Summary ......................................................... I
General structure ............................................... V
Table of contents .............................................. VI
List of abbreviations .......................................... IX
1 Current challenges in ecotoxicology .......................... 2
2 Relevance of aquatic macrophytes and metabolomics ............ 4
2.1 Ecological functions & Phytotoxicity assessment ......... 4
2.2 An omics perspective .................................... 6
2.3 The idea spreads into various research fields ........... 8
2.4 Dynamic methodological developments .................... 10
3 Introducing a multivariate metabolic endpoint into an
ecotoxicological effect assessment framework ................ 12
3.1 Monitoring tools & Effect diagnosis .................... 12
3.2 Concentration and time as variables in metabolomics
studies ................................................ 17
3.3 Aims & Experimental studies ............................ 20
3.4 Combining ecotoxicological approaches and metabolic
fingerprinting ......................................... 24
4 The metabolic effect level index links multivariate
metabolic fingerprints to ecotoxicological effect
assessment .................................................. 30
4.1 Introduction on biochemical responses of aquatic
plants ................................................. 31
4.2 Material & Methods of the bioassay investigation ....... 34
4.2.1 Test system and design .......................... 34
4.2.2 Metabolic fingerprinting analysis ............... 36
4.2.3 Multivariate statistics & Effect analysis ....... 38
4.3 Results of isoproturon exposure ........................ 42
4.3.1 Treatment selection based on functional and
apical endpoints ................................ 42
4.3.2 Metabolic fingerprints of M. spicatum after
isoproturon exposure ............................ 44
4.3.3 Effect estimation with projection analysis and
MELI ............................................ 47
4.3.4 Comparison of fingerprinting-based effect
estimation approaches ........................... 50
4.3.5 Effect comparison from different levels of
biological organization ......................... 51
4.4 Discussion on effect estimation and interpretation ..... 54
4.4.1 Effect observation in the macrophyte bioassay ... 54
4.4.2 Ecotoxicological effect estimation for
multivariate responses .......................... 56
4.4.3 Adverse outcome pathway for M. spicatum
exposed to isoproturon .......................... 59
4.4.4 Are metabolic fingerprints indicative for
stress or toxicity? ............................. 61
5 Uncovering subtle chemical-induced effects in metabolic
fingerprints using regression-based chemometric tools ....... 64
5.1 Introduction of environmental exposure and
information-rich fingerprints .......................... 65
5.2 Material & Methods of the microcosm investigation ...... 67
5.2.1 Test system and design .......................... 67
5.2.2 Metabolic fingerprinting analysis ............... 68
5.2.3 Statistical analysis & Compound determination ... 70
5.3 Results of metabolic effect detection .................. 73
5.3.1 Key figures of metabolic fingerprints ........... 73
5.3.2 Data quality inspection & Correction measures ... 74
5.3.3 Effect exploration with chemometric tools ....... 76
5.3.4 Confirming the concentration-dependent effect
as plant response ............................... 80
5.4 Discussion on systematic variation and
ecotoxicological effects ............................... 86
5.4.1 The uncovered metabolic fingerprint ............. 86
5.4.2 The sensitive metabolic fingerprint ............. 89
6 Spatial and temporal variation in metabolic fingerprints
of field-growing Myriophyllum spicatum ...................... 94
6.1 Introduction ........................................... 95
6.2 Material & Methods ..................................... 97
6.2.1 Field investigation ............................. 97
6.2.2 Metabolic fingerprinting analysis ............... 99
6.2.3 Statistical analysis ........................... 100
6.3 Results ............................................... 102
6.3.1 Key figures of GC-MS-based M. spicatum
metabolic fingerprints ......................... 102
6.3.2 Metabolic variation of biological replicates
of field-growing M. spicatum ................... 103
6.3.3 Pattern analysis of metabolic fingerprints of
M. spicatum .................................... 103
6.3.4 Metabolic fingerprints in relation to
environmental parameters ....................... 104
6.3.5 Stoichiometry and amount of low molecular
weight compounds ............................... 106
6.4 Discussion ............................................ 108
6.4.1 Metabolic fingerprinting on field-growing
individuals of M. spicatum ..................... 108
6.4.2 Spatial and temporal variation of metabolic
fingerprints ................................... 109
6.5 Conclusion ............................................ 112
7 Diagnosis of field-relevant exposure and organism
performance using metabolic fingerprints of M. spicatum .... 114
7.1 Concentration-fingerprint relationships - more than
the sum of its single components ...................... 115
7.2 From lab to field - metabolic responses at different
tiers, compared to environmental exposure & directed
by study design ....................................... 119
7.2.1 The influence of exposure-relevant and
confounding factors ............................ 119
7.2.2 Sensitivity - a matter of population state
and data evaluation? ........................... 124
7.2.3 Discriminative power through specific pattern
and study design ............................... 126
7.3 A broad response spectrum of metabolic fingerprints -
the basis for MELI-based effect extrapolation ......... 128
7.3.1 Characteristic metabolic responses along the
effect cascade ................................. 128
7.3.2 The MELI links effect progression across
different biological response levels............ 132
7.4 Reflecting the central hypotheses ..................... 135
8 A perspective for field and MELI applications .............. 137
8.1 Applicability of metabolic fingerprinting for
environmental monitoring .............................. 137
8.2 GC-MS-based fingerprints of M. spicatum as
monitoring tool? ...................................... 139
8.3 MELI and threshold concentrations as tools for
predicting adverse effects ............................ 141
8.4 Further applications .................................. 142
References ................................................. 143
A1 Facts about GC-MS-based metabolic fingerprinting
approaches - in general and applied to M. spicatum ......... 158
A2 Additional Figures and Tables for Chapter 5 & 7 ............ 172
List of publications .......................................... 177
Danksagung /Acknowledgements .................................. 179
Eidesstattliche Versicherung .................................. 181
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