rocha, Ruben Strecker, Dr Jan Wölz and Hanno
Zielke. Furthermore, I would like to thank Conny Bernecker, Sebastian Heger and Kerstin Winkens for
their professional support of my laboratory work.
Essential for good scientific research is reliable and inspiring collaboration. From the large number of
colleagues I had the pleasure to work with, my sincere thanks go to the following: Dr Werner Brack,
Tobias Schulze, Dr Georg Streck (UFZ Helmholtz Centre for Environmental Research, Leipzig), Dr
Katrin Schwab (Currenta, Leverkusen) and Dr Andrew C Rastall (Rifcon, Hirschberg) for
collaborating in the research on sediment extraction, PD Dr Wolfgang Ahlf (Technical University
Hamburg-Harburg, Hamburg), Dr Ute Feiler, Dr Peter Heininger, Denise Spira (Federal Hydrological
Institute, Koblenz), Daniel Gilberg (ECT Oekotoxikologie, Flörsheim), Dr Monika Hammers-Wirtz
(gaiac, Aachen), Dr Sebastian Höss (Ecossa, Starnberg), Dr Michael Meller (ecotox consult,
Ludwigshafen), Dr Helga Neumann-Hensel (Dr. Fintelmann & Dr. Meyer, Hamburg), Dr Richard
Ottermanns (RWTH Aachen University, Aachen) and Dr Jürgen Weber (Nordum, Rostock) for
cooperation in the SeKT joint research project on sediment contact tests. I am furthermore thankful to
the German Federal Ministry of Education and Research (BMBF) for funding the SeKT project.
I also thank the SETAC Europe Student Advisory Council, especially Markus Brinkmann, Dr. Amy
Brooks, Mirco Bundschuh, Marloes Caduff, Andre Dabrunz, Cornelia Kienle and Karen Van Hoecke,
for the pleasant time.
Always at my side were my family, my dearest friends, and Astrid.Content I
Content
Summary...................................................................................................................................12
Zusammenfassung.................14
1 Introduction........................18
1.1 The application of scientific research within ecotoxicology..........................................18
1.2 A necessity to understand the processes in aquatic ecotoxicology.................................19
1.3 Strategies for the ecotoxicological characterisation of sediments..................................20
1.4 Comparative investigations of sediment extraction and contact tests............................21
1.5 Objectives of the study...................................................................................................24
1.6 References......................................................................................................................25
2 Introductory Part A.............32
2.1 Abstract.......................32
2.2 Abbreviations..............32
2.3 Introduction....................................................................................................................32
2.4 A short outline of extraction...........................................................................................35
2.4.1 Geosorbents, accessibility, and extractability.........................................................35
2.4.2 Application of soil and sediment extracts...............................................................37
2.4.3 Fractions and the toxic potential.............................................................................38
2.5 Extraction and clean-up..................................................................................................38
2.6 Possible alterations affecting reliability of extracts.38
2.6.1 Extraction and extract testing.................................................................................39
2.6.2 Sample processing prior to extraction....................................................................41
2.7 Recommendations for research and technology.............................................................42
2.7.1 Quality of data........................................................................................................42
2.7.2 Future recommendations of extract preparation.....................................................43
2.8 Summary and conclusion...............................................................................................45
2.9 References......................................................................................................................46
3 Chapter 3............................54
3.1 Abstract.......................54
3.2 Introduction....................................................................................................................55
3.3 Materials and Methods...................................................................................................57
3.3.1 Sediment samples...................................................................................................57
3.3.2 Membrane dialysis extraction (MDE)....................................................................57
3.3.3 Soxhlet extraction...................................................................................................58
3.3.4 Cell cultures............................................................................................................59
3.3.5 Statistical analysis...................................................................................................59
3.3.6 Bioassays................................................................................................................60
3.4 Results and Discussion...................................................................................................61
3.4.1 Comparison to Soxhlet extraction..........................................................................62
3.5 Conclusions....................................................................................................................68Content II
3.6 References......................................................................................................................69
4 Chapter 4............................76
4.1 Abstract.......................76
4.2 Introduction....................................................................................................................76
4.3 Material and methods.....................................................................................................78
4.3.1 Chemicals...............................................................................................................78
4.3.2 Study area and sample collection...........................................................................78
4.3.3 Sample storage and preparation..............................................................................78
4.3.4 Grain size distribution and content of organic carbon............................................78
4.3.5 Extraction Methods.................................................................................................79
4.3.6 Extraction and hydrolysis of sediment residues.....................................................80
4.3.7 Extracts preparation................................................................................................80
4.3.8 Silica gel fractionation............................................................................................80
4.3.9 Gas chromatography / Mass spectrometry.............................................................80
4.3.10 Toxicity testing.....................................................................................................81
4.3.11 Data analysis.........................................................................................................81
4.4 Results and discussion.......82
4.4.1 Chemical analysis..............82
4.4.2 Comparison of different extraction approaches......................................................83
4.4.3 Compound specific extractability of PAH..............................................................85
4.4.4 Toxicity analysis.....................................................................................................87
4.5 Conclusions....................................................................................................................88
4.6 Acknowledgements........................................................................................................89
4.7 References......................................................................................................................89
5 Chapter 5............................98
5.1 Abstract.......................98
5.2 Abbreviations..............98
5.3 Introduction..................99
5.4 Material & Methods.....................................................................................................100
5.4.1 Samples and sampling..........................................................................................100
5.4.2 Extract treatment and storage...............................................................................101
5.4.3 Soxhlet extraction (SOX).....................................................................................101
5.4.4 Membrane dialysis extraction (MDE)..................................................................101
5.4.5 Methanol/Water extraction (MEOH)....................................................................101
5.4.6 Hydroxypropyl-β-cyclodextrin extraction (HBCD).............................................101
5.4.7 Tenax-TA extraction (TENAX)............................................................................102
5.4.8 Accelerated solvent extraction (ASE)...................................................................102
