Refractory Organic Substancesin the Environment

Edited byF. H. Frimmel, G. Abbt-Braun, K. G. Heumann, B. Hock,H.-D. Ludemann and M. Spiteller

WILEY-VCH

VII

Contents

Preface XIX

List of Authors XXI

List of Abbreviations XXXI

1 Setting the Scene 1

1.1 The Relevance of Reference Materials — Isolation and GeneralCharacterization 1

1.1.1 Introduction 11.1.2 Concept of Standard and Reference Material 31.1.3 Reference Materials in the ROSIG Priority Program 41.1.4 Experimental Details 51.1.4.1 Origin of the Samples 51.1.4.2 Isolation 71.1.4.3 Analytical Methods 101.1.5 Discussion 111.1.5.1 Mass Balance of the Isolates 131.1.5.2 Spectroscopic Characterization in the UV and Visible Range 161.1.5.3 Elemental Analysis 191.1.5.4 Proton and Complexation Capacities 251.1.5.5 Size-exclusion Chromatography 281.1.6 Conclusions 33

References 36Additional References for ROS Literature 38

1.2 Element Determination and its Quality Control in Fractions of RefractoryOrganic Substances and the Corresponding Original Water Samples 39

1.2.1 Introduction 391.2.2 Description of Analytical Methods 411.2.2.1 Sample Treatment 411.2.2.2 Flame and Graphite Atomic Absorption Spectrometry

(FAAS, GF-AAS) 421.2.2.3 Inductively Coupled Plasma Mass Spectrometry (ICP-MS) 42

VIII Contents

1.2.2.4 Inductively Coupled Plasma Isotope Dilution Mass Spectrometry(ICP-IDMS) 42

1.2.2.5 Instrumental Neutron Activation Analysis (INAA) 431.2.2.6 Total Reflection X-Ray Fluorescence Spectrometry (TXRF) 441.2.3 Results of the Interlaboratory Study 451.2.3.1 Element Concentrations in ROS Fractions and their Original

Samples 451.2.3.2 Relative Enrichment Factors of Elements in Separated FA and

HA Fractions 491.2.3.3 Quality Control of Analytical Data 49

References 53

2 Structural Investigations 552.1 Heavy Metal and Halogen Interactions with Fractions of Refractory

Organic Substances Separated by Size-exclusion Chromatography 552.1.1 Introduction 552.1.2 SEC/ICP-MS System and the Isotope Dilution Technique 562.1.3 Determination of Heavy Metal-ROS Complexes in Fractions Separated

by SEC 592.1.4 Correlation of Metal—ROS Complexes with other Analytical Data 622.1.5 Kinetically Stable Cr(III) and Co(III) Complexes of ROS 642.1.6 Determination of Halogen—ROS Compounds in Fractions separated

by SEC 682.1.7 Conclusions 71

References 72

2.2 Characterization of Refractory Organic Substances and their MetalSpecies by Combined Analytical Procedures 73

2.2.1 Introduction 732.2.2 Size Distribution and Stability of ROS-Metal Species Assessed by

Ultrafiltration, EDTA Exchange, and Atomic Spectrometry 742.2.3 On-site Characterization of Aquatic Refractory Organic Substances and

their Metal Species by Mobile Fractionation and ExchangeTechniques 79

2.2.4 Conclusions 86References 87

2.3 Application of Nuclear Magnetic Resonance Spectroscopy to StructuralInvestigations of Refractory Organic Substances — Principles andDefinitions 89

2.3.1 Potential of Nuclear Magnetic Resonance (NMR) Spectroscopy for ROSStructure Elucidation 89

2.3.2 Quantification of Solution-state and Solid-state NMR Spectra of ROS 912.3.3 Assignment of ROS NMR Signals 92

References 94

Contents IX

2.4 Structural Characterization of Refractory Organic Substances bySolid-state High-resolution 13C and 15N Nuclear Magnetic Resonance 96

