Effect-directed analysis and mechanism-specific bioassays to assess the toxicity of sediments of the Yangtze River (China)

Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH

Aachen University zur Erlangung des akademischen Gardes einer Dorktorin der

Naturwissenschaften genehmigte Dissertation

vorgelegt von

Hongxia Xiao, M.Sc. aus Henan, China Berichter: Univ. Prof. Dr. rer. nat. Henner Hollert Univ. Prof. Dr. rer. nat. Andreas Schäffer Prof. Ph.D Xiaowei Zhang

Tag der mündlichen Prüfung: 26.Juli 2016 Diese Dissertation ist auf den Internetseiten der Universitätsbibliothek online verfügbar.

“The highest good is like that of water

The goodness of water is that it benefits the entire world, yet it does not scramble

The superior man who has breadth of character carries the outer world

Efforts to benefit the future generations should never cease”

---China Three Gorges Corporation Annual Report (2007)

Summary ______Summary

The Yangtze River has been a source of life and prosperity for the Chinese people for centuries.

The river basin plays an important role for the economics of China and is habitat for a remarkable variety of aquatic species. Since 2009, the Three Gorges Dam (TGD) – located in the upper

Yangtze Reaches, is operating at full capacity, with the benefits of flood control, electrical energy production, and improvement of river navigation. However, as a consequence of TGD impoundment, large amounts of pollutants entered the newly established ecosytem – Three

Gorges Reservior (TGR). Thus, numerous questions in ecotoxicological research are raised.

With respect to the ecotoxciological challenges in the TGR, the Yangtze-Hydro project was cooperated among six Germany institutes, to study on sustainable water management of the

Yangtze River. The subproject “MICROTOX” was conducted at the Institute for Environmental

Research of RWTH Aachen University, in order to investigate the TGR ecosystem on ecology, ecotoxicology and environmental behavior of organic pollutants, and to assess the risk for human and the ecosystem. The thesis has been carried out as part of MICROTOX, aiming to (i) determinate the hazards potential of the Yangtze River Basin, (ii) provide a detailed insight into the ecological risks of TGR, and (iii) develop a monitoring strategy for future implementation for the Yangtze River. To achieve that, the thesis (1) investigated the ecotoxicological status in the

Yangtze River through a literature study, (2) evaluated the hazard potential of the TGR by applying the “triad approach”, and further (3) identified the causitive contaminants using effect directed anylsis (EDA) that were responsible for the environmental relevant endpoints at the regional “hot-spots” sites. Additionally, (4) an ecotoxicological study of the presence of the widely applied herbicides in TGR – propanil, and its metabolite 3,4-dichloroaniline (3,4-DCA), as well as 3,4,3’,4’-tetrachloroazobenzene (TCAB) was conducted with respect to support the

______I Summary ______regulatory decisions. Lastly, (5) an optimized work-flow for identification of dioxin-like compounds was developed with regard to rapid assessment of the AhR-agonists in the areas.

The literature study gave an comprehensive overview on the environmental pollution status from upstream of Chongqing until the Yangtze River mouth at the East China Sea. Persistent organic pollutants (POPs) levels such as polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and organochlorine pesticides (OCPs) showed comparable levels to other major Chinese and European river, while large share of measured pollutant levels in water and sediments reported in the literature met the current official criteria and related standards in China.

However, by considering the pollutant levels, mechanism-specific effect assessment, and adverse effects in aquatic organisms, the sections of Wuhan, Nanjing, and Shanghai Estuary presented a higher pollution level than other sections of the river, indicating a potential ecotoxicological impact on the surrounding areas.

Triad approach, integrates chemical analysis, bioassays, and in situ biomarkers, is a holistic assessment method to determine the potential risks associated with environmental samples. Based on the methodology of the triad approach, the ecotoxicological potential in TGR was evaluated.

Among the chemical pollutants, PAHs appeared to be the dominant pollutants for the TGR, ranging from 165 – 1,653 ng/g in 2011 and 127 – 590 ng/g in 2013. Adverse effects such as in vitro AhR-mediated activity, mutagenicity and in vivo embryotoxity/teratogenity were detected in the sediments. By applying in vivo bioassays with Danio rerio eggs, it was shown that sediments induced embryotoxic/teratogenic effects, particularly on the cardiovascular system to fish embryos. However, the bioavailability of particle bound pollutants was rather low. Significant genotoxic impacts could be observed on erythrocytes of Pelteobagrus vachellii sampled from

Chongqing, as well as from Hanfeng Lake. By accounting of chemical analysis, toxic effects in vitro and in vivo, and in situ biomarkers, it was concluded that adverse effects might be induced to the fish at long term at current environmental relevant concentrations, which will have

______II Summary ______influence on the overall fitness of fish and other aquatic organisms; Chongqing and Kaixian were taken as regional “hot-spots” with respect to mutagenicity, genotoxicity, and AhR-mediated activity.

EDA, featuring a combination of fractionation, chemical analysis and bioassays, is a useful tool to identify causative toxicants in complex environmental samples. Hence, EDA was implemented follow by the triad approach at Chongqing and Kaixian. High molecular weight PAHs accounted for 28 - 43% of the significant bioactive fractions in Chongqing section, thus presented to be significant AhR-active compounds at these sampling sites. The possible pollution source might come from urban traffic emissions and runoff, coal combustion, as well as intensified shipping activities. In Kaixian area, PAHs, methylated derivatives and heterocyclic polycyclic aromatic compounds (PACs) such as dinaphthofurans were identified as major contributor to the observed

AhR-mediated activity, which might come from incomplete combustion and industrial processes or fossil fuels. Biomass burning should be considered since the Kaixian area constitutes a rather rural area. Besides, benzothiazole and its derivatives were identified as suspected toxicants by non-target analysis, which most likely originated from an identified rubber factory nearby the sampling site. The identified compounds were recommended to be included in long-term monitoring strategies in TGR.

Moreover, the toxicity of propanil, 3,4-DCA, with a focus on TCAB in mechanism specific cell assays and early vertebrate development was investigated. As TCAB was considered as AhR agonist, an in silico tool has been applied to simulate the binding affinity of the three compounds to the AhR, as well as other cell-based receptors. The toxic equivalency factor (TEF) values of

TCAB in AhR-mediated activity were founded ranging four to five orders of magnitude less potent than TCDD based on fish liver cell line (RTL-W1) and rat hepatoma cell line (H4IIE).

Thereafter, the experimented TEF is comparable to those well-known dioxin-like chemicals like some mono-ortho coplanar PCBs and large PAHs. Besides, the three compounds were shown to

______III Summary ______be weak estrogenic compounds. The value of estradiol equivalence factor (EEF) and dihydrotestosterone equivalency factor (DEF) of TCAB were nine to ten orders of magnitude less potent than the corresponding standard compound. In FET, the exposure of Danio rerio indicated significant embryotoxicity and teratogenicity of TCAB. Together with recorded sub-lethal effects, under exposure of TCAB, it might result in adverse outcomes in aquatic systems on the long term.

Importantly, it demonstrated that TCAB has a higher toxicity than its parent compounds – propanil and DCA. Hence, risk assessment of pesticides are recommended to include the assessment of the toxicity and fate of their metabolites before they are deliberately brought to the environment.

Finally, to enlarge the application of EDA in environmental risk assessment and biological effects monitoring programs, a high-throughput EDA (ht-EDA) workflow – using reversed phase high-performance liquid chromatography (RP-HPLC) fractionation of samples into 96-well microplates, followed by toxicity assessment in the H4IIE Micro-EROD bioassay, and chemical analysis of biologically active fractions was developed. The approach was applied in the extracts of sediment samples collected at TGR the Yangtze River, China as well as the rivers Rhine and

Elbe, Germany successfully. Furthermore, by seeding previously adapted suspension-cultured

H4IIE cells directly into the microplate used for fractionation, the ht-EDA work-flow was further simplified, and could become the method of choice for the prioritization of environmental contaminants and support for regulatory decisions for TGR.

In conclusion, the present study might contribute to long-term monitoring strategies of the

Yangtze River Basin, and support the prioritization of contaminants and regulatory decisions in

TGR. The recommended monitoring strategies can be applied globaly to other similar river systems.

______IV Zusammenfassung ______

Zusammenfassung

Der Yangtse ist für Jahrhunderte eine Quelle des Lebens und des Wohlstands für die chinesische

Bevölkerung gewesen. Sein Einzugsgebiet hat großen Einfluss auf die Ökonomie Chinas und beherbergt eine große Anzahl an wassergebundenen Lebewesen. Seit 2009 arbeitet der am

Oberlauf des Yangtse gelegene Drei-Schluchten-Damm mit voller Kapazität, wodurch ein besserer Hochwasserschutz, eine beträchtliche Elektrizitätsgewinnung und eine bessere

Flussnavigation möglich sind. Es wird erwartet, dass das wirtschaftliche Potential des Yangtse durch die Eröffnung des Drei-Schluchten-Damms – des weltgrößten Damms, zunehmen wird.

Jedoch verursacht die Eröffnung des Drei-Schluchten-Dammes eine Zufuhr von großen Mengen an Schadstoffen in das neu geschaffene Ökosystem des Drei-Schluchten-Reservoirs. Daraus ergeben sich zahlreiche ökotoxikologische Fragestellungen.

Diese Fragestellungen wurden in einem gemeinsamen Forschungsprojekt mit sechs deutschen

Forschungseinrichtungen mit dem Ziel bearbeitet, ein nachhaltiges Wassermanagementsystem für den Yangtse zu erarbeiten. Diese Doktorarbeit wurde als Teil des Unterprojekts MICROTOX am

Institut für Umweltforschung an der RWTH Aachen durchgeführt. Das Ziel dieses Unterprojektes bestand darin, das Ökosystem des Drei-Schluchten-Reservoirs hinsichtlich der Ökologie und

Ökotoxikologie von organischen Schadstoffen zu evaluieren und das Risiko für Mensch und

Ökosystem zu bewerten. Der Fokus dieser Doktorarbeit lag dabei (i) auf der Analyse des

Gefährdungspotentials des Drei-Schluchten-Damms, (ii) darauf ein detailliertes Verständnis der

ökologischen Risiken des Drei-Schluchten-Reservoirs zu erlangen, (iii) und auf der Entwicklung einer Monitoringstrategie für eine zukünftige Anwendung entlang des Yangtses. Als Teil des

Projektes konzentrierte sich die vorliegende Doktorarbeit auf (1) die Untersuchung des

ökotoxikologischen Status des Yangtse durch eine Literaturstudie, (2) einer darauf aufbauenden

______V Zusammenfassung ______

Risikoanalyse für das Drei-Schluchten-Reservoir durch Anwendung des „Triad Approach“ und weitergehend (3) auf die Identifikation der Hauptschadstoffe mithilfe der „effect directed analysis“ (EDA), die für die ökologisch relevanten Endpunkte an den regionalen „Risiko-hot- spots“ verantwortlich sind. Zusätzlich wurde (4) zur Unterstützung regulatorischer

Rahmenbedingungen entlang des Yangtse ökotoxikologische Untersuchungen zur Konzentration des weit verbreiteten Herbizids Propanil und seiner Abbauprodukte 3,4-dichloranilin (3,4-DCA) und 3,4,3’,4’-tetrachloroazobenzene (TCAB) durchgeführt. Zuletzt wurde (5) ein optimierter

Arbeitsablauf zur Identifikation von dioxin-ähnlichen Komponenten entwickelt, der eine schnelle

Identifikation von AhR-Agonisten ermöglicht.

Die Literaturstudie konnte einen umfassenden Überblick über den Verschmutzungsstatus entlang des Yangtse von seinem Oberlauf in Chongqing bis zu seiner Mündung in das Chinesische Meer geben. Die Konzentration von persistenten organischen Schadstoffen wie polyzyklische aromatische Kohlenwasserstoffe (PAHs), polychlorinierte Biphenyle (PCBs) und chlororganische

Pestizide (OCPs) zeigten vergleichbare Level wie in anderen großen chinesischen und europäischen Flüssen. Ein großer Teil der in der Literatur angegebenen gemessenen

Schadstofflevel im Wasser und Sediment hielten die aktuellen offiziellen Kriterien und verwandte Standards in China ein. In den Regionen von Wuhan, Nanjing und Shanghai wurde, unter Beachtung der Schadstoffkonzentrationen, mechanismus-spezifischer Effektberwertungen und negativer Effeekte in aquatischen Organismen, eine höhere Schadstoffbelastung verglichen mit anderen Abschnitten des Yangtse festgestellt. Dies weist auf einen potentiellen

ökotoxikologischen Einfluss auf den Umkreis dieser Regionen hin.

Der Triad Approach ist ein ganzheitliches Bewertungsverfahren um potentielle Risiken anhand von Umweltproben zu ermitteln. Er kombiniert chemische Analysen, Bioassays und in situ

Biomarker. Basierend auf dem Triad Approach wurde eine Beurteilung des ökologischen

______VI Zusammenfassung ______

Potentials des Yangtse durchgeführt. Basierend auf den gemessenen Schadstoffen scheinen PAHs mit Konzentrationen von 165 – 1.653 ng/g in 2011 und 127 – 590 ng/g in 2013 die vorherrschende Schadstoffgruppe zu sein. In den Sedimenten konnten nachteilige Effekte wie in vitro AhR-vermittelte Aktivität, Mutagenität und in vivo Embryotoxizität und Teratogenität nachgewiesen werden. Durch die Verwendung von in vivo Bioassays mit Danio rerio Eiern konnten durch die Sedimente hervorgerufene embryotoxische/teratogenische Effekte, vor allem auf das kardiovaskuläre System der Fischembryos, nachgewiesen werden. Jedoch war die

Bioverfügbarkeit von partikelgebundenen Schadstoffen gering. Ein signifikanter genotoxischer

Einfluss konnte an Erythrozyten von Pelteobagrus vachellii aus Chongqing und aus dem Hafeng

Lake festgestellt werden. Unter Berücksichtigung der chemischen Analytik, den toxischen

Effekten in vitro und in vivo und in situ Biomarkern kann zusammenfassend gesagt werden, dass eine langfristig negative Beeinflussung von Fischen unter den gegebenen ökologischen

Bedingungen zu erwarten ist und eine Beeinträchtigung der Fitness von Fischen und anderen wassergebundenen Lebewesen nach sich zieht. Chongqing und Kaixian wurden als regionale

„Hot-Spots“ hinsichtlich der gemessenen Mutagenität, Genotoxizität und der AhR-vermittelten

Aktität identifiziert.

EDA ist eine nützliche Analysemethode um wirksame Schadstoffe in komplexen Umweltprogen mithilfe einer Kombination aus Fraktionierung, chemischer Analyse und Bioassays zu identifizieren. Daher wurde an diesen „Hot-Spots“ eine EDA und anschließend der Triad

Approach durchgeführt. PAHs mit hohem Molekulargewicht hatten einen Anteil von 28 – 43 % an den signifikanten bioaktiven Fraktionen in der Chongqing Region, und stellen demnach signifikant AhR-aktive Schadstoffe an den Entnahmestellen dar. Als mögliche Schadstoffquellen kommen Emissionen des Straßenverkehrs, von Abflüssen, aus Kohleverbrennung, der

Luftverschmutzung allgemein und des intensiven Schiffsverkehrs in Betracht. In der Region

______VII Zusammenfassung ______

Kaixian verursachen PAHs, methylierte Derivate und heterozyklische polyzyklische aromatische

Verbindungen (PACs) wie z.B. Dinaphtofurane den Großteil der gemessenen AhR-vermittelten

Aktivität. Als Quellen hierfür kommen unvollständige Verbrennungsprozesse und

Verarbeitungsprozesse der Mineralölindustrie in Frage. Auch das Verbrennen von Biomasse kommt in der ländlich geprägten Region Kaixian in Betracht. Zusätzlich wurde in einer non- target Analyse Benzothiazol und seine Derivate als möglicher Schadstoff identifiziert, die wahrscheinlich von einer Gummifabrik nahe der Probenentnahmestelle stammten. Daher wird empfohlen, die identifizierten Schadstoffe in eine langfristige Monitoringstrategie für den

Yangtse aufzunehmen.

Zusätzlich wurde in dieser Arbeit die Toxizität von Propanil und 3,4-DCA untersucht, wobei der

Fokus auf TCAB in mechanismus-spezifischen Zellassays und der Frühentwicklung von

Wirbeltieren lag. Da TCAB als AhR-Agonist angesehen wurde, wurde die Bindungsaffinität der drei Schadstoffe gegenüber speziellen Zellrezeptoren Mithilfe von in silico Methoden simuliert.

Die Toxizitätsäquivalenzfaktoren (TEF) für TCAB in AhR-vermittelter Aktivität lagen vier bis fünf Größenordnungen unterhalb der Faktoren für TCDD, basierend auf Fischleberzellen (RTL-

W1) und Hepatomazellen von Ratten (H4IIE). Daher ist der untersuchte TEF vergleichbar mit bekannten dioxinähnlichen Substanzen wie einigen mono-ortho coplanare PCBs und großen

PAHs. Zusätzlich konnte gezeigt werden, dass die drei Schadstoffe schwach estrogen wirksam sind. Die Werte des Estradiol-Äquivalenzfaktors (EEF) und des Dihydrotestosteron-

Äquivalenzfaktors (DEF) von TCAB waren neun bis zehn Größenordnungen weniger wirksam, als die zugehörige Standardmischung. Im FET mit Danio rerio wurde eine signifikante

Embryotoxizität und Teratogenität von TCAB identifiziert. Diese Eigenschaft kann zusammen mit bekannten sublethalen Effekten unter Einwirkung von TCAB nachteilige Langzeiteffekte auf aquatische Systeme haben. Wesentlich ist, dass TCAB eine höhere Toxizität aufweist als die

______VIII Zusammenfassung ______

Ausgangssubstanzen Propanil und DCA. Daher wird empfohlen, dass sollte die Risikobewertung von Pestiziden auch die Bewertung der Toxizität und des Verhaltens der Metabolite einschließeneinschließt, bevor diese Substanzen in der Umwelt freigesetzt werden.