5.4.9 Gel permeation chromatography (GPC)...............................................................102
5.4.10 Accelerated membrane-assisted clean-up (AMAC)...........................................103
5.4.11 Direct accelerated membrane assisted clean-up (DAMAC)...............................103
5.4.12 Process controls..................................................................................................103
5.4.13 Cell cultures........................................................................................................103Content III
5.4.14 Neutral red retention assay (NR)........................................................................103
5.4.15 EROD induction assay (EROD).........................................................................104
5.4.16 Zebrafish embryo assay (Fish embryo test; FET)...............................................104
5.4.17 Graphical evaluation and statistical analysis......................................................105
5.5 Results..........................................................................................................................105
5.5.1 Process controls....................................................................................................105
5.5.2 Comparability with respect to extraction power...................................................105
5.5.3 Ranking according extraction power and reproducibility....................................108
5.5.4 Correlations concerning strength of effect potential............................................109
5.6 Discussion.................111
5.7 Conclusion & Outlook..................................................................................................112
5.8 Acknowledgements.......................................................................................................113
5.9 References....................................................................................................................113
6 Chapter 6..........................122
6.1 Abstract.....................122
6.2 Abbreviations............123
6.3 Introduction..................................................................................................................123
6.4 Material & Methods.....................................................................................................126
6.4.1 Samples and sampling..........................................................................................126
6.4.2 Chemicals and solvents.........................................................................................126
6.4.3 Clean-up with gel permeation chromatography (GPC)........................................126
6.4.4 Accelerated membrane assisted clean-up (AMAC)..............................................127
6.4.5 Accelerated solvent extraction (ASE)...................................................................127
6.4.6 Membrane dialysis extraction (MDE)..................................................................127
6.4.7 Soxhlet extraction (SOX).....................................................................................128
6.4.8 Direct Accelerated Membrane Assisted Clean-up (DAMAC)..............................128
6.4.9 Tenax-TA extraction (TENAX)............................................................................128
6.4.10 Hydroxypropyl-β-cyclodextrin extraction (HBCD)...........................................129
6.4.11 Methanol/Water extraction (MEOH)..................................................................129
6.4.12 Ultrasonic extraction with hexane:acetone.........................................................129
6.4.13 Extraction with methanolic hydrolysis...............................................................129
6.4.14 Quality control....................................................................................................130
6.4.15 Instrumental analysis..........................................................................................130
6.4.16 Biological analysis.....130
6.4.17 Statistical analysis...............................................................................................130
6.4.18 Artificial mixtures and calculation of Toxic Equivalents (TEQs)
for EROD-data....................................................................................................131
6.5 Results..........................................................................................................................131
6.5.1 Total amounts of PAHs and chlorinated compounds – first extraction step.........131
6.5.2 Second and third extraction..................................................................................132
6.5.3 Compound specific composition of extracts.........................................................133
6.6 Discussion....................................................................................................................136Content IV
6.6.1 Ranking according to extraction power – first extraction step.............................136
6.6.2 Comparability with respect to extraction efficiency – first extraction step..........138
6.6.3 Comparison of all three extraction steps..............................................................140
6.6.4 Compound specific correlation analysis...............................................................140
6.6.5 Comparison with results from bioanalytical methods..........................................143
6.6.6 Confirmation of EROD-induction using artificial mixtures........143
6.7 Conclusion & Outlook..................................................................................................145
6.8 Supplemental data........................................................................................................146
6.9 Acknowledgements......................................................................................................146
6.10 References..................................................................................................................147
Supplemental data..............................................................................................................152
7 Chapter 7..............................................................................................................................162
7.1 Abstract.....................162
7.2 Introduction..................163
7.3 Material and Methods...................................................................................................165
7.3.1 Sediments, Substances and Spiking......................................................................165
7.3.2 Extractions............................................................................................................166
7.3.3 Fish embryo test with Danio rerio........................................................................169
7.3.4 Data processing and statistical analyses...............................................................170
7.4 Results......................171
7.5 Discussion....................................................................................................................173
7.6 Outlook.........................................................................................................................175
7.7 Acknowledgements .....................................................................................................176
7.8 References....................................................................................................................176
8 Chapter 8..........................184
8.1 Abstract.....................184
8.2 Introduction..................................................................................................................184
8.3 Material & Methods.....................................................................................................187
8.3.1 Samples and sampling..........................................................................................187
8.3.2 Extraction of sediment samples............................................................................188
8.3.3 Chemical analysis.................................................................................................188
8.3.4 Bioassays..............................................................................................................188
8.3.5 Graphical evaluation, calculation of potentials, and statistical analysis...............189
8.4 Results..........................................................................................................................189
8.5 Discussion....................................................................................................................192
8.6 Conclusions & Perspectives.........................................................................................195
8.7 References....................................................................................................................196
9 Introductory Part B...........204
9.1 Abstract.....................204
9.2 Introduction..................................................................................................................204
9.3 The significance of fish as sentinel species..................................................................207
9.4 The use of whole fish in sediment toxicity assessment................................................208