2.4.1 Introduction 962.4.2 Pulse Sequences Used 972.4.2.1 Cross Polarization Magic Angle Spinning (CPMAS) 972.4.2.2 Magic Angle Spinning (MAS) or Bloch-decay Spectra 972.4.2.3 Dipolar Dephasing Techniques (DD Techniques) 972.4.3 Lake Hohloh Samples 982.4.3.1 13C Solid-state Spectra 982.4.3.2 Proton Solution Spectra 1032.4.3.3 15N CPMAS Spectra 1042.4.4 Comparison of Samples from Different Locations 1072.4.4.1 13C Solid-state Spectra 1072.4.4.2 15N CPMAS Spectra 1102.4.5 Conclusions 1122.4.5.1 Methods 1122.4.5.2 Structural Results 113

References 114

2.5 Quantification of Substructures of Refractory Organic Substances byMeans of Nuclear Magnetic Resonance 225

2.5.1 Introduction 1152.5.2 Experimental Details 1162.5.2.1 Nuclear Magnetic Resonance 1162.5.2.2 Multistage Ultrafiltration 1162.5.3 Results from Solution-state NMR investigations 1172.5.3.1 General Considerations 1172.5.3.2 Aliphatic Structural Moieties 1182.3.3.3 Aromatic Structural Moieties 1182.5.4 Combination of Multistage Ultrafiltration and Two-dimensional

HMQC NMR 1252.5.5 Conclusions 227

References 228

2.6 Nuclear Magnetic Resonance Spectroscopy Investigations of SilylatedRefractory Organic Substances 129

2.6.1 Introduction 1292.6.1.1 Functional Group Analysis in Refractory Organic Substances 2292.6.1.2 Silylation of Refractory Organic Substances 2302.6.2 Materials and Methods 2322.6.3 Two-dimensional NMR Spectroscopy of Silylated Natural Organic

Matter 1322.6.3.1 Comparative Analysis of 2D NMR Spectra; Visualization of Conformity

and Variation by Calculation of Minimum and Difference Spectra 136

X Contents

2.6.4 NMR Functional Group Analysis of FA and HA Fractions Isolated fromSamples of Different Origin 137

2.6.4.1 Comparison of BS1 FA, HO10 FA, FG1 FA, HO13 FAand SUW FA 137

2.6.4.2 Comparison of BS1 HA, HO13 HA, and SUW HA 1402.6.4.3 Comparison of Results from NMR Functional Group Analysis with Data

Obtained from Complementary Methods 1412.6.5 Conclusions 143

References 244

2.7 Isotopic Evidence for the Origin and Formation of Refractory OrganicSubstances 246

2.7.1 Variations of Stable Isotope Abundances in the Course of the Synthesisand Diagenesis of Biomass 146

2.7.2 Bulk Isotope Analysis 1492.7.3 Pyrolysis-Gas Chromatography/Mass Spectrometry-Isotope-ratio

Mass Spectrometry (Py-GC/MS-IRMS) 2502.7.4 Bulk Isotopic Analyses of the Reference Samples 2522.7.4.1 Differences Between the Isotope Content of the Reference Samples 1522.7.5 Formation of Refractory Organic Substances in a Peat Profile of the

Hohlohsee 2542.7.5.1 Bulk Isotope Analyses on Hohlohsee Samples 1552.7.5.2 513C Values of Pyrolysis Products of Sphagnum Peat and DOM from

Bog Water 256References 160

2.8 Analytical Pyrolysis of Humic Substances and Dissolved Organic Matterin Water 263

2.8.1 Introduction 1632.8.2 Methods 1642.8.3 Characterization and Differentiation of Structural Properties 1652.8.4 Pyrolysis-Field Ionization Mass Spectrometry 2722.8.5 Structural Variations as a Result of Sampling Season and Year 2752.8.6 Quality Control and Changes During Sample Storage 1762.8.7 Correlations between 13C NMR and Py-FIMS 2792.8.8 Developments of Quantitative GC/MS 1802.8.8.1 Determination of Hexose Subunits of Cellulose by Py-GC/MS 2802.8.8.2 Determination of Solvent-extracted Fatty Acids by GC/MS 2832.8.9 Conclusions 284

References 286

2.9 Characterization of Refractory Organic Substances by HPLC/MS 2882.9.1 Introduction 1882.9.2 Experimental Details 1892.9.2.1 Mass Spectrometry 189

Contents | XI

2.9.2.2 Size-exclusion Chromatography (SEC) 1922.9.3 Results and Discussion 2922.9.3.1 Mass Spectra of Refractory Organic Substances 1912.9.3.2 Size-exclusion Chromatography of Refractory Organic Substances 297