Zuletzt wurde eine high-throughput EDA (ht-EDA) Workflow entwickelt, der die Anwendung von EDA in Umweltrisikobewertungen und Monitoringprogramme für biologische Effekte erhöhen kann. Der Ansatz schließt reversed phase Hochleistungsflüssigkeitschromatographie

(RP-HPLC), die Aufteilung der Proben auf 96-well Mikrotiterplatten, gefolgt von einer

Toxizitätsbewertung im H4IIE Micro-EROD Bioassay und einer chemischer Analyse der biologisch aktiven Anteile ein. Die Methode wurde für die Analyse von Extrakten von

Sedimentproben des Drei-Schluchten-Damms in China sowie aus Rhein und Elbe in Deutschland eingesetzt. Zusätzlich wurde der Arbeitsablauf der ht-EDA weiter vereinfacht, indem vorbereitete

Suspensionkulturen der H4IIE-Zellen direkt in die wells der Mikrotiterplatten, die für die

Fraktionierung genutzt wurden, ausgesäht wurden. ht-EDA könnte somit zur bevorzugten

Methode für die Priorisierung von Umweltschadstoffen werden und damit einen Beitrag zu zukünftigen regulatorischen Entscheidungen bezüglich des Drei-Schluchten-Dammes leisten.

Zusammenfassend kann gesagt werden, dass die vorliegende Doktorarbeit einen Beitrag für eine effektive und langfristige Monitoringstrategie des Yangtse leisten, die Identifikation von

Schadstoffen vereinfachen und regulatorische Entscheidungsprozesse verbessern kann. Die empfohlene Monitoringstrategie kann global für andere Flusssysteme angewendet werden.

______IX Zusammenfassung ______

______X Table of Contents ______

Table of Contents

1 General Introduction ...... 1

1.1 Environmental pollution and monitoring strategies ...... 3

1.2 Effect-based tools for monitoring the ecotoxicological effects in aquatic environment ... 4

1.3 EDA as an environmental monitoring strategy ...... 5

1.3.1 Components of EDA ...... 6

1.3.2 Current EDA study and limitations ...... 9

1.3.3 The development of high-throughput EDA (ht-EDA) ...... 10

1.4 The Yangtze River and the environmental challenges ...... 10

1.4.1 The Yangtze Basin ...... 11

1.4.2 Three Gorges Reservoir ...... 13

1.4.3 Environmental challenges ...... 14

1.5 Yangtze Hydro project ...... 15

1.5.1 MICROTOX project ...... 15

1.6 Toxic endpoints applied in the dissertation ...... 17

1.6.1 AhR-mediated activity ...... 17

1.6.2 Mutagenic and genotoxic activity ...... 19

1.6.3 Embryotoxic and teratogenic potential...... 19

1.7 Aim of the study and structure ...... 20

1.7.1 A comprehensive perspective of ecotoxicological status of the Yangtze River Basin ...... 21

1.7.2 Ecotoxicological evaluation of sediment and fish in the Three Gorges Reservoir .. 21

1.7.3 Effect-directed analysis of aryl hydrocarbon receptor agonists in sediments from the Three Gorges Reservoir, China ...... 22

______I Table of Contents ______

1.7.4 Ecotoxicology study of propanil, 3,4-dichloroaniline (3,4-DCA), and 3,4,3’,4’-tetrachloroazobenzene (TCAB) ...... 22

1.7.5 Optimized work-flow for identification of dioxin-like compounds in environmental samples combining High-throughput fractionation and bioassay ...... 23

2 Solution by dilution? – A review on the ecotoxicological status of the

Yangtze River ...... 25

2.1 Abstract ...... 27

2.2 Introduction ...... 29

2.3 Scope and aims ...... 29

2.4 Methodology and materials studied ...... 31

2.5 Pollutant levels in water and sediments ...... 32

2.5.1 Polycyclic aromatic hydrocarbons (PAHs) ...... 33

2.5.2 Polychlorinated biphenyls (PCBs) ...... 38

2.5.3 Organochlorine pesticides (OCPs) ...... 41

2.5.4 Polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDDs/DFs) ...... 46

2.5.5 Emerging pollutants ...... 47

2.5.6 Discussion: Pollutant levels in water and sediments ...... 53

2.6 Effect assessment of water and sediment (in vitro and in vivo) ...... 57

2.6.1 Genotoxicity and mutagenicity ...... 57

2.6.2 Endocrine disrupting activity ...... 60

2.6.3 Other endpoints ...... 62

2.6.4 Discussion: Effect assessment of water and sediment (in vitro and in vivo) ...... 65

2.7 Pollutant levels and adverse effects in aquatic organisms (in situ studies) ...... 71

2.7.1 Pollutant levels in aquatic organisms ...... 71

2.7.2 Adverse effects in aquatic organisms ...... 77

______II Table of Contents ______

2.7.3 Discussion: Pollutant levels and adverse effects in aquatic organisms (in situ studies) ...... 77

2.8 Overall conclusion ...... 79

2.9 Acknowledgements ...... 85

3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ... 87

3.1 Abstract ...... 89

3.2 Introduction ...... 91

3.3 Material and methods ...... 93

3.3.1 Sampling campaign ...... 93

3.3.2 Chemicals and materials ...... 96

3.3.3 Sediment extraction ...... 97

3.3.4 Triad A: Chemical analysis ...... 97

3.3.5 Triad B: In vitro/In vivo bioassays ...... 98

3.3.6 Triad C: In situ biomarkers ...... 102

3.3.7 Statistical analysis ...... 104

3.4 Results and discussion ...... 104

3.4.1 Chemical analysis ...... 104

3.4.2 In vitro bioassay ...... 113

3.4.3 In vivo bioassay ...... 117

3.4.4 In situ biomarkers ...... 119

3.4.5 Responsible compounds in the in vitro/in vivo assays ...... 132

3.4.6 Toxicity assessment in TGR ...... 133

3.5 Conclusion ...... 136

3.6 Acknowledgements ...... 139

______III Table of Contents ______

4 Effect-directed analysis of aryl hydrocarbon receptor agonists in sediments from the Three Gorges Reservoir, China ...... 141

4.1 Abstract ...... 143

4.2 Introduction ...... 145

4.3 Material and methods ...... 146

4.3.1 Sample collection and preparation ...... 146

4.3.2 Normal phase fractionation ...... 147

4.3.3 Ethoxyresorufin-O-deethylase (EROD) induction assay ...... 148

4.3.4 Targeted analysis of PACs by GC-MS ...... 148

4.3.5 Non-target analysis in polar fractions by LC-HRMS ...... 148

4.3.6 Processing of HRMS data for compound identification ...... 149

4.3.7 QSAR modeling usingVirtualToxLab ...... 150

4.3.8 Statistical analysis ...... 150

4.4 Results and discussion ...... 151

4.4.1 Bioassay-derived induction equivalent quantities ...... 151

4.4.2 Target chemical analysis ...... 153

4.4.3 Contribution of PACs to the EROD induction potency of fractions ...... 153

4.4.4 Non-target analysis of polar fractions ...... 155

4.4.5 Identification and selection of candidate AhR agonists ...... 155

4.4.6 Source of contamination and environmental significance ...... 158

4.5 Acknowledgements ...... 159

5 Ecotoxicological evaluation of 3,4,3’,4’-tetrachloroazobenzene (TCAB) in comparison to the parent compound 3,4-dichloroaniline and propanil ...... 161

5.1 Abstract ...... 163

______IV Table of Contents ______

5.2 Introduction ...... 165

5.3 Material and methods ...... 168

5.3.1 Chemicals and reagents ...... 168

5.3.2 EROD assay ...... 169

5.3.3 Micro-EROD assay ...... 169

5.3.4 CALUX assay ...... 170

5.3.5 Fish Embryo Toxicity (FET) test ...... 171

5.3.6 QSAR model according to VirtualToxLab ...... 171

5.3.7 Data analysis ...... 171

5.4 Results and discussion ...... 172

5.4.1 AhR-mediated activity ...... 172

5.4.2 Estrogen and androgen activity ...... 175

5.4.3 Lethal and sub-lethal effects in FET ...... 178

5.4.4 Prediction of toxicity potential using VirtualToxLab model ...... 181

5.4.5 Adverse outcome pathways ...... 183

5.5 Conclusion ...... 184

5.6 Acknowledgements ...... 185

6 Optimized work-flow for identification of dioxin-like compounds in environmental samples combining High-throughput fractionation and bioassays ...... 187

6.1 Abstract ...... 189

6.2 Introduction ...... 191

6.3 Material and methods ...... 193

6.3.1 Chemicals and solvents ...... 193

6.3.2 Sample collection and preparation ...... 193 ______V Table of Contents ______

6.3.3 Fractionation method ...... 194

6.3.4 Micro-EROD assay ...... 194

6.3.5 CDS-µEROD assay ...... 195

6.3.6 Statistical analysis ...... 196

6.4 Results and discussion ...... 196

6.4.1 Development and validation of the fractionation method ...... 196

6.4.2 Fractionation of single substances ...... 197

6.4.3 Fractionation of binary mixtures ...... 198

6.4.4 Fractionation of environmental samples ...... 200

6.4.5 Application potential of suspension bioassays in EDA...... 201

6.5 Conclusions and outlook ...... 203

6.6 Acknowledgements ...... 204

7 General discussion, conclusion and outlook ...... 205

7.1 General Discussion ...... 207

7.2 Environmental pollution status in the Yangtze River ...... 207

7.2.1 Overview of ecotoxicological status in the Yangtze River ...... 207

7.2.2 Ecotoxicological potential in the TGR ...... 208

7.2.3 Can the pollution problem be solved by dilution ...... 209

7.3 Identification of the causative pollutants and possible source of contamination in TGR ...... 210

7.4 Is EDA a suitable monitoring strategy? ...... 210

7.5 Recommend environmental monitoring strategies ...... 211

7.6 Conclusion and Outlook ...... 213

References ...... 215

Annex I – Solution by dilution? ...... 247 ______VI Table of Contents ______

I.1 Drainage basin definitions of the geographical regions ...... 247

Annex II – Effect-directed of aryl hydrocarbon receptor agonists ...... 259

II.1 Material and methods ...... 259

II.2 LC-HRMS ...... 259

II.2 Results and discussion ...... 261

II.2.1 Target chemical analysis ...... 261

II.2.3 Non-target analysis of polar fractions ...... 263

Annex III – High-throughput fractionation and bioassays ...... 271

III.1 Material and methods ...... 271

III.2 Results and discussion ...... 273

III.2.1 Development and validation of the fractionation method ...... 273

III.2.2 Fractionation of environmental samples ...... 274

Acknowledgments ...... 277

Curriculum vitae ...... 281

Scientific Contributions ...... 283

______VII Table of Contents ______

______VIII List of figures ______List of figures

Fig. 1.1 Scheme of Effect-directed analysis (EDA) (Redrawn from Simon (2013))...... 6 Fig. 1.2 Map of the Yangtze River (Changjiang) Drainage Basin...... 12 Fig.1.3 Three Gorges Reservoir (TGR)...... 14 Fig. 1.4 The sub-project “MICROTOX” is divided into three synergistic modules...... 16 Fig. 2.5 Flowchart demonstrating structure of the thesis ...... 21 Fig. 2.1 Holistic approach to evaluate ecotoxicity of water, sediment, soil and suspended particulate matter as well as state of contamination in local fish species according to the triad approach ...... 30 Fig. 2.2 Number of publications about the Yangtze River per year (1990-2012) ...... 31 Fig. 2.3 Minimum and maximum concentrations of PAHs in the Yangtze River...... 36 Fig. 2.4 Minimum and maximum concentrations of PCBs in the Yangtze River...... 39 Fig. 2.5 Maximum concentrations of OCPs, DDTs, HCHs in the Yangtze River...... 44 Fig. 3.1 Overview map of sampling locations in the Three Gorges Reservoir area...... 94 Fig. 3.2 Mutagenic activity of sediment extracts determined in the Ames fluctuation assay with Salmonella typhimurium tester strain TA98 with exogenous enzymatic S9 supplement for metabolic activation of promutagenic compounds...... 114 Fig. 3.3 Mean detected EROD inductions (Bio-TEQs) in comparison to calculated inductions of detected PAHs (Chem-TEQs) in sediment extracts from sampling campaigns September 2011 (A) and May 2013 (B)...... 116 Fig. 3.4 Overall effects on Danio rerio exposed for 96h to sediment extracts given as half maximal effective concentration (EC50)...... 118 Fig. 3.5 Micronucleus frequency in erythrocytes of Pelteobagrus vachellii from sampling campaign May 2012 and May 2013...... 120 Fig. 3.6 Hepatic EROD (A) and GST (B) activities in excised livers from Pelteobagrus vachellii determined for sampling campaign May 2012 and May 2013...... 126 Fig. 3.7 Plate of histological alterations of liver and kidney with surrounding tissue from Pelteobagrus vachellii from sampling campaign May 2012...... 130 Fig. 4.1 Flowchart of the applied EDA strategy...... 147

______IX List of figures ______

Fig. 4.2 Aryl hydrocarbon receptor-mediated activities of parent (par), reconstituted (rec) extracts, arithmetic sum of fraction BEQs (sum) and fraction activities (1-18) of four sediment extracts...... 152 Fig. 4.3 Chemical analyzed priority PAHs-TEQs, BEQs as well as the calculated contribution of PAHs (percentage) to the EROD induction in the selected fractions (based on EROD assay results)...... 154 Fig. 5.1 Dose − response curves and corresponding 95% confidence bands in the (a) EROD assay, (b) micro-EROD assay, (c) ER-CALUX assay, (d) AR-CALUX assay of TCAB, Propanil, 3, 4- DCA (○), and the standard curves (●), i.e.,TCDD in EROD and micro-EROD assay, E2 in ER-

CALUX, DHT in AR-CALUX, respectively...... 173 Fig. 5.2 Distribution of TEFs values for the dioxin-like compounds in comparison to TCAB and DCA as measured in the EROD assay and micro-EROD assay...... 175 Fig. 5.3 Concentration–response curve and corresponding 95% confidence bands in FET for TCAB after 72 h(○), 96 h(□), as well as negative control (NC●) and positive control (PC▪).178 Fig. 5.4 Observed sub lethal effects in Danio rerio embryo after 24, 48, 72 and 96 h exposure...... 180 Fig. 5.5 Adverse outcome pathway for TCAB resulting in fish population declining (cf. Ankley et al. 2010)...... 184 Fig. 6.1 HPLC-fractionation of single substances with subsequent micro-EROD bioassay...... 198 Fig. 6.2 HPLC-fractionation of a binary mixture of PCB 126 and β-naphthoflavone with subsequent micro-EROD bioassay...... 199 Fig. 6.3 Micro-EROD results of HPLC-fractionated sediment extract CNG-U...... 201 Fig. 6.4 HPLC-fractionation of single substances with subsequent CDS-µEROD bioassay...... 203 Fig. I.1 Map of the Yangtze Delta region ...... 248 Fig. II.1 Extracted ion chromatograms of benzothiazole and related compound in fraction HF-L- F13...... 265 Fig. II.2 MS/MS spectra of benzothiazole and related compounds in fraction HF-L-F13 acquired in APCI+ mode using HCD fragmentation (collision energy 100 a.u.)...... 266 Fig. II.3 Extracted ion chromatograms and MS/MS spectra of three probable benzothiazole derivatives (m/z 239.0669 at RT 16.2 min; m/z 239.0662 at RT 16.7 min; m/z 346.1658 at RT 34.0 min) in fraction HF-L-F13 acquired in ESI+ mode using HCD fragmentation (collision energy 100 a.u.)...... 267 ______X List of figures ______

Fig. II.4 Extracted ion chromatograms and MS/MS spectra of 2-(methylthio)benzothiazole derivatives (m/z 182.0091 at RT 24.3 min) and a related compound (m/z 224.0196 at RT 22.7 min) in fraction HF-L-F13 acquired in ESI+ mode using HCD and Cid fragmentation...... 268 Fig. II.5 MS/MS spectra of tribenzylamine and related compounds in fraction HF-L-F13 acquired in ESI+ mode using CID fragmentation (collision energy 35 a.u.)...... 269 Fig. III.1 Linear regression of chromatographic retention time versus the n-octanol/water partitioning coefficient (log Kow) for a total number of 33 chemicals...... 273 Fig. III.2 Micro-EROD results of HPLC-fractionated sediment extract HAN-D (Yangtze River, China)...... 274 Fig. III.3 Micro-EROD results of HPLC-fractionated sediment extract HAN-C (Yangtze River, China)...... 274 Fig. III.4 Micro-EROD results of HPLC-fractionated multilayer silica column-treated sediment extract (Ehrenbreitstein, Rhine, Germany)...... 276 Fig. III.5 Micro-EROD results of HPLC-fractionated multilayer silica column-treated sediment extract (Zollelbe, Elbe, Germany)...... 276

______XI List of figures ______

______XII List of tables ______List of tables

Table 3.1 Main morphological parameters of Pelteobagrus vachellii from sampling campaign May 2012 and 2013...... 96 Table 3.2 Target compounds in sediment extracts of campaign September 2011 (a) and May 2013 (b)...... 98 Table 3.3 PAH content in sediment extracts of sampling campaign September 2011 (ng/g) ..... 106 Table 3.4 PAH content in sediment extracts of sampling campaign May 2013 (ng/g) ...... 109 Table 3.5 Diagnostic ratios for PAHs in sediment from campaign September 2011 with related sources...... 111 Table 3.6 Diagnostic ratios for PAHs in sediment from campaign May 2013 with related sources...... 111 Table 3.7 Parameters for PAH metabolites in bile of Pelteobagrus vachellii from sampling campaign May 2012...... 121 Table 3.8 Fulton condition factors (k-values) of Pelteobagrus vachellii and other catfish species catched from fish stocks or as wildlife...... 124 Table 3.9 Overview of the temporal and spatial variations of different endpoints in sediment and fish at the respective sampling campaigns September 2011 (I; sediment), May 2012 (II; fish), and May 2013 (III; sediment and fish) given as means in relation to the reference site (REF)...... 135 Table 4.1 Nontarget compounds identified in the sediments of Kaixian ...... 157 Table 5.1 EEF and DEF of TCAB in comparison with other compounds based on ERα and AR CALUX assay...... 177 Table 5.2 The toxic potential of propanil, 3,4-DCA and TCAB predicated by VirtualToxLab using multi-dimensional QSAR ...... 182 Table I.1 Concentrations of PCDDs/DFs in water and sediments from the Yangtze River and other water bodies in the world...... 249 Table I.2 Concentrations of PBDEs in sediments from the Yangtze River and East China Sea.251 Table I.3 Concentrations of PFCs, PFOS, PFOA in water and sediments from the Yangtze River and other water bodies in China...... 252 Table I.4 Summary of other organic pollutants in water and sediment of the Yangtze River Drainage Basin...... 254 Table I.5 Summary of bioassay studies (in vitro and in vivo) in the Yangtze River...... 255 ______XIII List of tables ______

Table I.6 Summary of reported research about in situ studies in the Yangtze River...... 257 Table II.1 Settings of the LTQ Orbitrap XL instrument ...... 259 Table II.2 Processing steps and settings used for MZmine 2.10 ...... 260 Table II.3 Settings used for the R “nontarget” package ...... 260 Table II.4 Polycyclic aromatic hydrocarbons (PAHs), and hecterocyclic PAH concentrations together with AhR mediated activity in selected fractions of the TGR sediment samples ...... 261 Table II.5 Number of peaks detected in the active fractions and candidate filtering for potential AhR agonists based on retention time, mass defects, and absence in non-active fractions ...... 263 Table II.6 List of most intense peaks (>106 a.u. intensity) detected in the active fraction HF-LF13...... 264

Table III.1 List of single compounds used for establishing the relationship between log Kow and retention time (RT)...... 271 Table III.2 Characteristics of and concentrations of 17 PCDD/Fs and 12 dl-PCBs in sediments from Ehrenbreitstein (EBR) and Zollelbe (ZE)...... 275

______XIV

Chapter 1

General Introduction

______1

Chapter 1 General Introduction ______

Parts of this chapter have been published in peer-reviewed journals as:

Floehr, T*& Xiao, H*., Scholz-Starke, B., Wu, L., Hou, J., Yin, D., Zhang, X., Ji, R., Yuan, X., Ottermanns, R., Roß-Nickoll, M., Schäffer, A., Hollert, H. (2013) Solution by dilution? – A review on the pollution status of the Yangtze River. Environmental Science and Pollution Research 20: 6934-6971 (* Shared first authorship).

