Fakultät Umweltwissenschaften Institut für Hydrobiologie DISSERTATION Ecotoxicology of nanoparticles – effects on organisms from activated sludge in wastewater treatment plants zur Erlangung des akademischen Grades DOCTOR RERUM NATURALIUM (Dr. rer. nat.) vorgelegt am 29.02.2016 von Corinna Burkart, geboren am 11. Mai 1985 in Hannover, Deutschland Technische Universität Dresden, Fakultät Umweltwissenschaften, Institut für Hydrobiologie Gutachter: Prof. Dr. rer. nat. habil. Thomas U. Berendonk, TU Dresden Prof. Dr. rer. nat. habil. Jörg Oehlmann, Goethe Universität Frankfurt Dr. Marianne Matzke, Center for Ecology and Hydrology Wallingford, UK Ort und Datum der Verteidigung: Dresden, 21.11.2016 Ich bestätige hiermit die Übereinstimmung dieses Exemplares mit dem Original meiner Dissertationsschrift mit dem Titel „Ecotoxicology of nanoparticles – effects on organisms from activated sludge in wastewater treatment plants”, vorgelegt am 29.02.2016 von Corinna Burkart, mit Verteidigung am 21.11.2016 in Dresden. Ort, Datum Unterschrift Corinna Burkart Contents Contents ............................................................................................................................................ I Abbreviations and Symbols ............................................................................................................ IV 1 General introduction ............................................................................................................... 1 2 Nanomaterials .......................................................................................................................... 9 2.1 Silver nanoparticles .......................................................................................................... 9 2.1.1 Properties of silver nanoparticles ............................................................................ 9 2.1.2 Characterization methods for nAg........................................................................... 9 2.2 Titaniumdioxide Nanoparticles ...................................................................................... 15 2.2.1 Properties of titanium dioxide nanoparticles ........................................................ 15 2.2.2 Characterization of nTiO2 in test media ................................................................. 15 3 Single Species tests with Ciliates ........................................................................................... 16 3.1 Introduction - Ciliates ..................................................................................................... 16 3.2 Growth kinetics under experimental conditions for P. tetraurelia.............................. 18 3.2.1 Materials and Methods for P. tetraurelia growth kinetics ................................... 18 3.2.2 Results and Discussion for P. tetraurelia growth kinetics .................................... 20 3.3 Toxicity tests with P. tetraurelia and nAg ..................................................................... 23 3.3.1 Materials and Methods for P. tetraurelia toxicity tests with nAg ........................ 23 3.3.2 Results and discussion of nAg toxicity tests with P. tetraurelia ........................... 28 3.4 Toxicity tests with P. tetraurelia and nTiO2 ................................................................... 42 3.4.1 Materials and methods for nTiO2 toxicity tests .................................................... 42 3.4.2 Results and discussion of nTiO2 toxicity tests ....................................................... 44 4 Single species tests with bacteria ......................................................................................... 52 4.1 Introduction – Bacteria in WWTPs and tested species ................................................ 52 4.2 Experiments with Raoultella planticola ........................................................................ 53 4.2.1 Introduction – R. planticola .................................................................................... 53 4.2.2 Growth kinetics of R. planticola at experimental conditions ............................... 53 4.2.3 Toxicity tests with Raoultella planticola and nAg ................................................. 60 4.3 Experiments with Hyphomicrobium sp. ........................................................................ 74 I 4.3.1 Introduction – Hyphomicrobium and denitrification 74 4.3.2 Growth kinetics of Hyphomicrobium under experimental conditions ................ 75 4.3.3 Toxicity tests with Hyphomicrobium and nAg ....................................................... 78 4.4 Experiments with Nitrobacter vulgaris ......................................................................... 88 4.4.1 Introduction – Nitrobacter and nitrification ......................................................... 88 4.4.2 Growth kinetics of Nitrobacter vulgaris ................................................................ 89 4.4.3 Toxicity tests with Nitrobacter vulgaris and nAg .................................................. 95 5 Accumulation tests with nAg in a simple food chain ......................................................... 102 5.1 Introduction - accumulation of nanoparticles ............................................................ 102 5.2 Materials and Methods for accumulation experiments ............................................ 103 5.2.1 Characterization of nAg in conditioned CM at exposure concentrations ......... 105 5.2.2 Acute biomagnification experiments with R. planticola and P. tetraurelia ....... 105 5.2.3 Chronic biomagnification experiments with R. planticola and P. tetraurelia ... 108 5.3 Results and discussion for acute accumulation experiments .................................... 110 5.3.1 Comparison of single species tests with nAg in conditioned CM and ABC ....... 110 5.3.2 UV-vis characterization of nAg in conditioned CM at exposure concentrations of final accumulation assay...................................................................................................... 111 5.3.3 Results and discussion of acute accumulation experiments with R. planticola and P. tetraurelia......................................................................................................................... 112 5.3.4 Results and discussion of chronic accumulation experiments with R. planticola and P. tetraurelia ................................................................................................................. 117 6 Activated Sludge Community Tests .................................................................................... 121 6.1 Introduction – Activated sludge .................................................................................. 121 6.2 Acute toxicity test with activated sludge community ................................................ 125 6.2.1 Materials and Methods for acute toxicity test with activated sludge ............... 125 6.3 Results and Discussion of acute toxicity test with activated sludge community...... 136 6.4 Chronic toxicity test with activated sludge community ............................................. 151 6.4.1 Materials and Methods for chronic toxicity test ................................................ 151 6.4.2 Results and Discussion of chronic toxicity test ................................................... 153 7 Conclusions & Prospects ..................................................................................................... 155 8 Summary .............................................................................................................................. 161 9 References ............................................................................................................................ 163 II 10 Danksagung ...................................................................................................................... 173 List of figures ................................................................................................................................ 176 List of tables ................................................................................................................................. 178 Erklärung zur Eröffnung des Promotionsverfahrens ................................................................. 179 Übereinstimmungserklärung ...................................................................................................... 180 References to own original publications used in this thesis ..................................................... 181 Curriculum Vitae .......................................................................................................................... 182 Annex I: Protocol for Axenic Broth for Ciliates (ABC) ..................................................................... i ANNEX II: Protocol for 0.25% Cerophyl-Medium (CM) .................................................................... iii ANNEX III: Protocol for R2A Medium ................................................................................................ iv ANNEX IV: Protocol for Nitrobacter medium 756.c ..........................................................................v ANNEX V: Protocol for aniline violet staining ................................................................................... vi III Abbreviations and Symbols ABC axenic broth for ciliates %v/v volume percent µL microliter µg microgram µm micrometer Ag+ silver ion AMO ammonium monooxygenase