References 299

2.10 UV-Visible Spectroscopy and the Potential of Fluorescent Probes 2002.10.1 UV-Visible Absorption Spectroscopy 2002.10.2 Colorimetric Analysis 2052.10.3 Fluorescence of Refractory Organic Substances 2082.10.4 Aging of Refractory Organic Substances 2092.10.5 Complexing of Heavy Metals 212

References 214

2.11 Stationary and Time-resolved Fluorescence for Refractory OrganicSubstances Characterization 215

2.11.1 Introduction 2152.11.2 Experimental Details 2172.11.3 Results and Discussion 2182.11.3.1 Steady-state Fluorescence of Humic Substances 2182.11.3.2 Time-resolved Fluorescence of Humic Substances 2212.11.3.3 Fluorescence of Chemically Altered Humic Substances 2252.11.4 Conclusions 227

References 229

2.12 Structural Characterization of Refractory Organic Substancesby Pyrolysis-GC/FTIR 232

2.12.1 Introduction 2322.12.2 Experimental Details 2332.12.2.1 Sample Preparation 2332.12.2.2 Curie-point Pyrolysis 2332.12.2.3 Flash Pyrolysis 2342.12.2.4 GC/FTIR Spectroscopy 2342.12.3 Results and Discussion 236

References 238

2.13 X-ray Microscopy Studies of Refractory Organic Substances 2392.13.1 Introduction 2392.13.2 X-ray Microscopy 2402.13.3 Results 2432.13.3.1 Refractory Organic Substances in Soils 2432.13.3.2 Flocculation of Refractory Organic Substances by Cations 2442.13.3.3 Spectromicroscopy of Refractory Organic Substances 2442.13.4 Conclusions 246

References 247

XII I Contents

2.14 Fractionation of Refractory Organic Substances by Electrophoresis 2492.14.1 Introduction 2492.14.2 Experimental Details 2502.14.3 Results and Discussion 2522.14.4 pH-dependence 2542.14.5 Electrophoresis with Organic Modifiers 2552.14.6 Micellar Electrokinetic Chromatography 2552.14.7 Electrophoresis in Sieving Media 2562.14.8 Isoelectric Focusing 2572.14.9 Characterization of Degradation Products of Refractory Organic

Substances 258

2.14.10 Hydrothermolysis 2602.14.11 Pyrolysis 2612.14.12 Conclusions 262

References 263

2.15 Occurrence of Amino Acids, Carbohydrates, and Low-molecular-weightOrganic Acids in Refractory Organic Substances 264

2.15.1 Introduction 2652.15.2 Experimental Details 2672.15.2.1 Samples 2672.15.2.2 Determination of Amino Acids 2682.15.2.3 Determination of Carbohydrates 2682.15.2.4 Determination of Low-molecular-weight Organic Acids 2692.15.3 Results and Discussion 2702.15.3.1 Acid-hydrolyzable Amino Acids and Carbohydrates 2702.15.3.2 Enzymatically Hydrolyzable Amino Acids and Carbohydrates 2772.15.3.3 Alkaline-hydrolyzable Low-molecular-weight Organic Acids 2782.15.4 Conclusions 279

References 280

2.16 Serological Characterization of Refractory Organic Substancesby Serotyping 282

2.16.1 Introduction 2822.16.2 Experimental Details 2832.16.2.1 Material 2832.16.2.2 Antibody Production 2832.16.2.3 Precipitation Test 2842.16.2.4 Dot Blot Test 2842.16.2.5 Enzyme Immunoassay 2842.16.2.6 Fractionation of Refractory Organic Substances 2862.16.2.7 Serotyping 2872.16.3 Results 2872.16.3.1 Immunochemical Reactions of Refractory Organic Substances 2872.16.3.2 Fractionation of Refractory Organic Substances 293