Xiao, H., Kuckelkorn, J., Nüßer, L-K., Floehr, T., Henning, M-P., Roß-Nickoll, M., Schäffer, A., Hollert, H. (2016) The metabolite 3,4,3’,4’-tetrachloroazobenzene (TCAB) exerts a higher ecotoxicity than the parent compounds 3,4-dichloroaniline (3,4-DCA) and propanil. Science of the Total Environment 551: 304-316.

______2 Chapter 1 General Introduction ______

1.1 Environmental pollution and monitoring strategies

Since human societies benefit from the industrial advancement and prosperity, the development of economic and industry also comes along with the production, usage, and emission of a constantly increasing number of chemicals. Nowadays, 108 chemicals are registered in the Chemical Abstracts System (CAS) (American Chemical Society, 2016), among which about 9 × 106 chemicals are in daily use (Daughton, 2005; Brack et al., 2016). This extensive use of chemicals unfortunately results in considerable amounts of hazardous compounds into the environment, which cause many challenges to the environmental sustainability, like water pollution. Chemical pollution in freshwater systems is a crucial environmental problem all over the world (Schwarzenbach et al., 2006). Around 1.2 billion people, or almost one-fifth of the world’s population, do not have access to safe water, and two-fifths are suffering health crises without adequate sanitation (United Nations 2003; US Department of State 2011). A recent Chinese government report has shown that within China around 36% of the 956 monitored river sections were unfit for human consumption (Ministry of Environmental Protection of the People´s Republic of China 2015), as a result of industrial and municipal wastewater discharges, agricultural and aquaculture fertilizer runoffs, pesticides and manure, and widespread eutrophication (Johnson et al., 1997; Wu et al., 1999; Zhang et al., 2004). River deterioration and pollution can pose serious threat to human health. Besides lacking of safe drinking water, human health is also threatened by other routes. For instance, humans may potentially be exposed to pathogens or chemical toxicants via the food chain, which can lead to bioaccumulation of toxic compounds through the ingestion of contaminated aquatic organisms (seafood and fish); or during recreation like swimming in polluted waters (Schwarzenbach et al., 2010). Additionally, water pollution can impair the biodiversity of aquatic ecosystems (Moyle and Leidy, 1992; Dudgeon et al., 2006) and further affect humankind (Gabsi, 2014). The prerequisite for appropriate pollution control is to initiate an effective and sustainable monitoring strategy. Chemical analyses are convential methods in environmental monitoring. Both target and nontarget analysis might provide valuable insights into known or unknown contaminants. However, the large number and great structural variety of possible pollutants in the environmental matrices make chemical analysis very difficult and prohibit to achieve complete information about the concentration of all inorganic and organic residues. Even if the complete

______3 Chapter 1 General Introduction ______information about all compounds is available, the overall toxic effects of the contaminants still cannot be characterized (Hecker and Giesy, 2011). Considering the possibility of synergistic effects of exposure to mixtures, the overall toxic effects can excess the sum of adverse impacts caused by individual exposures (Venkatesan and Halden, 2015). Biological analyisis can integrate response to all contaminants as well as to other environmental stress, thus indicating the overall effects for environmental matrices (Bartram and Ballance, 1996). Still, biological monitoring makes the integration of observed effects to a relevant group of chemicals difficult, and thereby hamper pollution control. In summary, either chemical or biological analysis alone is not sufficient to comprehensively monitor samples and chemicals contained in the environment matrix (Chapman and Hollert, 2006).

1.2 Effect-based tools for monitoring the ecotoxicological effects in aquatic environment

Since either chemical or biological monitoring face challenges when assessing the environmental risk in aquatic systems, approaches that integrate the two approaches are required to link ecological and chemical information, and to improve the environmental relevance of risk assessment (Connon et al., 2012). Effect-based tools with response detection on the molecular, subcellular or cellular levels of environmental samples (Altenburger et al., 2015) have been recommended as suitable tools in monitoring programs by the subgroup Chemical Monitoring and Emerging Pollutants (CMEP) under the Common Implementation Strategy (CIS) (Wernersson et al., 2014; Wernersson et al., 2015) for the European Water Framework Directive (WFD) (EWFD Directive 2000/60/EC, 2000). With the conceptual framework, several projects founded by the European Union are carried out for improving water monitoring management (Brack et al., 2012; Dulio and von der Ohe, 2013; Altenburger et al., 2015). While effect-based monitoring provides information on toxicological endpoints of concern, current approaches focus on the evaluation of toxic effects through in vitro assays, in vivo assays and biomarkers (Connon et al., 2012; Wernersson et al., 2015). For instance, the triad approach is a holistic assessment method to determine the potential risks associated with contaminated environmental samples (Chapman, 1990). The method integrates three components simultaneously: (i) chemical analysis, indicating the contamination level of detected compounds; (ii) bioassays (in vitro and in vivo), for measuring biological effects also regarding the bioavailability of environmental samples; (iii) in situ biomarkers, that can imply the alterations in ______4 Chapter 1 General Introduction ______the field (Chapman, 1990; Chapman and Power, 1992; Chapman and Hollert, 2006; Rocha, 2009). With a combination of the three individual parts, the triad approach is able to link ecological and chemical information, and can be useful for a comprehensive assessment of environmental conditions. Morever, effect-based tools that detect apical organism responses can be linked to toxicologically adverse effects to organism, or population, possibly leading to adverse outcomes to community (Altenburger et al., 2015). Thus, they can be applied in chemical environmental hazard assessment, and support to establish early warning systems (Wernersson et al., 2015). For example, Adverse outcome pathways (AOPs) are conceptual frameworks that summarize existing knowledge, and directly link a molecular-level initiating event to an adverse outcome at the biological organization level relevant to hazard assessment (Ankley et al., 2010; Volz et al., 2011). The aim of the AOP framework is to identify alternative, high-throughput predictive assays and testing strategies for risk assessment, facilitate identification of chemicals responsible for adverse effects or toxicity in a system. Importantly, the approach provides to a flexible method, which can reduce the costs and animal experiments in research on the basis of risk assessment (Ankley et al., 2010; Yozzo et al., 2013; Villeneuve et al., 2014). Hence, AOP is a promising method to link mechanism responses on the cellular levels with whole organism, population, community, and potentially ecosystem effects (Connon et al., 2012).

1.3 EDA as an environmental monitoring strategy

Numerous studies have shown that priority pollutants often contribute only to a minor extent to the adverse effects detected in the environmental samples (Brack and Schirmer, 2003; Woelz et al., 2008; Kaisarevic et al., 2009). Hazard and risk assessment of complex environmental mixtures can be significantly underestimated, if the focus is just on priority pollutants (Brack and Schirmer, 2003). The concept of effect-directed analysis (EDA), featuring a combination of fractionation, chemical analysis and bioassays, is able to identify unknown toxicants in complex environmental samples (Brack, 2003; Weller, 2012; Brack et al., 2016). The approach, which is applied in environmental sciences, is based on biotesting of environmental mixtures in combination with a sequential reduction of mixture complexity by fractionation procedures (Brack, 2003; Simon et al., 2015). EDA is a suitable tool to reduce the complexity of the samples and to detect the toxicants in organic chemical mixtures that contribute to an observed and measureable toxic effect (Connon et al., 2012; Brack et al., 2016). Thereby, the approach can be ______5 Chapter 1 General Introduction ______applied in environmental monitoring programes in order to address the effects on ecological health and envorinmental management of contamination source (Burgess et al., 2011), thus further supporting the prioritization and regulation of environmental contaminants (Brack et al., 2016).

1.3.1 Components of EDA

The basic assumption of EDA is that, although ecosystems and humans are exposed to complex mixtures of compounds, only few toxicants dominate the adverse effects (Weller, 2012). An overview on the EDA procedure is shown in Fig. 1.1. This approach is directed by the (i) biotests (in vitro or in vivo assays), through the assignment of toxicity to several groups of toxicants by (ii) separation steps including extraction, clean up, and fractionation. The aim is to remove compounds without significant contribution to sample toxicity and to identify the predominat toxicants using (iii) chemical analytical tools.

Fig. 1.1 Scheme of Effect-directed analysis (EDA) (Redrawn from Simon (2013)).

______6 Chapter 1 General Introduction ______

Biological analysis: The effects in biological analysis (Fig. 1.1), which ranging from small scale biological systems on a molecular, cellular (in vitro) or whole organism (in vivo) level, provide the direction to the iterative procedure (Brack et al., 2016). Thus, the toxicological endpoints are crucial for EDA study. The chosen endpoints largely predetermine the character of pollutants in EDA study (Simon, 2013). In principle, any toxicological, ecotoxicological or biological endpoint that can be detected and quantified with sufficient throughput can be applied in EDA study (Burgess et al., 2013b). Early EDA studies performed in the late 1970s focused on the identification of mutagenic compounds in cigarette smoke, diesel exhaust particles, drinking water and consumer products (Tabor and Loper, 1980; Wilson et al., 1980; Bjorseth et al., 1982; Schuetzle and Lewtas, 1986). While mutagenicity identification still remains a research topic nowadays (Lübcke-von Varel et al., 2012; Hug et al., 2015b), many other toxicological endpoints have been applied in EDA studies. Burgess et al. (2013b) partly reviewed the biological endpoints applied in EDA of surface waters, sediments, groundwater, landfill materials, and fish bile. The most frequently investigated toxic endpoints associated with sediment and surface waters are endocrine disruption (estrogenic activity, androgenic activity), dioxin and dioxin-like activity (cytochrome P-450 activity), mutagenicity, bioluminescene, algal growth, and daphnid toxicity. More endpoints, such as gene expression alterations (Scholz et al., 2008), thyroid hormone disruption (Simon et al., 2011), neurotoxicity (Qu et al., 2011b), androgenicity (Thomas et al., 2002), and anti-androgenicity (Weiss et al., 2009) have been applied in EDA study for emerging environmental problems. In addition, regarding the lack of realism in in vitro systems and bioavailability of contaminated samples, in vivo tests like Fish Embryo Toxicity (OECD, 2013) and Sediment Contact Test (Hollert et al., 2003) are encouraged to be applied in future EDA study (Hecker and Giesy, 2011). Separation: Sample separation is a prerequisite for biological diagnosis and compound identification in complex mixtures, since it reduces the interference of non-toxic substances and effectively supports the further EDA (Brack et al., 2011; Qu et al., 2011a). The initial step of EDA is extraction, which removes soluble compounds from the matrices and makes them feasible for subsequent biotesting and chromatograph analysis (Schwab and Brack, 2007). The chosen method mostly depends on the matrices. Liquid-liquid extraction (LLE) and solid phase extraction (SPE) are widely used for aqueous phase samples, while ultrasonic extraction, Soxhlet extraction and pressurized liquid extraction (PLE) are frequently applied for solid phase samples (Qu et al., 2011a). For biotic compartment samples, the pre-treatments are more challengeable ______7 Chapter 1 General Introduction ______since the co-extracted lipids and proteins would hamper biological and chemical analysis in further EDA. The methods have been reported by Simon and co-workers (Simon et al., 2010; 2011). Environmental matrices often contain target chemicals with a mixture of minerals, salts, water, and large biogenic organic molecules such as humic compounds, proteins, lipids, and polysaccharide (Brack et al., 2011), which are natural compounds and not of interest for organic toxicants identification. Thus, a clean-up procedure after extraction is often required. Several methods were reported (Streck et al., 2008; Simon et al., 2011), as reviewed in Brack et al. (2016). Fractionation is one of the key procedures in EDA. Compounds are separated according to their physicochemical properties, e.g. polarity, hydrophobicity, molecular size, planarity, and the presence of specific functional groups (Brack et al., 2003a). Fractionation in EDA is predominantly based on preparative reversed phase (RP) and normal phase high performance liquid chromatography (NP-HPLC) (Brack et al., 2003b; Lübcke-von Varel et al., 2008; Woelz et al., 2010a). Other fractionation methods, like hydrophilic interaction chromatography, size exclusion chromatography, affinity chromatography, planar chromatography, and gas chromatography were reviewed in Brack et al. (2011). The fractionation method choice usually depends on the matrix, expected compounds, applied bioassay, as well as the availability of equipment (Simon, 2013; Brack et al., 2016). Sometimes one single fractionation is not sufficient for EDA, and multistep fractionation procedures are required (Brack and Schirmer, 2003; Lübcke-von Varel et al., 2012). Identification and confirmation strategies: Chemical identification and confirmation are still the major challenges in EDA studies (Leonards et al., 2011; Simon et al., 2015). The most frequently used technique is gas chromatography coupled to mass spectrometry (GC-MS). The major advantage is that large mass libraries (e.g. Wiley, National Institute of Standard Technology [NIST]) can be used to identify unknown compounds. However, this application of GC-MS in EDA is limited, because compounds that are not included in the library cannot be identified correctly (Simon, 2013). Within the last decade liquid chromatography coupled with high-resolution mass spectrometry (LC–MS) is more frequently used than GC-MS (Hogenboom et al., 2009; Bataineh et al., 2010; Leonards et al., 2011), due to the increasing importance of polar contaminants and thermolabile compounds in the environment which are unfit for analysis by GC-MS. A series of analytical methods coupled with GC and LC are reviewed in Leonards et al. (2011). The type of compounds to be analysed is a prerequisite to choose a suitable analytical method (Simon, 2013). ______8 Chapter 1 General Introduction ______

After the suspected compounds are identified, toxic confirmation is the crucial final step to perform. A tiered approach has been suggested by Brack et al. (2008), and recently categorized in Schymanski et al. (2014a). Typically, suspect toxicants should be evaluated with the same bioassay and their contribution to the mixture of overall effects should be quantified. Moreover, toxicological data from literature, database or QSAR modeling can be helpful for qualitative confirmation (Brack et al., 2016).

1.3.2 Current EDA study and limitations

EDA has achieved a steep development since the 1970s, particularly in the past decade. The approach has been proven to be a powerful strategy for the identification of emerging compounds that contribute significantly to the toxic effects in the environment. The identified compounds have not only raised the concern to emerging contaminants, but also to some “old compounds” that have been ignored before (Lübcke-von Varel et al., 2012; Burgess et al., 2013a; Hug et al., 2015a). The approach can be used as a tool for pollution-source tracking, thus to improve the environmental quality (Qu et al., 2011a). However, EDA is not widely applied in environmental routine monitoring programs. Several aspects can be the reasons that limit a wider applicability of EDA. Fractionation steps that reduce the complexity of environmental mixtures and separate toxic fractions from non- or less-toxic fractions is one of the key procedures in EDA (Section 1.3.1) Due to the complexity of environmental samples, lower resolution fractionation is often not sufficient, and thus multistep fractionation procedures are required (Brack and Schirmer, 2003; Lübcke-von Varel et al., 2012). Moreover, the solvents used in fractionation usually interfere with bioassays and chemical analysis. Thus, after each fractionation step, a time-consuming evaporation procedure is necessary. Sometimes, several liters of solvents may need to be evaporated during the process, some of which have negative environmental impacts (e.g. chlorinated solvents). Moreover, multiple evaporation steps can lead to a decrease in recovery of compounds (Booij et al., 2014). Importantly, the iterative fractionation and subsequent handling procedures hinder straightforward biotesting, and thus hamper identification of bioactive fractions or chemicals. Furthermore, to perform a complete EDA study (Fig. 1.1), it also requires interdisciplinary knowledge of the researcher (Simon et al., 2015). In summary, the process is very laborious, costly, and time consuming; thus, conventional EDA is difficult to apply in routine monitoring programs (Simon et al., 2015; Brack et al., 2016).