Contents | XIII

2.16.3.3 Serotyping 2942.16.4 Discussion 297

References 302

2.17 Chemical and Spectroscopic Data of the Reference Samples —Comparison and Evaluation 302

2.17.1 Evaluation of the Isolation Procedure by Combining 13C NMR Spectrawith DOC Concentration Results 302

2.17.2 Comparison of NMR Results with Data from Other Methods ofStructural Characterization 304

2.17.2.1 NMR and UV Spectroscopy 3042.17.2.2 NMR and Analysis of the Content of Carbohydrates and Amino

Acids 3052.17.3 Complementary Results from Data Comparisons 3062.17.3.1 Carboxylic Groups Bound in Esters, Amides and Free Carboxylic Acid

Groups 3062.17.3.2 Phenolic Groups 3072.17.3.3 - Exchangeable Hydrogens - Comparison of *H and 13C NMR Data 3082.17.4 Conclusions 309

3 Biochemical and Biological Characterization 322

3.1 Formation, Utilization, and Transformation of Some Refractory OrganicSubstances by Aquatic Microorganisms 312

3.1.1 Introduction 3113.1.2 Experimental Details 3123.1.2.1 Materials and Methods 3123.1.3 Results and Discussion 313

References 320

3.2 Effect of Microorganisms on the Formation and Transformationof Iodine Species of Refractory Organic Substances 322

3.2.1 Introduction 3213.2.2 Formation of Iodine—ROS Species from Inorganic Iodine 3223.2.3 Microbiological Influence on the Iodine—ROS Formation 3243.2.4 Transformation of Iodine—ROS Species by Aging 3263.2.5 Conclusion 328

References 329

3.3 The Influence of Refractory Organic Substances on BacterialColonization and Diversity Patterns 330

3.3.1 Introduction 3303.3.2 Materials and Methods 3313.3.2.1 Experimental Design 3323.3.2.2 Physicochemical Investigations 332

XIV Contents

3.3.2.3 Microbiological Monitoring 3323.3.2.4 Molecular Methods 3333.3.2.5 Cluster Analysis 3333.3.2.6 Scanning Electron Microscopy 3343.3.3 Results 3343.3.3.1 Physicochemical Investigations 3343.3.3.2 Microbiological Monitoring 3363.3.3.3 Molecular Methods 3393.3.3.4 Cluster Analysis 3393.3.3.5 Scanning Electron Microscopy 3413.3.4 Discussion 342

References 344

3.4 Influence of Refractory Organic Substances on Enzyme Activity in-vivoand DNA Damage of Aquatic Microorganisms 346

3.4.1 Introduction 3463.4.2 Methods 3483.4.2.1 Detection and Evaluation of Microbial Enzyme Activity in Batch

Experiments 3483.4.2.2 Flow Injection Analysis of Microbial Enzyme Activity (FAME) 3483.4.2.3 Detection of Genotoxicity by Use of the umu Test 3493.4.2.4 Detection of Genotoxicity by Use of the Comet Assay 3503.4.2.5 Origin of Water Samples and Microorganisms 3503.4.2.6 Refractory Organic Substances 3503.4.2.7 Spiking of Refractory Organic Substances and Pollutants 3523.4.2.8 Influence of pH and Conductivity on Refractory Organic Substances 3513.4.3 Results 3523.4.3.1 Effects of Different Concentrations of Refractory Organic Substances

on Enzyme Activities in Batch Experiments 3513.4.3.2 Interaction of Refractory Organic Substances with Pollutants in Batch

Experiments 3533.4.3.3 Application of the FAME System Looking for Effects of Different

Refractory Organic Substances on Enzyme Activity 3543.4.3.4 Addition of HgCl2 and its Influence on Peroxidase Activity Using the

FAME System 3543.4.3.5 Detection of Genotoxic Effects by Use of the umu Test 3563.4.3.6 Detection of Genotoxic Effects by Use of the Comet Assay 3573.4.4 Discussion 3583.4.4.1 ROS as Nutrients or Toxicants or as a Trap for Nutrients and

Toxicants 3583.4.4.2 Interactions of ROS with Added Toxicants 3593.4.4.3 Do pH and Electrical Conductivity Influence the Effects of ROS and

Toxicants? 3593.4.4.4 Are ROS Genotoxic or Can They Mask Genotoxins? 359

References 360

Contents | XV

3.5 Effects of Dissolved Organic Matter on the Bioconcentration of OrganicContaminants and on Reproduction in Aquatic Invertebrates 361

3.5.1 General Introduction 3613.5.2 Influence of Dissolved Organic Matter on the Bioconcentration of

Hydrophobic Organic Contaminants — Quantitative Aspects 3623.5.2.1 Introduction 3623.5.2.2 Influence of the Concentration of Dissolved Organic Matter 3633.5.2.3 Influence of the Origin of Dissolved Organic Matter 3653.5.2.4 Influence of the Type of Contaminant 3693.5.2.5 A General Model for Describing the Effect of Dissolved Organic Matter

on the Bioconcentration of Hydrophobic Organic Contaminants 3703.5.3 Effect of Refractory Organic Substances on the Reproduction of a

Nematode (Caenorhabditis elegans) 372

3.5.3.1 Introduction 3723.5.3.2 Experimental Details 3733.5.3.3 Results 3733.5.3.4 Discussion 3763.5.3.5 -- Ecological Implications 377

References 378

4 Molecular Interactions 383

4.1 Sorption of Dissolved Organic Matter on Soil Particles and itsDependence on their Surface-charge Properties 383

4.1.1 Introduction 3834.1.2 Materials and Methods 3844.1.2.1 Samples 3844.1.2.2 Methods 3854.1.3 Results and Discussion 3864.1.3.1 Charge of Dissolved Organic Matter 3864.1.3.2 Charge of the Soil Samples 3874.1.3.3 Sorption of Dissolved Organic Matter 3884.1.4 Conclusions 392

References 393

4.2 Dissolved Organic Carbon in Seepage Water —Production and Transformation During Soil Passage 394

4.2.1 Introduction 3954.2.2 Materials and Methods 3964.2.2.1 Experiment (1) — Laboratory Experiments with Forest Soils — DOC

Production as a Function of Litter Quality and Ash Addition 3964.2.2.2 Experiment (2) — DOC Production in Agricultural Soils — Continuous

Rye and Silage Maize Fields 3984.2.2.3 Experiment (3) — Field and Laboratory Studies with Forest Soils —

Transformation of DOC in Seepage Water During Passage ThroughSoil 399

XVI Contents

4.2.3 Results and Discussion 4004.2.3.1 Experiment (1) — Laboratory Experiments with Forest Soils — DOC

Production as a Function of Litter Quality and Ash Addition 4004.2.3.2 Experiment (2) — DOC Production in Agricultural Soils - Continuous

Rye and Silage Maize Fields 4034.2.3.3 Experiment (3) — Field and Laboratory Studies with Forest Soils —

Transformation of DOC in Seepage During Soil Passage 406References 409

4.3 Refractory Organic Substances in Aggregated Forest Soils — Retentionversus Translocation 411

4.3.1 Introduction 4224.3.2 Materials and Methods 4234.3.2.1 Sorption Experiments with Homogenized Soil Samples and Intact

Soil Cores 4234.3.2.2 Field Experiment on Dissolved Organic Matter in Slightly Developed,

Shallow Forest Soils 4164.3.3 Sorption of Dissolved Organic Matter in Aggregated Forest Soils 4274.3.3.1 Sorption of Dissolved Organic Matter to Homogenized Soil Samples

and Intact Cores 4174.3.3.2 Ionic Strength and Competitive Inorganic Anions Effects on the

Sorption of Dissolved Organic Matter 421

4.3.3.3 Competition of Dissolved Organic Matter Constituents DuringSorption 422

4.3.3.4 Dissolved Organic Matter Adsorption to Surface and Interior Materialof Soil Aggregates 423

4.3.3.5 Sorption of Dissolved Organic Matter in Aggregated Soils —Synopsis 424

4.3.4 Dissolved Organic Matter in Slightly Developed,Shallow Forest Soils 424

4.3.4.1 Release of Dissolved Organic Matter from the Forest Floor 4244.3.4.2 Dissolved Organic Matter in the Mineral Soil 4264.3.4.3 Fluxes of Organic Matter and Nutrients — Rainstorm Events 4284.3.4.4 Chemical Characteristics of Leached Organic Matter 4294.3.4.5 Dissolved Organic Matter in Slightly Developed Shallow Soils —

Synopsis 430References 432

4.4 Refractory Organic Substances Derived from Organic Amendmentsin Soil — Formation, Translocation, and Interaction withXenobiotics 435