______9 Chapter 1 General Introduction ______

1.3.3 The development of high-throughput EDA (ht-EDA)

To overcome the limitations of current study and enlarge the application of EDA in environmental monitoring programs, different studies have focused on technologies to improve online fractionation method and high-throughput biological screening. Pieke et al. (2013) presented an optimized micro-fractionation of compounds after GC separation. The GC-fractionation setup was able to collect fractions in second range in 96-well plates. In another study, Booij et al. (2014) and co-workers carried out a 96-well plate ultra-performance LC microfraction of passive sampler extracts, in combination with a pelagic marine algae test. Subsequently, the bioactive fractions were directly analyzed by chemical analysis on the same plates. Also, the contributors to estrogenic activity and androgenic activity were identified by a combination with gradient LC fractionation, estrogen and androgen bioassay respectively, and correlated to electrospray quadrupole time of flight MS (QTOFMS) analysis (Nielen et al., 2004; 2006). Jonker et al. (2015) combined a human cell (BG1.Luc) gene reporter assay with LC nanofractionation procedure to detect and identify the estrogenic and anti-estrogenic compounds in environmental water samples. A recent study has successfully identified acetylcholinesterase (AChE) inhibitors in a wastewater treatment plant by employing a two-dimensional liquid chromatography (LC × LC) micro-fractionation in parallel with high resolution Time of Flight (HR-ToF) mass spectrometric detection combined with the AChE assay (Ouyang et al., 2016). Such online fractionation methods can accelerate the EDA procedure without additional evaporation and solvent exchange. Moreover, high-throughput EDA (ht-EDA) requires relatively smaller volumes of sample in comparison to conventional EDA. The methodology, which can be used as a rapid screening tool, is also less complex for researchers. Therefore, as an efficient, simplified and environmental-friendly technology, ht-EDA is a promising method in environmental monitoring programs in future application, and requires more research.

1.4 The Yangtze River and the environmental challenges

Yangtze River (Chinese pronunciation: Changjiang) has been a source of life and prosperity for the Chinese people for centuries. The river is an important habitat for a remarkable variety of aquatic species (Fu et al., 2003). The potential benefit is expected to enlarge since the Three Gorges Dam (TGD) – the largest dam in the world – started operating at full capacity in 2009, including flood control, energy production and improvement of river navigation (Fu et al., 2010).

______10 Chapter 1 General Introduction ______

With such a huge project, numerous questions in ecotoxicological research are raised. Thus, the Yangtze River as well as the Three Gorges Reservoir (TGR) is selected as research model, with the purpose to develop a suitable monitoring strategy for other similar river systems in China as well as for global systems.

1.4.1 The Yangtze Basin

The Yangtze River, the longest river in Asia (6,300 km), rises from the Qinghai-Tibetan Plateau in the west and crosses the country through 11 provinces. Usually, the river is divided into three major sections: Upper Yangtze Reaches, Middle Yangtze Reaches and Lower Yangtze Reaches (Fig. 2.2). The Upper Yangtze (4,300 km) originates from the Geladandong Mountains down to Yichang, comprising a catchment area of roughly 100,000 km2. This section can be divided into Upstream Three Gorges Reservoir (Qinghai-Tibetan Plateau to Chongqing – 3,640 km) and the Three Gorges Reservoir (Chongqing to Sandouping/Yichang – 660 km). The Middle Reaches (950 km) stretch down to Hukou (outlet of Poyang Lake) with a catchment area of about 68,000 km2 (Bergmann et al., 2012). Downstream Hukou, the final 930 km, constitutes the lower Yangtze River with a total drainage area of 120,000 km2 (Chen et al., 2001) until the Yangtze River eventually empties into the East China Sea at the city of Shanghai. The Yangtze River has over 700 tributaries, with major tributaries such as Jialing River, which enters the main stream at the city of Chongqing, and Han River, which has its inlet at the city of Wuhan. It also connects with important freshwater lakes like Dongting Lake, Poyang Lake and Tai Lake in the Middle and Lower Reaches (Fig 1.2). These tributaries and interlaced lakes form a complete riverine- lacustrine network together with the Yangtze River (Fu et al., 2003).

______11 Chapter 1 General Introduction ______

Fig. 1.2 Map of the Yangtze River (Changjiang) Drainage Basin. TGR - Three Gorges Reservoir; TGD - Three Gorges Dam; (1) Jinsha River; (2) Yalong River; (3) Dadu River; (4) Ming River; (5) Jialing River; (6) Han River; (7) Wu River; (8) Dongting Lake; (9) Poyang Lake; (10) Taihu Lake.

The Yangtze River drains one-fifth of the land area of China and the river basin is home to 400 million people, one-third of the Chinese population. The river basin accounts for 40% of China’s freshwater resources (China Statistical Yearbook 2004, Wong et al. 2007). The Yangtze River’s annual runoff is about 9.5×1011 m3 (mean water discharge: 30,200 m3/s), accounting for 52% of the national total runoff (National-conditions; Zhang, 1995), which makes the river the major artery of China’s inland water transportation and the most important source for drinking water, supplying 186 cities (Greenpeace, 2010). The river basin also plays a significant role in fishery. It has been reported to account for about 60% of China’s freshwater fishery production (Liu and Cao, 1992; China Statistical Yearbook 2004; Liu et al., 2005), with a fish fauna that is considered to be one of the richest world-wide (Fu et al., 2003). It provides habitats for about 387 fish species, among which 146 species are unique to the Yangtze River (Yang, 2009). Due to its rich natural resources the river contributes to China’s economic development to a large extent. Known as the “Golden Channel”, it serves as a key factor in inter-province business navigating and the

______12 Chapter 1 General Introduction ______regional economy. The river is also one of China’s industrial and agricultural key locations. In 2006, over 10,000 chemical enterprises – about half of the total number in China – were located by the river (Yang et al., 2008). The Yangtze River basin provides more than 70% of the country’s rice and 50% of grain production, accounting for 40% of China’s gross domestic product (GDP) (China Statistical Yearbook 2004).

1.4.2 Three Gorges Reservoir

The Three Gorges Reservoir (TGR), created in consequence of the Yangtze River’s impoundment by the Three Gorges Dam (TGD), spreads over a distance of 663 km between the town of Sandouping, Hubei Province, and the Jiangjin district of Chongqing Municipality. The total area of TGR region is about 79,000 km2, involving 20 counties of Chongqing city and Hubei Province. Among the area, Chongqing municipality (82,400 km2) is the largest city in TGR. It had a population of more than 29 million people in 2011 (National Bureau of Statistics - China, 2012) with 6 – 7 million in the urban area in 2012 (BBC, 2012) (Fig. 1.3). The TGR is the largest river–type reservoir and freshwater resource reserve in China. TGR contains more than 400 secondary rivers, among which 42 main tributaries areas are above 100 km2, like Jialing River, Wu River, Pengxi River, Daning River, Xiangxi River etc. The total storage capacity is 39.3 billion cubic meters while having a 22.15 billion cubic meters flood control capacity (Ji et al., 2013). After the completion of the TGD, a 30 m high fluctuation zone between 145 and 175 m above sea level water will seasonally appear along the banks, and will form a seasonal wetland ecosystem (Fig. 1.3). Subsequently, particle bound pollutants, water, and sediments can be relocated on agriculturally used areas of the TGR’s water fluctuation zone (Scholz-Starke et al., 2013). Moreover, the elevation of water level improves the navigation of large container ships between Sandouping and Chongqing.

______13 Chapter 1 General Introduction ______

Fig. 1.3 Three Gorges Reservoir (TGR). Chongqing municipality (upper left), the Daning River at Wushan (upper right), a local dam at Hanfeng Lake in Kaixian (lower left) and its sea levels in May.2013 (lower right).

1.4.3 Environmental challenges

The construction of TGD poses great challenges to the unique ecosystem of TGR (Shen and Xie, 2004), particularly when facing numerous anthropogenic impacts, e.g., overpopulation, navigation wastes, and industrialization (Floehr et al., 2015a; 2015b). Furthermore, construction of the dam reduced the river’s flow velocity (Chen et al., 2005; Wang et al., 2009), and consequently increased the sedimentation rate of suspend particles and adhering contaminants (Hu et al., 2009a). Since the operation of TGD, annually 151 – 172 megatons of sediment have been trapped in the TGR (2003–2008) (Yang et al., 2007; Hu et al., 2009a). Sediments may function as the final sink of persistent and lipophilic pollutants in the environment (Hollert et al., 2003; Hilscherova et al., 2007). They can also become a secondary source of pollutants through resuspensions of particulate matter, e.g., during flood events

______14 Chapter 1 General Introduction ______

(Gerbersdorf et al., 2007; Woelz et al., 2009; Woelz et al., 2010b). Frequently occurring floods in the water fluctuation zone of TGR may increase the pollutants bioavailability through remobilization and direct exposure of benthic organisms (Brinkmann et al., 2010; Woelz et al., 2010b). To ensure the environmental and public health, suitable and effective monitoring strategies in TGR are urgently demanded.

1.5 Yangtze Hydro project

With respect to the environmental challenges in the TGR, and the concern in technical, ecological and social problems raised by the construction of TGD, German and Chinese groups from various scientific fields collaborated to provide knowledge for the sustainable management of the reservoir within the Sino-German joint research project “Sustainable management of the newly created ecosystem at the Three Gorges Dam” (Yangtze Project). The German Federal Ministry of Education and Research (BMBF) has been providing financial support for five German research institutes to perform applied research on changing land use, soil erosion, mass movements, and matter fluxes in the highly dynamic ecosystem since 2008/2009. Between August 2010 and July 2014, six more German partners (Karlsruhe Institute of Technology, IWW Water Centre, the Environmental Research Institute of RWTH Aachen University, Technische Universität München (TUM), Research Center of Jülich, Water Technology Center of Karlsruhe (TZW)) received funding from BMBF to research on sustainable water management as Yangtze-Hydro project (Bergmann et al., 2012). The Yangtze-Hydro Project has a close cooperation with Chinese research groups from a number of universities, institutes of the Chinese Academy of Sciences, the China Research Academy of Environmental Sciences, and other research centers. On the subject of understanding the most relevant changes in water and sediment quality in TGR, the research consisted of four work packages: (i) analyse the dynamics of physico-chemical parameters within the TGR; (ii) investigate the pollution levels in TGR; (iii) identify the behavior and transformation of contaminants; and (iv) study the degradation of contaminants (Bergmann et al., 2012).

1.5.1 MICROTOX project

The Institute of Environmental Research, RWTH Aachen University, Germany with a close corporation with Nanjing University, Chongqing University and Tongji University in China, worked on the subproject “Transformation, Bioaccumulation and Toxicity of Organic

______15 Chapter 1 General Introduction ______

Micropollutants in the Yangtze Three Gorges Reservoir (MICROTOX)”. The subproject aimed at the investigation of the TGR ecosystem on ecology, ecotoxicology and environmental behavior of organic pollutants (Fig. 1.4), to enable a sustainable management for the impacted area. Model “Fate” aims to investigate the fate of relevant model pollutants and its environmental behavior. The commonly used rice herbicide – propanil in TGR was applied as a model substance for mimicking the alteration of rice field soil between unsubmerged and submerged over a temporal course. The bioaccumulation of parent and metabolized substances was evaluated in a realistic simulation environment (Bioaccumulation). Propanil and metabolized substances were chosen as model substances for the simulation of the bioaccumulation in worst-case situations at TGR (Scholz-Starke et al., 2013). The present study focused on an integrated assessment of ecological aspects of TGR, with the objective of study on ecotoxicity of sediments and analysis of biomarkers of toxic stress (Ecotoxicity).

Fig. 1.4 The sub-project “MICROTOX” is divided into three synergistic modules. The Model-“Ecotoxicity” is applied with two methodologies: Triad Approach and Effect-directed analysis.

Ecotoxicity: In order to evaluate the ecotoxicity of TGR and assess the risk for human and the ecosystem, the model “Ecotoxicity” was conducted in the present study. The evaluation of ecotoxicity of sediments and water of the TGR was performed with several lines of evidence (Fig. 1.4). On the subjection of (i) evaluation the ecotoxicological status of TGR, and (ii) determination the regional “hot-spots” paired with the ecological relevant endpoints in the study area, the concept of triad approach (Chapman and Power, 1992) was coordinated with Floehr (2015), with a study on (1) chemical record on 54 organic pollution status based on the EWFD, (2) potential ecotoxicological effects of particle bound contamination by a batteries of in vitro/in vivo bioassays – mutagenicity, EROD induction, and embryotoxicity/teratogenicity. Furthermore, the ______16 Chapter 1 General Introduction ______approach was extended in a more detailed investigation of (3) in situ markers in the model fish species Pelteobagrus vachellii, as detailed described in in Floehr (2015). By accounting all the investigation results, the present study further applied EDA (Brack, 2003) in TGR, in purpose of (iii) identification of the unknown substance responsible for the biological effects in the identified “hot-spots” area. The overall results of these two approaches are used (iv) to develop a merged monitoring strategy to prevent the environment from excess exposure and effects of xenobiotics.

1.6 Toxic endpoints applied in the dissertation

Previous research has shown a significant pollution of persistent organic pollutants (POPs) in TGR, such as polychlorinated polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs), and polycyclic aromatic hydrocarbons (PAHs) (Floehr et al., 2013; Deyerling et al., 2014; Floehr et al., 2015a; 2015b). The groups of toxicants that are of particular interest relative to potential environmental health effects are dioxin-like, mutugenic, genotoxic, and embryotoxic chemicals. Such compounds are known to induce cytochrome P450 1A (CYP1A) by ligand- activation of the aryl hydrocarbon receptor (AhR) (Van den Berg et al., 2006; Sorg, 2014), genotoxicity and mutagenicity even at low concentrations (Ohe et al., 2004; Grund, 2011; Umbuzeiro et al., 2011; Sorg, 2014). The toxicants may cause increased mortality in early life stage fish (Walker and Peterson, 1994; Andreasen et al., 2002) and adverse effects in wild fish populations (Niimi, 1983; Gilbertson, 1992; Whyte et al., 2000; Van der Oost et al., 2003). This is of particular concern, as the Yangtze River plays an important role for fishery production in China (Section 1.4.1). Thereby, the following endpoints are of particular interest for the present study.

1.6.1 AhR-mediated activity

The AhR is a member of the basic helix-loop-helix protein family (Zacharewski et al., 1995; Mocarelli et al., 2008). It is a ligand-dependent transcription factor, that is localised in the cytoplasm (Hecker and Giesy, 2011). The receptor can be activated by a variety of xenobiotic compounds that can diffuse through plasma membranes because of their lipophilic properties (Sorg, 2014). With the presence of AhR agonists in the cytoplasm, the agonists bind to the AhR and form AhR-agonist complex. After the binding of ligand and receptor, the complex translocates into the nucleus, where it forms a heterodimer with the AhR nuclear translocator protein (ARNT) (Hahn, 1998; Hecker and Giesy, 2011). The ligand – ARNT complex has high

______17 Chapter 1 General Introduction ______affinity to specific DNA sequences, and thus binding modulates the expression of downstream genes, like phase I (e.g. CYP1A1) and phase II (e.g. UGT1A1) enzymes (Schmidt and Bradfield, 1996; Sorg, 2014). The strength with which ligands bind to the AhR is proportional to the toxicity, the transcriptional activity, and the AhR-mediated enzyme activities (Safe, 1995). Thus, in vitro bioassays were developed for the characterization of AhR activation potency of environmental samples. 7-ethoxyresorufin-O-deethylase (EROD) assays are frequently used in vitro to exemplarily determine the AhR-mediated dioxin-like activity of environmental samples. These assays can be conducted with different cell lines, such as fish cell lines, and rat hepatoma cell lines, where the deethylation reaction is catalyzed by CYP1A1 and CYP1A3, as well as CYP1A1 and CYP1A2, respectively (Goksøyr and Förlin, 1992; Whitlock Jr, 1999; Bols et al., 2005). The AhR agonists are assumed to have quantitative structure-activity relationships to the AhR (Safe, 1998). Based on such relationships, the relative equivalent potency (REP) of individual compound relative to a standard (e.g., TCDD) can be experimentally determined. Consequently, when the concentrations of individual compounds in the environment are known, the overall effects on activated receptors by different agonists can be estimated, which is expressed as toxic equivalents (TEQs). This approach has been extensively applied as a probabilistic methodology to assume the associated burden of overall toxicity in environmental matrices, such as water, soil, sediment (Andersson et al., 2009; Brinkmann et al., 2014), and to evaluate the potential hazard and risk assessment of human exposure to chemicals (Van den Berg et al., 2006; Meyer et al., 2014; Brinkmann et al., 2015; Eichbaum et al., 2014; 2016). AhR agonists are known as hydrophobic aromatic compounds with planar structure of a particular size, such as PCDDs/PCDFs, PCBs, PAHs and polychlorinated napthalenes (PCNs) as well as other structurally similar organic compounds (Poland and Knutson, 1982; Lewis et al., 1986; Giesy et al., 1994; Blankenship et al., 2000; Van den Berg et al., 2006). Some other compounds, like heterocyclic aromatic compounds (Hinger et al., 2011), natural compounds (Jia and Hu, 2010), benzothiazole derivatives (Noguerol et al., 2006; He et al., 2011a), pesticides and metabolites (Poland et al., 1976) were reported to bind to the AhR, thus have been suggested as potential AhR agonists. So far, only limited research was done on bioassays to determine AhR- mediated activity in TGR (Cui et al., 2009; Wang et al., 2014b; Floehr et al., 2015a). To our knowledge, no research has been performed the EDA approach to identify the AhR agonists in TGR. ______18 Chapter 1 General Introduction ______

1.6.2 Mutagenic and genotoxic activity

Chemicals that can interact with the genetic material (DNA) of living organisms causing different types of structural modifications are termed as genotoxins (Eastmond et al., 2009). If the damage cannot be repaired, a permanent change in the DNA like a mutation occurs (Umbuzeiro et al., 2011). Due to their impact on genetic material, mutagenic and genotoxic effects can lead to adverse effects at the individual level such as aging, cancer, genetic and development disorders, and at the population level by alterations in population fitness and offspring, and finally showing effects on the ecosystem level (Brusick et al., 1992; Dearfield et al., 2002; Diekmann et al., 2004). There are genotoxicity assays that detect DNA damage (e.g., comet assay, umu-test, chromotest, levels of DNA adducts, micronuclei assay) and mutagenicity assay whose endpoints are mutations and chromosome damage. The mutagenicity assays can be divided into those that detect point mutations (e.g., bacterial Salmonella/microsome assay) and those that detected chromosome aberrations, including micronuclei (Umbuzeiro et al., 2011). The Salmonella mutation test has been widely used to assess mutagenic activity in environmental samples, such as sediments and water. The most frequently used strains for environmental applications are TA98 and TA100 that can detect mutagens that cause frameshift and base pair mutations, respectively (Ohe et al., 2004). In the present study, the Ames fluctuation assay (ISO 11350 2012) was used to detect the mutagenicity of sediment extracts from the TGR. The micronucleus assay is an important component of bioassay batteries that aim to assess the genotoxic and mutagenic potencies of chemicals and environmental samples. Fish blood has been proven to be a sensitive compartment for genotoxicity measurement in fish (Rocha, 2009; Boettcher et al., 2010), which was applied as in situ micronucleus assay in the study area.