4.4.1 Introduction 4354.4.2 Material and Methods 4364.4.2.1 Crop Residues 4364.4.2.2 Xenobiotics 436

Contents XVII

4.4.2.3 Soil-column Experiments 4374.4.2.4 Sampling and Analysis 4384.4.2.5 Extraction of the Soil Samples 4394.4.2.6 Calorimetric Studies of the Interaction of Refractory Organic Substances

and Methabenzthiazuron 4404.4.2.7 Biosurfactant Properties of Refractory Organic Substances from Crop

Residues 4404.4.3 Results and Discussion 440

References 445

4.5 Analysis of the Binding of Amitrole and Anilazine to Aquatic andTerrestrial Refractory Organic Substances 446

4.5.1 Introduction 4464.5.2 Materials and Methods 4484.5.2.1 Chemicals 4484.5.2.2 Synthesis 4484.5.2.3 Refractory Organic Substances and Dissolved Organic Matter 4504.5.2.4 Chromatographic Methods 4514.5.2.5 NMR Spectroscopy 4524.5.2.6 Selective Reaction Monitoring (SRM) of Bound Residues 4524.5.2.7 Formation and Clean-up of Dissolved Organic Matter—Amitrole

Complexes 4524.5.2.8 Formation of Bound Residues 4544.5.2.9 Interaction of Anilazine with Dissolved Organic Matter 4544.5.2.10 Determination of kf Values According to the Freundlich Equation for

Amitrole and Dissolved Organic Matter 4554.5.2.11 Incubation of Amitrole with Five Different Soils 4554.5.2.12 Water — Sediment Systems with Anilazine and Dihydroxyanilazine

4564.5.2.13 Water — Sediment Systems with Amitrole 4574.5.2.14 Quantification of Mineralization 4574.5.2.15 Release of Amitrole from DOM—Amitrole Complexes 4574.5.2.16 Extraction Methods for Purified Bound Residues 4584.5.2.17 Extraction of Soil Samples 4584.5.2.18 Silylation of Soil Samples 4584.5.2.19 Matrix Effects 4584.5.2.20 Biotests 4594.5.3 Results and Discussion 4594.5.3.1 Pesticides in Soil 4594.5.3.2 Pesticides in Water - Sediment Systems 4604.5.3.3 Pesticides and Dissolved Organic Matter 462

References 472

XVIII Contents

4.6 Sorption and Chemical Reactions of Polycyclic Aromatic Hydrocarbonswith Dissolved Refractory Organic Substances and Related ModelPolymers 475

4.6.1 Introduction to the Problems 4754.6.2 The Solid-phase Microextraction Technique 4774.6.3 Kinetics of the Interaction of Dissolved Organic Matter and PAH 4844.6.4 Reversibility of Sorptive Interactions in the Aqueous Domain 4894.6.5 Relationships between Structural Properties of HOC and their Sorption

Affinity 4914.6.6 The Modified Flory-Huggins Concept 4924.6.7 Relationships between Properties of Dissolved Organic Matter and their

Sorption Potential 4974.6.8 Poly(Acrylic Acid) Esters as Surrogates of Dissolved Organic Matter

for Sorption Studies 4994.6.9 Long-term Interactions between Dissolved Organic Matter and PAH

under Strictly Abiotic Conditions 5064.6.10 Summary 510

References 522

4.7 Investigation of the Interactions between Polycyclic AromaticCompounds and Refractory Organic Substances with Stationary andTime-resolved Fluorescence and Absorption Spectroscopy 526

4.7.1 Introduction 5164.7.2 Experimental Details 5184.7.2.1 Samples 5184.7.2.2 UV-Visible and Fluorescence Spectroscopy 5294.7.3 Results and Discussion 5214.7.3.1 Absorption and Fluorescence Spectroscopic Characterization of

Refractory Organic Substances 5214.7.3.2 Interactions between Refractory Organic Substances and Pyrene in

Electronic Singlet States 5244.7.3.3 Interactions between Refractory Organic Substances and Polynudear

Aromatic Components in Electronic Triplet States 5274.7.3.4 Strong Interactions of Refractory Organic Substances and Polycyclic

Aromatic Compounds — Triplet Quenching of Methylene Blue 5294.7.4 Conclusions 531

References 532

Index 535

Author index 545