1.6.3 Embryotoxic and teratogenic potential

The aquatic system can be considered as a sink for various pollutants. Fish are important in the aquatic food webs by top-down and bottom-up regulation of nutrient and energy flow, function as sentinels for the water quality, and food resources for humans (Lammer et al., 2009). In conventional ecotoxicity testing strategies, fish are an indispensable component of integrated toxicity testing strategies for the aquatic environment (Lammer et al., 2009). Additionally, chemicals should be subjected to fish acute toxicity examinations before releasing them to the environment, in support of environmental risk assessment and its hazard classification. Nonetheless, fish are hypothesized to suffer severe distress and pain in acute tests with their ______19 Chapter 1 General Introduction ______exclusive endpoint of mortality (Nagel, 2002; Braunbeck et al., 2005), which is in conflict with current animal rights legislations in many regulatory jurisdictions (Lammer et al., 2009). The in vivo Fish Embryo Toxicity (FET) test with eggs of Danio rerio (OECD, 2013) has been proven to be a suitable tool in ecotoxicological risk assessment (Peddinghaus et al., 2012; Floehr et al., 2015b). The FET can serve as a replacement to the acute toxicity test with adult fish, and also allows to record sublethal impacts that might affect the overall fitness of the fish (Nagel, 2002; Braunbeck et al., 2005; Lammer et al., 2009; Embry et al., 2010; Strähle et al., 2012). Moreover, Sediment Contact Assay (SCA), which allows the assessment of sediment toxicity including the bioavailability of sediment contaminants (Hollert et al., 2003), can be applied for the evaluation of vertebrate toxicity to contaminated sediments. Thus, FET and SCA were used as in vivo tool to evaluate the embryotoxic and teratogenic potential of the sediments in TGR.

1.7 Aim of the study and structure

The objective of this PhD thesis is to determine the hazards potential, and to provide detailed insights into ecotoxicological risks of the Yangtze River Basin in particular of TGR. The thesis aims to (1) evaluate the ecotoxicological status of the Yangtze River (Chapter 2), (2) investigate the general ecotoxicological situation, and provide a holistic insight into ecotoxicological risks of contaminated sediments from the TGR (Chapter 3), and (3) apply the EDA concept to the sediments to identify the main pollutants responsible for the observed ecological effects (Chapter 4). Moreover, (4) the ecotoxicological effects of widely used herbicide in the study area – propanil and the metabolized compounds – are investigated (Chapter 5). Furthermore, (5) a rapid work-flow for identification of AhR-agonists for environmental samples is developed (Chapter 6). The structure of the thesis is shown in Fig. 1.5.

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Fig. 1.5 Flowchart demonstrating structure of the thesis

1.7.1 A comprehensive perspective of ecotoxicological status of the Yangtze River Basin

The Yangtze River Basin is particular important for the Chinese people. However, it suffers from serious pollution due to the anthropogenic actives along the River Basin, such as wastes from urban sewage, agricultural effluents, and industrial wastewater as well as ship wastes along its course. With respect to the vast amounts of water and sediments discharged by the Yangtze River, Chapter 2 reviewed the published research about the Yangtze River for the past two decades, with a focus on organic pollutants and potential effects of water and sediments on wildlife and humans in the Yangtze River, as well as several adjacent water bodies connected to the main stream. The chapter gave an overview of the ecotoxicological status of the Yangtze River, pointed out highly polluted sections that have potential environmental risk. Based on the relevant data, we answered the question whether the pollution problem of the Yangtze River can be solved by simple dilution.

1.7.2 Ecotoxicological evaluation of sediment and fish in the Three Gorges Reservoir

The Three Gorges Reservoir (TGR), created in consequence of the Yangtze River’s impoundment by the Three Gorges Dam, faces numerous anthropogenic impacts that challenge

______21 Chapter 1 General Introduction ______its ecosystem. In order to record organic contamination, find links to ecotoxicological impacts and to serve as reference for ensuing monitoring, the triad approach, with several lines of evidence (chemical analysis, in vitro/in vivo bioassays, and in situ biomarkers) was applied for a holistic assessment in the TGR area. Chapter 3 presented the organic pollution of sediments, toxicological effects via in vitro/ in vivo bioassays and relevant impacts on fish for the field study. With comparison of a broad range of effects, the regional pollution “hot-spot” areas and relevant environment endpoints were identified.

1.7.3 Effect-directed analysis of aryl hydrocarbon receptor agonists in sediments from the Three Gorges Reservoir, China

Numerous studies pointed out that risks may be significantly underestimated if monitoring strategies only focus on a limited number of priority compounds, thereafter the regulatory decisions may potentially be misled. To avoid to overlook the relevant toxicants in TGR, effect- based monitoring and toxicant identification were performed in Chongqing and Kaixian, which were identified as regional hot-spots paired with the AhR-mediated activity in TGR from Chapter 3. Chapter 4 represented the EDA research – in combination of effect assessment, fractionation procedure, and target and non-target analyses – in order to characterize aryl hydrocarbon receptor (AhR) agonists in the sediments of the TGR. Individual AhR agonist in Chongqing and Kaixian are were characterized.

1.7.4 Ecotoxicology evaluation of propanil, 3,4-dichloroaniline (3,4-DCA), and 3,4,3’,4’-tetrachloroazobenzene (TCAB)

Propanil (3,4-dichloropropionaniline), which is applied to control barnyard grass in the cultivation of rice, is one of the most extensively used herbicides worldwide, as well as in TGR. The herbicide is not persistent in environment (Tanaka et al., 1981; WHO, 2004), and can be transformed to 3,4-dichloroaniline (3,4-DCA) and further converted to 3,4,3’,4’- tetrachloroazobenzene (TCAB). To assess the impact of these compounds on the environment and to support regulatory decisions, Chapter 5 investigated the ecotoxicological effects of propanil, 3,4-DCA and with a focus on TCAB using in vitro, in vivo assays, and in silico tool. The possible adverse outcomes pathways (AOP) of TCAB were conducted.

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1.7.5 Optimized work-flow for identification of dioxin-like compounds in environmental samples combining High-throughput fractionation and bioassay

EDA has been demonstrated to be a powerful strategy to identify biologically active compounds in environmental samples. However, in current EDA studies, fractionation and subsequent handling procedures are usually laborious. These procedures involving multiple steps are prone to contamination and may decrease recovery of the compounds. These drawbacks limit the application in environmental risk assessment and biological effects monitoring programs. Hence, in Chapter 6, we developed a high-throughput EDA (ht-EDA) work-flow combining reversed phase high-performance liquid chromatography (RP-HPLC) fractionation of samples into 96-well microplates, followed by toxicity assessment in the micro-EROD bioassay, and chemical analysis of biologically active fractions. The work-flow was further optimized by seeding previously adapted suspension-cultured H4IIE cells directly into the microplate used for fractionation. The optimized work-flow aimed to simplify the conventional EDA for wider application in ecotoxicology. Finally, Chapter 7 summarizes the important findings from the above chapters and explains how the research can assist in environmental monitoring programs and thus support regulatory decisions.

______23 Chapter 1 General Introduction ______

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Chapter 2 2 Solution by dilution? – A review on the ecotoxicological status of the Yangtze River

______25

Parts of this chapter have been published in a peer-reviewed journal as:

Floehr, T*& Xiao, H*., Scholz-Starke, B., Wu, L., Hou, J., Yin, D., Zhang, X., Ji, R., Yuan, X., Ottermanns, R., Roß-Nickoll, M., Schäffer, A., Hollert, H. (2013) Solution by dilution? — A review on the pollution status of the Yangtze River. Environmental Science and Pollution Research 20: 6934-6971 (* Shared first authorship).

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Chapter 2 Solution by dilution? ______

2.1 Abstract

The Yangtze River has been a source of life and prosperity for the Chinese people for centuries and is habitat for a remarkable variety of aquatic species. But the river suffers from huge amounts of urban sewage, agricultural effluents and industrial wastewater as well as ship navigation wastes along its course. With respect to the vast amounts of water and sediments discharged by the Yangtze River it is reasonable to ask whether the pollution problem may be solved by simple dilution. This chapter reviewed the past two decades of published research on organic pollutants in the Yangtze River and several adjacent water bodies connected to the main stream, according to a holistic approach. Organic pollutant levels and potential effects of water and sediments on wildlife and humans, measured in vitro, in vivo and in situ, were critically reviewed. The contamination with organic pollutants, including polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs), polychlorinated dibenzo-p- dioxins/polychlorinated dibenzofurans (PCDDs/PCDFs), polybrominated diphenyl ethers (PBDEs), perfluorinated compounds (PFCs) and others, of water and sediment along the river was described. Especially Wuhan section and the Yangtze Estuary exhibited stronger pollution than other sections. Bioassays, displaying predominantly the endpoints mutagenicity and endocrine disruption, applied at sediments, drinking and surface water indicated a potential health risk in several areas. Aquatic organisms exhibited detectable concentrations of toxic compounds like PCBs, OCPs, PBDEs and PFCs. Genotoxic effects could also be assessed in situ in fish. To summarize, it can be stated that dilution reduces the ecotoxicological risk in the Yangtze River, but does not eliminate it. Keeping in mind an approximately fourteen times greater water discharge compared to the major European river Rhine the absolute pollution mass transfer of the Yangtze River is of severe concern for the environmental quality of its estuary and the East China Sea. Based on the review further research needs have been identified.

Keywords: Yangtze River, ecotoxicity, triad approach, organic pollutants, bioassay, mutagenicity, fish

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Chapter 2 Solution by dilution? ______

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Chapter 2 Solution by dilution? ______

2.2 Introduction

The Yangtze River basin plays a significant role in industry, agricultural and fishery for Chinese economic development (cf. Section 1.4.1). However, benefits always go with consequences. The anthropogenic disturbances of the Yangtze River basin leads to increased soil erosion and growing risks for landslides due to deforestation and the impoundments of reservoirs created by overall 45,000 dams in the Yangtze watershed (Wu et al., 2004; Mueller et al., 2008). These dams and the increase of river navigation alter the original ecosystems and pose threats to the local biodiversity. Beyond that the river suffers huge amounts of industrial wastewater, urban sewage discharge, ship navigation and oil containing wastewater discharge from ships. It was rated by the World Wildlife Fund (WWF) among the top 10 rivers in the world at risk (Wong et al., 2007). In the 1990s, the organic pollutants of the Yangtze River were monitored by the Yangtze Valley Water Environment Monitoring Center (YVWEMC). Two hundred and six hazardous organic chemicals were detected in waters and 106 in sediments, 17 of them belonging to the priority controlled pollutants of America (Wang, Peng et. al 2002). Some organic compounds, like the so-called persistent organic pollutants (POPs), which resist photolytic, biological and chemical degradation (Ritter. L. et al., 1995), pose great ecotoxicological risks to aquatic ecosystems. Later in 2006, the growing fraction of wastewater in the Yangtze River was confirmed by Mueller et al. (2008), which induces rising levels of nutrients, heavy metals and dissolved organic carbon (DOC) accompanied by an astonishing discharge of 500 to 3,500 kg industrial organic chemicals per day (Mueller et al., 2008).

2.3 Scope and aims

The question “How polluted is the Yangtze River?” was previously raised by Mueller et al. (2008). In an extended sampling campaign they studied the concentrations of major ions, nutrients, trace elements and organic pollutants in the middle and lower reaches of the Yangtze River. They concluded that the concentrations of many anthropogenic compounds were comparable to other major rivers in the world, because high dilution rates mask the enormous discharge of pollutants. Yet they pointed out that economic growth, population and living standards suggest that the concentrations of important water quality constituents are rising and that especially the Yangtze Estuary might face disastrous effects (Mueller et al., 2008).

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Chapter 2 Solution by dilution? ______

It is reasonable to ask whether there is actually a pollution problem due to the high mass transport of water and sediment of the Yangtze River despite all contamination sources. Is the pollution problem solved by simple dilution? Is the Yangtze River capable to dilute pollutant levels to a non-toxic degree, without considerable consequences for wildlife and humans? To answer this question, on the one hand data about pollutant levels are required, on the other hand it should be considered to acquire knowledge about possible effects which might be induced by these (even low) concentrations. Even smallest concentrations of certain contaminants are capable to induce toxic effects, and bioaccumulation as well as chronic toxicity is not negligible. In order to complement the work of Mueller and coworkers (Mueller et al., 2008), this review summarizes the current state of knowledge, comprising published data of the last two decades, regarding ecotoxicological investigations on organic pollutants in the Yangtze River. The information was divided into three major chapters according to the conceptual strategy of the triad approach (Chapman, 1990) (Fig. 2.1). First, the concentrations of organic pollutants, including POPs, like polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), organochlorine pollutants (OCPs) and other hazardous substances, in water and sediments were critically reviewed. Second, a series of screening bioassays were referred to with the endpoints genotoxicity, mutagenicity and estrogenic activity. Third, available in situ studies were reviewed according to pollutant levels and adverse effects in aquatic organisms, like fish, mollusks and crabs. In this way, we attempt to link chemical pollution to ecotoxicological effects in the Yangtze River to provide a clear and comprehensive view on the pollution status of this unique water body.

Fig. 2.1 Holistic approach to evaluate ecotoxicity of water, sediment, soil and suspended particulate matter as well as state of contamination in local fish species according to the triad approach (redrawn from Chapman et al. 1992). ______30

Chapter 2 Solution by dilution? ______

2.4 Methodology and materials studied

The literature research was carried out based on the triad approach (Chapman, 1990; Chapman and Hollert, 2006) (Fig. 2.1), which combines exposure (chemical analyses) and effect analyses (acute and mechanism-specific toxicity). Furthermore in situ biological assessment can be used to assess effects on organisms in the field (Chapman, 1990). The data used for this literature review were obtained from a broad variety of platforms such as Web of Knowledge, PubMed, Google scholar, Wanfang and CNKI (China National Knowledge Infrastructure). Some of the papers were published in Chinese. The time frame was set to browse the recent literature of the last two decades (1990-2012). The chosen keywords included Yangtze River/Changjiang (Fig. 2.2) and Yangtze River/Changjiang in combination with “sediment”, “water”, “toxicity”, “ecotoxicity”, “biotest”, “organic pollutants” or “persistent organic pollutants”. Overall the number of scientific publications on the Yangtze River has grown significantly during the past years (source: Web of Science, searching for Yangtze River/Changjiang, in article’s topic) (Fig. 2.2). In total 84 articles fit into the conceptual approach of this review and have been evaluated. The predominant research on the ecotoxicological status of the river focuses on chemical concentrations in the compartments water and sediment. Research applying bioassays in this area has just started up and only limited literature was found. Similarly, limited information was identified for in situ studies in context of concentrations in aquatic biota and toxicological effects on these organisms.

Fig. 2.2 Number of publications about the Yangtze River per year (1990-2012) (source: Web of Science, searching for Yangtze River/Changjiang, in article’s topic). ______31

Chapter 2 Solution by dilution? ______

In order to limit the scope of this review the focus lies on the Yangtze River mainstream and organic pollutants. Some of the reviewed studies include adjacent water bodies that are linked to the Yangtze River (Annex I, I.1), e.g., tributaries like the Jialing River at Chongqing section or freshwater lakes like Dong Lake in Wuhan. Information about these water bodies are given for comparability reasons. It should also be noted that values have been rounded to whole numbers for reasons of better illustration and comparability. Values below 0.5 have not been set to zero to avoid misinterpretations. Guidelines values and some exceptions have not been rounded. All comparisons between the concentrations of pollutants at different sections are based on the reported results, and differences between the laboratories as well as variations in the analysis methods were neglected. Moreover, to assess the worst pollution scenario at each section, not only the average but also the maximum values were used. Additional information is given in Annex I.

2.5 Pollutant levels in water and sediments

The water quality in the Yangtze River has deteriorated since the 1990s due to the anthropogenic activities in China (Yang et al., 2008). To ensure the health of aquatic biota and human beings in surrounding areas, the Chinese scientists and international communities pay close attention to the pollution status in the Yangtze River. For instance, the YVWEMC investigated micro-organic pollutants in water, sediments, and fish at the mainstream of the Yangtze River in 1990s (Wang, Peng et. al 2002). In order to study the water quality in the Yangtze River, a sampling campaign was carried out by Swiss and Chinese scientists in 2006. They sampled hundreds of water and sediments from below the Three Gorges Dam (TGD) to the river mouth at Shanghai, to determine major anions and cations, nitrogen and phosphorus, dissolved and particulate trace elements, and some organic pollutants (Mueller et al., 2008). In addition, a number of studies were conducted to characterize the pollution of different environmental matrices with organic contaminants such as OCPs (Liu et al., 2011a) and perfluorinated compounds (PFCs) in water (So et al., 2007), as well as perfluorooctane sulfonyl fluoride (PFOS) in fish (Greenpeace, 2010) have been conducted along the river. Several pollutants, such as PAHs, PCBs and OCPs, in the Yangtze River are studied for a long time with sufficient information at some reaches. Apart from those “typical pollutants”, new emerging contaminants like polybrominated diphenyl ethers (PBDEs), perfluorinated compounds (PFCs) have recently attracted much attention in China (Wang et al.,

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Chapter 2 Solution by dilution? ______

2010a) as well as their occurrence in the Yangtze River (So et al., 2007; Xian et al., 2008). This part describes and compares the available data on the occurrence of the predominant organic pollutants which were reported in water and sediments of the Yangtze River during the last two decades: PAHs, PCBs, OCPs, PCDDs/DFs, PBDEs, PFCs in addition to phthalic acid esters (PAEs), nonylphenol (NP), bisphenol A (BPA).

2.5.1 Polycyclic aromatic hydrocarbons (PAHs)

PAHs are a class of compounds that consist of three or more fused benzene rings with only carbon and hydrogen atoms (ATSDR, 2009). PAHs are usually produced by incomplete combustion or high-pressure processes, and mainly originate from natural (oil seepage, biomass burning, volcanic eruptions and diagenesis) and anthropogenic sources (fossil fuel combustion and industrial processes) (Yunker et al., 2002; Wang et al., 2007). PAHs mostly act as carcinogens and mutagens, e.g., certain PAHs metabolites are genotoxic and may interact with DNA, causing malignancies and heritable genetic damage in humans (ATSDR, 2009). The 16 PAHs, chosen as priority pollutants by the U.S. Federal Water Pollution Control Act (1972) and the US Clean Water Act (1977) (Huang et al., 2003; Tobiszewski and Namieśnik, 2012), have been more commonly studied, among which, benzo[α]pyrene (BaP) is used as an environmental indicator for PAHs (ATSDR, 2009). In the investigation of the YVWEMC, PAHs were one of the main organic pollutants in the Yangtze River during the 1990s, while Chongqing and Nanjing section presented more serious PAHs pollution than other sections (Wang, Peng et. al 2002). The PAHs in water and sediments of the Yangtze River were summarized in Fig. 2.3. Water: The highest concentration of PAHs was reported at Panzhihua section in the Jinsha River, ranging from 2.1 × 104 to 3.83 × 105 ng/L with an average concentration of 1.06 × 105 ng/L (Huang et al., 2003). The lowest concentration of PAHs was observed at the TGR (14 – 97 ng/L) (Wang et al., 2009). Higher concentration of PAHs in the Yangtze River were detected at the Wuhan section (mainstream: 322 – 6,235 ng/L; tributaries: 242 – 1,379 ng/L) (Feng et al., 2007a) and Jiangsu section (40 – 3,345 ng/L) (Martins et al., 2011). In comparison to other rivers in China such as the Yellow River (97 – 477 ng/L) (Zhang, 2010) and the Tai Lake (469 ng/L) (Zhang et al., 2012), the concentration of PAHs at Wuhan section was higher in the Yangtze River. Concentrations in the South Branch of the Yangtze Estuary (Annex I, Fig. I.1) ranged from 478 to 5,027 ng/L (mean: 1,727 ng/L) in high water period (August) and 972 to 6,273 ng/L (mean: 1,988 ng/L) in low water period (February) (Ou et al., 2009). The level of PAHs in the ______33

Chapter 2 Solution by dilution? ______

Yangtze Estuary share the similar level with the Pearl Estuary (987 – 2,879 ng/L, mean: 1,796 ng/L) (Luo et al., 2004), while they are lower than that at Macao harbor (944 – 6,655 ng/L, mean: 4,124 ng/L) (Luo et al., 2004). The PAHs pollution status has significantly increased in the past decades, in comparison to PAHs levels along the river in 1990s (n.d. – 135 ng/L, mean: 22 ng/L) (Wang, Peng et. al 2002). So far, no regulatory limit has been imposed on any PAHs, except BaP (2.8 ng/L), for the surface water in China (Ministry of Environmental Protection - China, 2002). Fig. 2.3a presents the concentrations of total PAHs and BaP reported at each section. It is, however, interesting that BaP was not detected in any sampling station in the Jinsha River (Huang et al., 2003), while the predominance of benzo[k]fluoranthene (BkF) and indeno[1,2,3-cd]pyrene (InD) was clearly observed in this area (Huang et al., 2003). This is quite different from the results of other sections in the Yangtze River reported in the literature. The concentrations of BkF (16 – 296 µg/L) and InD (4 – 83 µg/L) in the Jinsha River exceeded the criteria recommended by the European Union (sum of BkF and BbF: 30 ng/L, and sum of benzo[g,h,i]perylene and InD: 2 ng/L) (European Union, 2008). In addition, the maximum concentrations of BaP in Wuhan section (n.d. – 214 ng/L) (Feng et al., 2007a) and Jiangsu section (n.d. – 768 ng/L, mean: 256 ng/L) (He et al. 2011) exceeded both the Chinese regulatory limit (2.8 ng/L) and EC Directives for surface water (50 ng/L) (European Union, 2008). The highest concentration of BaP was observed at Nanjing section, ranging from n.d. to 768 ng/L (mean: 256 ng/L). The pollution was attributed to some heavy chemical industry in the districts of Nanjing (Martins et al., 2011). Sediment: Compared to the Lower Reaches, especially the Yangtze Estuary, sediments in the Upper Reaches of the Yangtze River are not well investigated (Fig. 2.3b). The upper reaches of the river contain few sediments due to the rapid flow and special local geographical features (Huang et al., 2003). According to the present studies at Chongqing section (257 – 723 ng/g, mean: 359 ng/g) (Tang et al., 2011) and the Jialing River (132 – 349 ng/g, mean: 240 ng/g) (Tang et al., 2011), the levels of PAHs in the Upper Reaches were lower than those in Middle and Lower Reaches. The concentration of PAHs in sediments at Wuhan section showed a higher level, ranging from 303 to 3,995 ng/g (mean: 2,032 ng/g) in the main stream and 4,121 to 4,262 ng/g in the tributaries (Feng et al., 2007a). The highest concentration of core sediments in the Yangtze Estuary (11,740 ng/g) was found near one sewage discharge point of Shanghai (Liu et al., 2000), in which sewage discharges were a significant input source. However, in comparison to the

______34

Chapter 2 Solution by dilution? ______

Yellow Estuary (11 – 252 ng/g, mean: 91 ng/g) (Hui et al., 2009a), the total PAHs levels of the Yangtze Estuary in surface sediments (263 – 6,372 ng/g, mean: 1,662 ng/g) was significantly higher (Liu et al., 2001), but was lower in other urban areas in China, such as the Pearl Estuary (323 – 14,812 ng/g) (Mai et al., 2002) or elsewhere in the world, like San Francisco Bay (2,653 – 27,680 ng/g) (Pereira et al., 1996). There have not yet been any official related criteria or standards to evaluate biological effects of Total PAHs in sediments. Two guideline values, “effect range-low (ERL)” and “effect range- medium (ERM)”, developed by Long et al. (1995), were commonly used to assess the adverse biological effects of contaminants in sediments. The assessment criteria are as follows: concentration < ERL: biological effects rarely occur; ERL ≤ concentration < ERM: biological effects occasionally occur; concentration ≥ ERM: biological effects frequently occur (Long et al., 1995). The effect range values for PAHs (ERL: 4,022 ng/g dry weight, ERM : 44,792 ng/g dry weight) demonstrate that the maximum concentrations of PAHs in the tributaries at Wuhan section (4,121 – 4,262 ng/g) (Feng et al., 2007a), one discharge point of Shanghai at the Yangtze Estuary (11,740 ng/g) (Liu et al., 2000), and the Yangtze Estuary (6,372 ng/g) (Liu et al., 2001) exceeded the ERL value, but were significantly lower than ERM value. It indicates that the PAHs may cause toxic effects. This is in accordance to a guidance proposal for the assessment of sediment quality presented by Ahlf et al. (2002), which gives a quality goal of 1,000 – 4,000 ng/g for EPA-PAHs in sediments. Values that exceed this goal require further analysis or immediate action like remediation and excavation of the contaminated sediments. However, based on the available studies and according to a classification by the International Commission for the Protection of the Rhine (ICPR, 2009), which is based on the European Water Framework Directive (EUWFD) (EWFD Directive 2000/60/EC), an integrated river basin management to improve the quality of European water bodies, all of the BaP levels in sediments Fig. 2.3b were below a “relevant contamination” (> fourfold ICPR target value) (800 – 1,600 ng/g) (ICPR, 2009).

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Chapter 2 Solution by dilution? ______

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Sources: PAHs diagnostic ratios, such as ANT/(ANT+PHE), FLA/(FLA+PYR), IP/(IP+Bghi), ƩLMW/ƩHMW (Total Low Molecular Weight: sum of two and three-ring PAHs; Total High Molecular Weight: sum of four and five ring PAHs), provide an important tool for the

______36

Chapter 2 Solution by dilution? ______identification of the responsible pollution emission sources (Mai et al., 2002; Wang et al., 2009; Tobiszewski and Namieśnik, 2012). Likely due to the development of the coal chemical industry at Panzhihua section, Huang et al. (2003) discovered that five- and six- ringed PAHs were abundant at most sampling stations in the Jinsha River, and serious PAHs pollution could mainly be attributed to the waste discharge of some local coking plants and many coal chemical industries in that area (Huang et al., 2003). The largest steel-making center in Southwest China and the largest national titanium and vanadium producing center is located in Panzhihua city and is considered to be the most important input source of hydrocarbons in Panzhihua region. According to the molecular ratio of ANT/(ANT+PHE), BaA/(BaA+CHR) and FLA/(FLA+PYR), the combustion of coal, grass and wood, as well as wastewater runoff were the main sources of PAHs at Chongqing section (Wang et al., 2009). Meanwhile, with the construction of the Three Gorges Dam, the intensification of ship traffic and decreased water flow, which results in higher sedimentation rates of potentially contaminated dissolved organic matter, may elevate levels of PAHs near the dam. The PAHs contamination of sediments at Wuhan section was mainly dominated by three-, four-, and five- ringed PAHs. The ratios suggested that the contamination was mainly caused by combustion, including wood, coal and petroleum (Feng et al., 2007a). The PAHs composition in the Yangtze Estuary, in which four- to six- ringed PAHs were dominant, showed that they mainly derived from petroleum combustion, vehicle emission, and biomass combustion (mainly coal) in the near- shore area (Wang et al., 2012b). Whereas two- to three- ringed PAHs were chiefly presented in the farther shore zone, originating from petroleum combustion of shipping processes and shore side discharges (Wang et al., 2012b). Chongming Island, the largest alluvial island in the world, has been rapidly developing its town industry, agriculture, tourism and shipping (Li et al., 2009). The PAHs ratios of ANT/(ANT+PHE), FLA/(FLA+PYR) and IP/(IP+Bghi) indicated that the sources of this site were a mixture of petroleum combustion, sewage, biomass and coal combustion. But the island is also influenced from the outside. Additional sources were particle bound PAHs discharged into the estuary from upstream sources, which accumulated around the island. Also, atmospherically transported contaminants from Shanghai played an important role for the PAHs input into the Yangtze Estuary (Feng et al., 2006).

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Chapter 2 Solution by dilution? ______

2.5.2 Polychlorinated biphenyls (PCBs)

Polychlorinated biphenyls (PCBs), which are composed of 209 possible congeners, are “a subset of chlorinated hydrocarbons” (UNEP, 1999). Exposure to different levels of PCBs can lead to acute toxicity like skin rashes, itching, burning, eye irritation, immune system disorders, chronic effects including liver damage, reproductive and developmental defects, and possibly cancer (UNEP, 1999; ATSDR, 2000). Because of these characteristics, PCBs were designated as typical POPs on Stockholm Convention in 2001. There are two main input sources of PCBs in China: one was the commercial production of PCBs (approximately 104 tons, including 9×103 tons trichlorobiphenyl mixture and 1,000 tons pentachlorobiphenyl mixture) from 1965 to 1974, and the other is the large quantity of imported PCBs transformers accompanied by other electrical equipment since 1970s (Xing et al., 2005; Bao et al., 2012). The PCBs usage in eastern China, including most of the Lower Yangtze Reaches, was reported to account for 45% of the whole national PCB contamination, with an average gross use density of 2.9 kg/km2 (Xing et al., 2005). PCBs were reported to be the major organic pollutants of the Yangtze River in the investigation of YVWEMC in 1990s (Wang, Peng et. al 2002). Water: The total concentrations of PCBs in the Yangtze River ranged from undetectable levels to 44 ng/L (Fig. 2.4a) (Chen et al., 2008; Martins et al., 2011). The levels of PCBs (0.01 – 1 ng/L) at TGR in 2008 (Wang et al., 2009) present an increase in comparison to the concentrations reported in 2004 (n.d. to 0.01 ng/L, mean: 0.002 ng/L) (Chen et al., 2008). The highest level of PCBs in water was found at the outlet of Nanjing city, ranging from 0.2 to 44 ng/L with an average concentration of 11 ng/L (Martins et al., 2011). Compared to the data from 1999 at Nanjing section (up to 3 ng/L) (Sun et al., 2002), it is found that the concentrations of PCBs increased significantly in that area. Taking the quality standards of surface water in China (20 ng/L) into account (Ministry of Environmental Protection - China, 2002), attention needs to be paid to the risk posed by PCBs at Nanjing section to human health as well as to the surrounding environment. Ye and coworkers attributed the existence of PCBs in Nanjing section mainly to discharged wastewater from a hormone-producing plant (Ye et al., 2009). The concentration of PCBs in the Yangtze Estuary ranged from 1 to 17 ng/L (Zhang et al., 2011). Compared to the levels of PCBs in the Pearl Estuary (0.1 – 2 ng/L) (Guan et al., 2009), this concentration was significantly higher. Concentrations of PCBs in the Yangtze River decreased significantly in comparison to the levels in 1990s (n.d. to 53.5 × 103 ng/L, mean: 869 ng/L) ______38

Chapter 2 Solution by dilution? ______

(Wang, Peng et. al 2002). PCB levels increased in most areas of China during the 1980s and 1990s, what can be attributed to improper disposal and leakage from PCB-containing equipment (Xing et al., 2005).

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Fig. 2.4 Minimum and maximum concentrations of PCBs in the Yangtze River. (a) PCBs in water compared to quality standards for PCBs in surface water (20 ng/L) recommended by the Ministry of Environmental Protection of the People’s Republic of China (MEP). TGR (2004) (Chen et al., 2008); TGR (2008) (Wang et al., 2009); Jiangsu (2004-2005) (Martins et al., 2011); Nanjing (1999) (Sun et al., 2002); Yangtze Delta (2009) (Zhang et al., 2011); (b) PCBs in sediments compared to effect range-low (ERL) value (PCBs: 22.7 ng/g) (Long et al., 1995). TGR (2010) (Zhao et al., 2013); Wuhan (2005) (Yang et al., 2009); Yangtze Delta (1998) (Xu et al., 2000c); Yangtze Delta (2004) (Shen et al., 2006); Yangtze Estuary (2003) (Cheng et al., 2006); Yangtze Estuary (2004) (Hui et al., 2009b); Yangtze Estuary (2006) (An et al., 2009); Yangtze Estuary (2007) (Yang et al., 2012). Numbers in brackets represent the sampling time; the locations are arranged in flow direction from left to right.

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Chapter 2 Solution by dilution? ______

Sediment: Zhao et al. (2013) recently reported the levels of PCBs in sediments at TGR, ranging from 0.5 to 4 ng/g. Compared to other reaches of the Yangtze River, PCB levels in TGR are extremely low. The concentrations of PCBs in sediments at Wuhan section ranged from 1 to 45 ng/g (mean: 9 ng/g) (Yang et al., 2009), which were higher than those found in the upper and middle reaches of the Yellow River (up to 6 ng/g) (He et al., 2006). The sampling site was close to discharge points of wastewater treatment plants and various chemical companies in the tributaries. In comparison to urban areas in other parts of the world, such as the Elbe River (45 – 64 ng/g) (Kiersch et al., 2010), the Rhine River (1 – 32 ng/g) (Woelz et al., 2008), the Upper Danube River (n.d. – 0.2 ng/g) (Keiter et al. 2008) and the Neckar River (1 – 14 ng/g) (Woelz et al., 2008), the detected levels in Yangtze River were in the medium range. The concentration of PCBs in Yangtze Estuary ranged up to 51 ng/g (Cheng et al., 2006), which is far less than in the Pearl Estuary (10 – 486 ng/g) (Mai et al., 2002), and the Yellow Estuary (0.004 – 180 ng/g) (Hui et al., 2009a). The higher levels in sediment than in water can be attributed to the characteristics of PCBs which are less soluble in water and have high octanol-water partition coefficients, and thus can ultimately accumulate in bottom sediments due to their strong affinity to particulate matter. The ERL and ERM guideline values have been frequently applied to assess the associated biological risks growing from PCB pollution in sediments. PCB levels above the ERL value (22.7 ng/g) suggest toxic effects on aquatic organisms, while a comparably high ERM value (180 ng/g) indicates a high possibility of detrimental effects (Long et al., 1995). All mean concentrations of PCBs in the Yangtze River were lower than the ERL values. However, the maximum concentrations of PCBs in the tributaries at Wuhan section (45 ng/g) (Yang et al., 2009), Hangzhou Bay in Lower Yangtze Reaches (52 ng/g) (Cheng et al., 2006), and the South Branch of Yangtze Estuary (30 ng/g) (Hui et al., 2009b) exceeded the ERL limit (Fig. 2.4b), suggesting that these levels of PCBs may cause biological impairments. However, according to a classification by ICPR (2009), the PCB levels were all below the “relevant contamination” level

(> fourfold ICPR target value) (ƩPCBs7, fourfold target value: 56 – 122 ng/L) (ICPR, 2009). Sources: There are no natural sources of PCBs and the major input into the aquatic environment is via atmospheric deposition, runoff from the land, and transport through food chains (Yang et al., 2009). Trichlorobiphenyl mixtures were the predominant PCB congeners observed in sediment of TGR, suggesting that transformer or capacitor oils were the main sources, which

______40

Chapter 2 Solution by dilution? ______have been carried via runoff into the water body. Furthermore, atmospheric depositions also contributed large loads of PCBs at TGR. PCBs at Wuhan section originated mostly from land runoff during floods and heavy rains (Yang et al., 2009), in addition to wastewater from municipal and industrial sources, atmospheric deposition, and upstream sources (Morrison et al., 2002). The conclusion is in agreement with the study by Yang et al. (2012), who claimed that agricultural soils might be a crucial input source for PCBs in the sediments of the Yangtze Estuary. Moreover, the pattern of PCBs in core sediment profiles of the Yangtze Estuary and the adjacent East China Sea demonstrated an increasing pollution level from 1980s to 2000. This can be explained by PCB-containing equipment imported from industrialized countries over the last two decades. A decreasing tendency could be shown after the Chinese Government prohibited the import of electronic wastes at the beginning of 2000s (Yang et al., 2012).

2.5.3 Organochlorine pesticides (OCPs)

Well-known organochlorine pesticides (OCPs) comprise hexachlorocyclohexanes (HCHs, sum of α-, β-, γ-, δ-HCH), dichlorodiphenyltrichloroethane (DDT) and its primary metabolites (the sum of o,p’-DDT, p,p’-DDT, dichlorodiphenyldichloroethane (DDD), and dichlorodiphenyldichloroethylene (DDE); designated as DDTs), aldrin, chlordane, dieldrin, endrin, heptachlor, hexachlorobenzene, mirex and toxaphenes (Bodo, 1996; Bao et al., 2012), among which DDT and HCHs have been widely used as pesticides to control insects on agricultural crops worldwide in the 1940s and the 1950s. DDT and its metabolites may increase the risks for breast cancer and play a role in endocrine disruptions (Turusov et al., 2002; Cohn et al., 2007). The toxicological effects of various isomers of HCHs can be as diverse as renal and liver failures, blood circulation disorders, imbalance in biochemical homeostasis, and reproductive defects in laboratory animals (Bodo, 1996; Willett et al., 1998). To protect the health of living organisms in the environment, DDT was subsequently limited for malaria control under the Stockholm Convention (UNEP, 2001); while HCHs (α-, β-, γ- HCH) were added as three of nine new POPs in the Stockholm Convention (UNEP, 2009). China banned the production of DDT and HCHs in 1983 and 1984, while large amounts of DDT (0.4 million tons) and HCHs (4.9 million tons) were produced during the 1950s to early 1980s (Bao et al., 2012). Though the residues of OCPs in the environment have considerably declined in the past decades (Wang, Peng et. al 2002), the levels of OCPs can still be detected in various environmental matrixes along the Yangtze River. DDTs and HCHs have been observed in Tuotuo River (the ______41

Chapter 2 Solution by dilution? ______origin of the Yangtze River), even though OCPs have never been used in that area (Liu et al., 2011a). A recent study reported the bioaccumulation of DDTs and HCHs in sediment-dwelling animals like mollusks and crabs in the Yangtze River (Yang et al., 2006), while another recent study indicated new input sources of HCHs in the Wuhan area (Tang et al., 2007), to which attention should be paid. Furthermore, Pentachlorophenol (PCP) has been recognized to be bio-accumulative and cause endocrine disrupting effects, mutagenicity and carcinogenicity (Seiler, 1991; Vom Saal and Hughes, 2005), and is often contaminated with dioxins and furans (Zheng et al., 2012). Fig. 2.5 summarizes the occurrence of OCPs in the Yangtze River. Water: Currently available data suggest that the Yangtze River from headstream to estuary is contaminated by organochlorine pesticides (Fig. 2.5a). To investigate the levels of DDTs and HCHs in the Yangtze River, Liu and colleagues sampled 37 natural water samples from Tibet (headstream) to the estuary along the Yangtze River (Liu et al., 2011a). The total HCHs (sum of α-, β-, γ-, δ-HCH) were in the range of 0.1 to 14 ng/L (mean: 3 ng/L). The DDTs (sum of p,-p’-DDT, o,-p’-DDT, p,-p’-DDE, p,-p’-DDD) ranged up to 21 ng/L (mean: 4 ng/L). The total concentration of OCPs (sum of HCHs and DDTs) could be detected in a range of 0.1 to 27 ng/L (mean: 3 ng/L) (Liu et al., 2011a). Levels of OCPs were reported to be stable along the Yangtze River except some areas near industrial sites (Liu et al., 2011a). It is also interesting to find that OCP levels in Tuotuo River, which is the source of the Yangtze River, were similar to those of the Yangtze Delta (Liu et al., 2011a). The highest concentrations of HCHs (0.1 – 28 ng/L), DDTs (0.1 – 17 ng/L) and total OCPs (0.3 – 47 ng/L) were detected by Tang and colleagues in the tributaries at Wuhan section (Tang et al., 2008). These tributaries pass areas of intensive agricultural activities, where HCHs had been widely used (Wu et al., 1997; Tang et al., 2008). OCP levels of the Yangtze River were lower, compared to the concentrations measured in the Yellow River (3 – 109 ng/L) (Sun et al., 2009) and the Pearl River (3 – 41 ng/L) (Guan et al., 2009), and were much lower than those in Macao Harbor (25 – 68 ng/L) (Luo et al., 2004). None of the concentrations in the Yangtze River exceeded the quality standards of HCH (lindane) (2,000 ng/L) and DDTs (1,000 ng/L) in surface water recommended by Ministry of Environmental Protection - China (2002), as well as quality standards in drinking water (DDTs; 1,000 ng/L, lindane: 2,000 ng/L) (Ministry of Health - China, 2006). Taking the regulatory limit (DDTs: 25 ng/L) for drinking water set forth by the European Union (European Union, 2008) into consideration, the maximum concentration of DDTs at Wuhan section would are also below

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Chapter 2 Solution by dilution? ______the limit (Fig. 2.5a). Han et al. (2009) investigated PCP levels in water of the Yangtze River at Jiangsu section and found that the related PCP levels ranged from undetectable to 220 ng/L, which is below the regulatory value (400 ng/L) recommended by European Union (2008). The levels of heptachlor epoxide along the Yangtze River were lower than the Chinese surface quality standards (200 ng/L) (Ministry of Health - China, 2006). However, the concentration of heptachlor epoxide in some area was higher than the Criterion Continuous Concentration (CCC) (3.8 ng/L) recommended by the USEPA (USEPA, 2009). For instance, one high concentration of heptachlor epoxide (18 ng/L) was reported at Wuhan section, which was nearly 5 times higher than the USEPA guideline (Tang et al., 2008). The OCPs in surface water were generally within safe levels according to environmental standards in China. However, it does not mean that the potential adverse effects on ecosystems and human health should be neglected at particular sections. Sediment: Levels of DDTs (2 – 16 ng/g) and HCHs (0.1 – 12 ng/g) in main stream sediments at Wuhan section were lower than the levels in tributaries (DDTs: 1 – 36 ng/g; HCHs 0.2 – 21 ng/g) (Tang et al., 2007). These results are in accordance with concentrations of DDTs and HCHs in the water of this area (Tang et al., 2008). The levels of DDTs in the tributaries at Nanjing section (4 – 32.6 ng/g) (An et al., 2006) and the South Branch of the Yangtze Estuary (1 – 33 ng/g) (Liu et al., 2008a) were higher than other sections. Lower concentrations were found in the East China Sea (DDTs: 0.1 – 6 ng/g, mean: 3 ng/g, HCHs: 0.1 – 2 ng/g, mean: 1 ng/g) (Yang et al., 2005), which are likely due a to very high dilution in the sea. The PCP levels in sediments of the Yangtze River at Wuhan section (1 – 2 ng/g, mean: 0.4 ng/g) were lower than those at Nanjing section (1 – 5 ng/g) (Xu et al., 2000b) and in Donghu Lake and Moshui Lake (n.d. – 13 ng/g), which are also located at Wuhan section (Tang et al. 2007a). Based on current sediment quality criteria for DDTs in sediments, the concentrations of DDTs show that levels in all the sampling sections have exceeded ERL values (DDTs: 1.6 ng/g), but are lower than the ERM value (46.1 ng/g) (Fig. 2.5b), suggesting that potential adverse biological risks exist in those areas. However, there is a lack of guidelines value about HCHs and PCP in sediments.

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Fig. 2.5 Maximum concentrations of OCPs, DDTs, HCHs in the Yangtze River. (a) OCPs, HCHs and DDTs in water compared to the regulatory limit (DDTs: 25 ng/L) for drinking water set forth by the European Union (EC Directives 2008). TGR (2008) (Wang et al., 2009); Sichuan (2007) (Liu et al., 2011a); Wuhan (2005) (Tang et al., 2008); Nanjing (1998) (Xu et al., 2000b); Jiangsu (2004-2005) (He et al. 2011); Jiangsu (2007) (Liu et al., 2011a); (b) OCPs, HCHs, DDTs in sediments compared to ERL value (1.58 ng/g of DDTs) (Long et al., 1995). Wuhan-MS (2005) (Tang et al., 2007); Wuhan-TB (2005) (Tang et al., 2007); Nanjing-MS (1998) (Xu et al., 2000a); Nanjing-TB (2004) (An et al., 2006); Yangtze Estuary (2001) (Liu et al., 2003); Yangtze-SB (2002) (Liu et al., 2008a); Yangtze-SB 2006 (Liu et al., 2007); ECS-East China Sea (2002) (Yang et al., 2005). Numbers in brackets represent the sampling time; MS – Main Stream; TB – Tributary; SB – South Branch; the locations are arranged in flow direction from left to right.

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Chapter 2 Solution by dilution? ______

Sources: Concentrations of OCPs have been detected in the Tuotuo River, though DDTs and HCHs have never been used in that area. Transport of OCPs to the origin of the river were explained by “cold condensation” and “the grasshopper-effect”, which means that OCPs are transported from the high temperature areas (Middle and Lower Yangtze Reaches) to the low temperature area (source region of the river) and precipitate there through cold condensation (Liu et al., 2011a). Both lindane (“pure” γ-HCH) and technical HCH mixtures have been used in China from the 1950s to 1980s. The ratio of α-HCH/γ-HCH can be used to diagnose the input sources of HCHs in the environment (Liu et al., 2011a; Bao et al., 2012). If the ratio varies between 0.2 to 1, this would mean that lindane is the typical input source in that area, while a ratio in the range of 4 to 7 would suggest that the HCHs in the environment originate from the usage of technical HCH mixtures (McConnell et al., 1993). The value of α-HCH/γ-HCH ranged from 1.2 to 8.4 in the mainstream and 0.3 to 15.9 in the tributaries at Wuhan section, indicating that most of the HCHs were mainly derived from the technical HCH usage over the past decades. Lower ratios of α-HCH/γ-HCH ( <1 ) were observed in the tributaries at Wuhan section, suggesting that there could be possible input sources of lindane at those sections (Tang et al., 2008). Furthermore, high levels of γ-HCH in the Yangtze Estuary indicated that the HCHs derived from a continuous use of lindane rather than technical HCH usage in the Yangtze Delta area (Liu et al., 2008a). DDT can be biodegraded to DDE under aerobic conditions and to DDD under anaerobic conditions. The diagnostic indexes of DDE/DDT, (DDE+DDD)/DDTs are used to differentiate the historical and recent inputs of DDTs (Metcalf, 1973; Guo et al., 2009). According to the distribution of DDTs in core sediments studied by Yang and coworkers, the decrease of DDTs concentration in the sediment occurred significantly after the official ban of DDTs in 1980s (Yang et al., 2005). Besides, Wang and colleagues found that DDTs levels in water of the Yangtze River in early 1990s were an order of magnitude lower than those in late 1990s (Wang, Peng et. al 2002). The ratio of (DDE+DDT)/DDTs in suspended particulate matter varied from 0.4 to 0.96 (mean: 0.64) at Wuhan section, indicating that most of DDTs residues might be mainly aroused from the usage of technical DDTs over the past decades (Tang et al., 2008). Other studies in the Yangtze Estuary also confirmed this conclusion (Xu et al., 2000a; Liu et al., 2008a). In addition, the ratio of o,p’-DDT/p,p’-DDT can be used as indicative indices for the input of technical DDT mixtures or dicofol-related sources (Guo et al., 2009). The “dicofol type DDT

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Chapter 2 Solution by dilution? ______pollution” is defined as the DDT pollution caused by dicofol use and is characterized by higher o,p’-DDT/p,p´-DDT concentration ratio than that of technical DDT. The DDTs reported by Liu et al. (2008a) in SPM and Tang et al. (2007), can be traced to the dicofol usage in some agricultural areas of the Middle and Lower Yangtze Reaches. PCP and its sodium salt (Na-PCP) are strong pesticides, which were used to control the spreading of schistosomiasis disease (Zheng et al., 2008). It is reported that Na-PCP with impurities of PCDDs/DFs was sprayed over the vast agricultural areas in the Middle and Lower Reaches of the Yangtze River since the 1960s. Zheng et al. (2012) assumed an increase of PCP contamination in environmental matrices, due to a growing use of Na-PCP to control the reemergence of schistosomiasis. The authors also emphasized that a double cancer risk from independent and joint effects of PCP and PCDD/DFs may raise further concerns (Zheng et al., 2012).

2.5.4 Polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDDs/DFs)

Polychlorinated dibenzo-p-dioxins/dibenzofurans: PCDDs/DFs represent groups of halogenated polycyclic aromatics, comprising 75 PCDD congeners and 135 PCDF congeners (Van den Berg et al., 1994; Zheng et al., 2008). There is no commercial use for PCDDs/DFs and they are unintentional byproducts of manufacturing processes. PCDDs/DFs can generate as impurities during the production of polychlorophenol under certain conditions (Zheng et al., 2008) or in combustion processes, e.g., solid waste burning in municipal incinerators, forest fires and volcanic eruptions (Van den Berg et al., 1994). PCDDs/PCDFs elicit a broad spectrum of biological and toxicological effects. Adverse effects on reproduction, development, endocrine functions and several types of dermal lesion have been observed in laboratory animals as well as wildlife species exposed to these compounds (Van den Berg et al., 1994; Luebke et al., 2001; Wen et al., 2008). Based on the contract of “Stockholm Convention on Persistent Organic Pollutants”, China and other countries in the world are taking measures to eliminate the possible sources of PCDDs/DFs. The sources of PCDDs/DFs in China were mainly derived from industrial wastes, production and usage of polychlorophenols, PCBs, chlorinated pesticides and triclosan which contained PCDDs/DFs as impurities (Zheng et al., 2008). Because of limited instrumentation and restricted trained personnel (UNEP, 2002), the pollution status of PCDDs/DFs in water and sediments in China, especially the Yangtze River are rarely studied (Annex I, Table I.1).

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Chapter 2 Solution by dilution? ______

Water: Chen et al. (2008) reported the occurrences of PCDDs/DFs at TGR in the Upper Yangtze Reaches. The concentrations of total PCDDs/DFs in water ranged from 2 to 96 pg/L (mean: 12 pg/L), and the toxic equivalency (WHO-TEQ) of PCDDs/DFs in the TGR ranged from 0.001 to 0.3 pg/L (mean: 0.1 pg/L) (Chen et al., 2008). The concentration of 2,3,7,8-TCDD was undetectable, which is hard to compare with the tolerable limit of 2,3,7,8-TCDD (50 × 10-3 pg/L) for surface and drinking water proposed by Di Domenico (1990). As there are no regulatory limits for surface water about PCDDs/DFs in China, this recommendation could be applied in the Yangtze River to improve analysis methods and risk control. Sediment: Two studies reported about PCDDs/DFs in surface sediments of the Yangtze Estuary. Wen et al. (2008) detected the concentrations of total PCDDs/DFs and WHO-TEQs, ranging from 25 to 374 pg/g (mean: 170 pg/g), and from 0.4 to 1 pg/g, respectively. The latter were similar to those in the study of Sun et al. (2005), where WHO-TEQs of PCDDs/DFs ranged from 0.3 to 1 pg/g. The most abundant congeners of PCDD in sediment was OCDD in the Yangtze Estuary (Sun et al., 2005; Wen et al., 2008), which accord with the PCDDs/DFs pattern in water of other places, like Jiangxi Province and Hubei Province in China (Zheng et al., 2008). The occurrence of PCDDs/DFs in TGR and Yangtze Estuary was attributed to the usage of Na-PCP in 1960s, which contains PCDD/DFs as impurities, have been widely used in the middle and lower Yangtze River to control the spread of schistosomiasis disease (Zheng et al., 2008). The levels of PCDDs/DFs were lower than those in the Pearl River Estuary (Zhang et al., 2009a) and Mai Po Wetland (Müller et al., 2002), but marginally higher than that in the Yellow Estuary (Hui et al., 2009b) and Mondego Estuary (Spain) (Nunes et al., 2011) (Annex I, Table I.1). TEQ concentrations for the Yangtze River were below the value of 21.5 pg WHO-TEQ/g dw (1998) as the probable effect level on aquatic organisms suggested by Canadian Sediment Quality Guideline (CCME, 2002).

2.5.5 Emerging pollutants

Concerning the organic contamination status of the Yangtze River the main attention was paid to the typical priority pollutants PAHs, PCBs and OCPs (including DDTs and HCHs). However, within the last two decades environmental risks of so called “emerging pollutants” raise increasing concern. So far, there is no international consensus with respect to a clear definition of “emerging pollutants”. Many constituents described as emerging pollutants are pharmaceuticals or personal care products (PPCPs) including endocrine disrupting compounds (EDCs) (Da Silva ______47

Chapter 2 Solution by dilution? ______et al., 2012). Examples are polybrominated diphenyl ethers (PBDEs), perfluorinated compounds (PFCs) and phthalates (USEPA, 2008). This chapter summarizes and describes the detected levels of emerging pollutants (PBDEs, PFCs, PAEs, NP, BPA), which were obtained from the available articles in the scope of this review (Annex I, Table I.2-I.4). These pollutants have been reported to be harmful to the environment and also been hypothesized to be harmful to human health. They mainly act as endocrine disruptors and carcinogens, or can be bioaccumulated in the aquatic system (Seiler, 1991; Staples et al., 1998; Vom Saal and Hughes, 2005; Demers et al., 2006; Heudorf et al., 2007; Soares et al., 2008). Polybrominated diphenyl ethers (PBDEs): PBDEs are a class of organobromine compounds that are used as flame-retardant additives. PBDEs have been used in a large variety of products, e.g., building materials, plastics, electrical appliances, television sets, computer circuit boards and casings (De Wit, 2002; McDonald, 2002). The environmental sources, metabolic relationships, and relative toxicities of PBDEs and their analogs, especially estrogenicity and androgenicity, effects on the thyroid hormone system has been reviewed by Wiseman et al. (2011). Long-time exposure to low concentrations of PBDEs can cause neurobehavioral deficits and might cause carcinogenicity (McDonald, 2002). In addition, lower molecular weight congeners of PBDEs (tri- to hexa-BDEs) are highly bioaccumulated. Potential risks will come up when sensitive populations, like pregnant women, the developing fetuses or infants, are exposed to these substances (WHO/IPCS, 1994; McDonald, 2002). The industrial usages of some PBDEs are prohibited by the European Union and the United States of America. However, no such restrictions do apply in China to date (Alexandra Mcpherson, 2004; Bao et al., 2012). In addition to the continuous usage of technical BDE mixtures, another main source of PBDEs in China is imported electronic waste (e-waste) (Ni and Zeng, 2009). A report set forth by Ni et al. (2010) estimated that about 70,600 tons of PBDEs were annually imported to China. The potential harm of PBDEs to human health and ecosystems, in addition to biotransformation of PBDEs to metabolites with even greater toxicities, has caused recently great concern (Wang et al., 2010a; Bao et al., 2012; Chen et al., 2012). Water: It should be noted that there is no monitoring data of PBDEs in water of the Yangtze River. However, Federal Environmental Quality Guidelines (FEQGs) for PBDEs have been developed by Environment Canada to assess the ecological significance of PBDE levels

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Chapter 2 Solution by dilution? ______

(Environment Canada, 2012). These guidelines can be utilized as references to control the presence of PBDEs in the Yangtze River. Sediment: There is little information concerning the levels of PBDEs in sediments in the Upper Reaches of the Yangtze River except one recent publication reported by Zhao et al. (2013). Zhao and colleagues measured PBDEs at TGR, with total PBDEs (except BDE 209) and BDE 209 levels from n.d. to 0.15 ng/g and n.d. to 0.5 ng/g, respectively. Shen et al. (2006) investigated PBDE levels in sediments of the Yangtze River Delta (Yangtze Estuary, Hangzhou Bay and Qiantang River). Thirteen out of 32 sediment samples contained PBDEs, ranging from 2 to 4 ng/g (mean: 3 ng/g). These low levels of PBDEs were attributed to dilution effects. Huge amounts of fresh water and associated suspended matter from the Upper Reaches diluted the organic contaminants in the lower reaches of the Yangtze River. However, higher concentrations were reported by Chen et al. (2006). The concentrations of Ʃ12PBDEs (sum of 12 PBDEs congeners without BDE 209) and BDE 209 in sediment of the Yangtze Delta varied up to 1 ng/g (mean: 0.2 ng/g) and from 0.2 to 95 ng/g (mean: 13 ng/g), respectively. BDE 209 is a component of the commercial deca-BDE mixtures mostly used in China (Bao et al., 2012) and contributed 90-100% to the total PBDEs (Chen et al., 2006). In addition, the levels of lightly brominated congeners indicated that the atmospheric deposition was also a significant input source in the study area. Li and colleagues studied PBDEs in sediments of the near-shore East China Sea, sampling from the Yangtze Estuary to about 1,000 km southward (Li et al., 2012b). The levels of BDE-29 and

ƩPBDE7 ranged from 0.3 to 45 ng/g and up to 8 ng/g, respectively. Relatively higher PBDEs concentrations were reported in the Yangtze Estuary and the south of Hangzhou Bay. PBDEs in the near-shore sediments of East China Sea were partly attributed to the input of the Yangtze River, because the areas with higher levels are the deposition center for fine-grained sediments (Li et al., 2012b). The maximum concentrations of BDE-209 in the Yangtze Estuary (Chen et al., 2006; Li et al., 2012b) exceeded the FEQGs of decaBDE as given by Environment Canada (19 ng/g) (Environment Canada, 2012). Perfluorinated compounds (PFCs): PFCs are a category of organofluorine compounds, consisting of two well-studied compounds: perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS). This group also contains compounds like perfluorononanoic acid (PFNA), perfluorobutanesulfonic acid (PFBS), perfluorooctanesulfonyl fluoride (POSF) and perfluorooctanesulfonamide (PFOSA). PFCs have been applied as

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Chapter 2 Solution by dilution? ______surfactants and surface protectors in carpets, leather, paper, packaging, fabric, and upholstery as well as in or as aqueous film fire-fighting foams (AFFFs), mining and oil well surfactants, alkaline cleaners, floor polishes and photographic films (OECD, 2002; Ahrens, 2010). Stahl et al. (2011) reviewed the toxicodynamics of PFCs, such as acute toxicity, subacute and subchronic toxicities, chronic toxicity like immunotoxicity, carcinogenesis, genotoxicity and epigenetic effects, reproductive and developmental toxicities, neurotoxicity etc. Within the class of PFCs, PFOS and PFOA are generally considered as reference substances, and they can accumulate in serum and liver, causing hepatotoxicity (damage to the liver), forma adenomas (non-cancerous tumours) in liver and thyroid tissues, and decrease the serum cholesterol in rodents (OECD 2002, Giesy et al. 2010, Suja et al. 2009). Thus, PFOS, PFOA and its salts are proposed as a new class of POPs at the Stockholm Convention in May 2009 (Wang et al., 2010a; Vierke et al., 2012). Due to the potential negative effects of PFCs on human health, the United States of America and the European Union formed a series of directives to prohibit to the production of PFCs since 2000 (Ahrens, 2010; Cai et al., 2012a). For instance, a major American Company, was once the most important global producer of PFCs, especially PFOS in 1940s to 1990s, but phased out its production in 2002 under the PFC reduce program launched by the USEPA (Ahrens, 2010; Wang et al., 2010a). The major production of PFOS was shifted from North America and Europe primarily to China in the last decades (Cai et al., 2012a). The production of PFOS increased from less than 50 tons in 2003 to 247 tons in 2006 (Bao et al., 2010; Wang et al., 2010a). There were twelve PFC manufactures in China, according to the authors four have stopped the production and eight of them still produced. Three of those eight were located in Hubei Province, two in Fujian and the others in Beijing, Shanghai and Wuhan (Wang et al., 2010a). Since the beginning of 2000s Chinese scientists began to collect information of PFCs in the Yangtze River (Jin et al., 2006). Water: Jin et al. (2006) have measured PFOS and PFOA in the Yangtze River at TGR and Wuhan section. They reported high levels of PFOS and PFOA in TGR, of which the maximum concentrations reached to 38 ng/L and 298 ng/L, respectively. The concentrations of PFOA upstream of Chongqing (0.2 - 0.4 ng/L) were far lower than downstream of Chongqing to Yichang (2 - 298 ng/L). High levels were detected at Xituo section (PFOA: 111 ng/L) and Fengdu section (PFOA: 298 ng/L). This indicated that input sources were located around these areas (Jin et al., 2006). However, the low average concentration at TGR can be explained by the

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Chapter 2 Solution by dilution? ______dilution with enormous amounts of water. High concentrations of PFOA were also detected at Wuhan section (298 ng/L) (Jin et al., 2009) and Shanghai (260 ng/L) (So et al., 2007). This is in accordance with locations of PFC factories (Wang et al., 2010a). The highest concentration of PFOS (144 ng/L) was found in the South Branch of the Yangtze Estuary (Pan and You, 2010). The sampling site is located at Baozhen Port, which serves as a freight terminal and passenger wharf. Significant amounts of domestic sewage, which have been reported to contain high levels of PFOS, were also considered as important sources (Pan and You, 2010). PFCs pollution in two interior lakes of the Yangtze River (Dian Lake and Tai Lake) has been recently reported (Yang et al., 2011; Zhang et al., 2012). The PFC contamination in Tai Lake was different to other studies, with PFOS being the most abundant compound. This indicates a different origin compared to Chongqing, Wuhan and Shanghai. The authors attributed this to the production of paints, plastic pipes and plastic anticorrosion products, containing fluorinated chemicals, in Wuxi city (Yang et al., 2011). In 2009, the USEPA set short-term provisional health advisory values for PFOA and PFOS of 400 ng/L and 200 ng/L, respectively. According to the available data, the levels of PFOA and PFOS in the Yangtze River are still below the USEPA guidelines (Environmental Protection Agency - USA, 2009). Sediment: No research has been performed on PFCs in sediments of the Yangtze River except in the Yangtze Estuary. Li et al. (2010) reported a maximum concentration of PFOA (203 ng/g) in sediment from Huangpu River, which exceeded PFOA levels from many other studies (Bao et al., 2010; Pan and You, 2010). Concentrations of PFOS were even higher in the South Branch of the Yangtze Estuary (73 - 537 ng/g) (Bao et al., 2010; Li et al., 2010). The sampling location was situated in the near shore region of Chongming Island. Correlation analysis between salinity and distribution coefficient of PFOS indicated that the affinity of PFOS to sediment was higher during salt intrusion. This means that PFOS can be carried from upstream sources for a long distance and will accumulate in the sediments of the Estuary, due to the dramatic change in salinity (Pan and You, 2010). In comparison to other water bodies in China, like the Pearl River (Bao et al., 2010), Liao River (Yang et al., 2011), Tai Lake (Yang et al., 2011) and Dianchi Lake (Zhang et al., 2012), the Yangtze River was more severely polluted with PFCs. Phthalic acid esters (PAEs): Phthalates are used as plasticizers in polyvinyl chloride (PVC) plastics, which are applied in the production of electrical cords, films, glues, paints, ink, varnishes, coatings, adhesives, cosmetics, pesticides, repellents, dielectric media (Jarosova, 2006).

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Chapter 2 Solution by dilution? ______

In recent years toxicological concerns of PAEs were raised with regard to their potential endocrine disrupting potencies (Heudorf et al., 2007). Shi et al. (2012) studied thyroid hormone (TH) disrupting activities associated with phthalate esters in water of the Yangtze River Delta. The results indicated that di-n-butyl phthalate (DBP) was the primary TH receptor (TR) antagonist in water sources in the Yangtze River Delta, followed by di(2-ethylhexyl) phthalate (DEHP), di-n-octyl-phthalate (DnOP), di-isononyl phthalate (DiNP). This is in accordance with reports that describe DBP and DEHP as the predominant PAEs at the following sections. The concentrations of DBP and DEHP in Chongqing section (DBP: n.d. - 42 µg/L, DEHP: n.d. - 12 µg/L) (Luo et al., 2009 ), Wuhan section (DBP: n.d. - 35 µg/L, DEHP: 4 - 54 µg/L) (Wang et al., 2008) and Yangtze Delta (DBP: n.d. - 7 µg/L, DEHP: 4 - 28 µg/L) (Zhang et al., 2012) all exceeded the surface water quality standard of China (DBP: 3 µg/L, DEHP: 8 µg/L) (Ministry of Environmental Protection - China, 2002) as well as the regulatory limit of European Union (DEHP: 1.3 µg/L) (European Union, 2008). Nonylphenol (NP) and bisphenol A (BPA): NP is mainly used for the synthesis of nonylphenol ethoxylates (NPEOs), which are applied as surfactants, e.g., in industrial cleaners, or as emulsifiers, e.g., in paints and lacquers, adhesives and pesticides. NPEOs are degraded to nonylphenol in the environment. NP is known to be slowly degradable and bioaccumulative with a far greater toxicity than NPEO (German Environmental Specimen Bank, 2012). BPA is a synthetic monomer with one of the highest production yields worldwide, normally used in the production of polycarbonate plastics and epoxy resins (Diamanti-Kandarakis et al., 2009). NP and BPA are linked to a wide variety of endocrine dysfunctions. NP is known as an endocrine disruptor causing harmful effects including feminization and carcinogenesis in various organisms (Soares et al., 2008). Exposure to BPA can increase the risk of mammary cancer, obesity, diabetes, and reproductive and neuroendocrine disorders (Diamanti-Kandarakis et al., 2009). Water: Shao et al. (2005) detected NPEOs at a range of 6.9 × 103 to 97.6 × 103 ng/L in April and 2.5 × 103 to 52.7 × 103 ng/L in December, as well as NP from 1.7 to 7.3 × 103 ng/L in July in Yangtze River and Jialing River at Chongqing section. But corresponding drinking water samples derived from river water sources suggested that conventional water treatment processes had a significant removal efficiency of NPEOs (> 99%) and NP (62 -9 5%). However, the drinking water still exhibited levels of NP in a range of 0.1 × 103 to 2.7 × 103 ng/L, to which attention needs to be paid (Shao et al., 2005). Shao et al. (2002) reported NP concentrations in Yangtze

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Chapter 2 Solution by dilution? ______

River also at Chongqing section (20 - 6.9 × 103 ng/L), which were far greater than levels in the Yangtze Estuary and adjacent areas, ranging from 13 to 186 ng/L (Ping, 2011). The concentrations of NP at Jialing River and Yangtze River at Chongqing section were above the regulatory limit (300 ng/L) set forth by European Union (2008). Wang et al. (2012a) investigated NP and BPA concentrations in surface water from Yangtze River, Suzhou River and Huangpu River, as well as drinking water sourcing from surface water of Huangpu River and Yangtze River in Shanghai section. No phenols were detected in surface water from Yangtze River, while BPA was positive in all surface water samples from Suzhou River and Huangpu River. The concentrations of BPA in surface water of Huangpu River ranged from 184 to 782 ng/L. BPA was also detected in some drinking water and barreled water samples, with concentrations of 6 to 432 ng/L, and 14 to 280 ng/L, respectively. The authors emphasized that more attention needs to be paid to the water quality surveillance in the city of Shanghai (Wang et al., 2012a). BPA is subject to review for possible identification as priority substance or priority hazardous substance by the European Union (2008). However, there is still a lack of regulatory limits for BPA in drinking water and surface water. Sediment: Bian et al. (2010) reported the distribution characteristics of NP and BPA in surface sediments of the Yangtze River Estuary and the adjacent East China Sea. The contents of NP and BPA in surface sediments ranged from 2 to 36 ng/g and 1 to 13 ng/g, respectively. The contents of NP in the sediment core ranged up to 21 ng/g in layers from 1971 to 2001 with a pattern that reflected the traces of economic development history in China during this period. The deposition fluxes of NP varied from 1 to 18 ng/(cm2•a). BPA was detected in sediment layers deposited from 1973 to 2001 with contents of up to 4 ng/g. The fluxes of BPA varied from 1 to 3 ng/(cm2•a) exhibiting a similar pattern as NP.

2.5.6 Discussion: Pollutant levels in water and sediments

This chapter summarizes and describes the levels of PAHs, PCBs OCPs, PCDDs/DFs, emerging pollutants (PBDEs, PFCs PAEs, NP, and BPA), in water and sediment of the Yangtze River. The concentrations of organic pollutants in the environment reflect the rapid industrialization and increased urbanization seen in the vicinity of the Yangtze River in China over the last two decades. Some areas such as Wuhan section and the South Branch of the Yangtze Estuary posed a potential ecotoxicological risk according to current guidelines and require a long-term

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Chapter 2 Solution by dilution? ______monitoring. Specific strategies should be employed to restrict the pollutants discharge in those areas. Compared to the 1990s, the PAHs pollution in the Yangtze River has dramatically increased due to anthropogenic activities. The concentrations of PAHs in water of the Yangtze River were significantly high at Panzhihua section of the Jinsha River and comparably low in the TGR region. The concentration of BaP in water, which is one of the most toxic PAHs, peaked at the sections of Jiangsu and Wuhan, but was not detected at all at Jinsha River. The highest concentration of PAHs in the sediments of the Yangtze River was found in the tributaries of Wuhan section and the Yangtze Estuary near Shanghai. Based on the comparison of two guideline values (ERL and ERM), PAHs detected at Wuhan section may cause toxic effects to aquatic life. The serious PAHs pollution at Panzhihua section in Jinsha River could mainly be attributed to the waste discharge of some local coking plants and many coal chemical industries in the area. PAHs in Upper and Middle Reaches of the Yangtze River seemed to originate from the combustion of coal and biomass. The combustion of petroleum and vehicle emissions mainly contributed to the PAHs contamination of the Yangtze Estuary and near-by shore areas. Furthermore, sewage discharge from cities like Chongqing, Wuhan, and Shanghai are also an important input source of PAHs into the surrounding environment. All these regions should be supervised to minimize the output of PAHs. PCBs are toxic chlorinated hydrocarbons, which mainly originated from the import of contaminated electronic devices and former national production. Except at the outlet of Nanjing city, the concentration of PCBs in water of the Yangtze River is below the quality standards enacted by China’s Ministry of Environmental Protection. High concentrations of PCBs were found in sediments at Wuhan section. Due to low solubility and high octanol-water partition coefficients, they can easily accumulate in sediments. The guideline value (ERL) suggests that most of the PCB levels in sediments of the Yangtze River at Wuhan section and the Yangtze Estuary may cause adverse biological effects, though they are below the “relevant contamination” recommended by ICPR (2009). Most PCBs in the sediments of the Middle and Lower Reaches seemed to originate from land runoff and contaminated electronic wastes during the past two decades. DDTs and HCHs are important constituents of OCPs which cause severe health problems. DDTs and HCHs could be detected in Tuotuo River, which is the origin of the Yangtze River, but

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Chapter 2 Solution by dilution? ______neither of these pollutants has been used in that area. This could be explained by “cold condensation” and “the grasshopper-effect” of OCPs atmospheric transport and sedimentation. In all sections of the Yangtze River, the levels of DDTs and HCHs are below the quality standards for surface water enacted by the China’s Ministry of Environmental Protection and DDTs regulatory limits imposed by the Europe Union. Moreover, the concentrations of DDTs in sediments of the Yangtze River at Nanjing section and the Yangtze Estuary are above the guideline value (ERL), indicating that this may have ecotoxicological impacts. The α-HCHs/γ-HCH ratio suggests that HCHs, prevalent at Wuhan section, derived mainly from the usage of technical HCH in the past decades. However, there may be other input sources of lindane (γ-HCH) in this area. Moreover, DDTs residues mostly derived from the usage of technical DDT over the past decades in the middle and lower reaches of the Yangtze River. Furthermore, the usage of dicofol in some agricultural areas should be carefully regulated to reduce the input of DDTs into the surrounding environment. There were only limited studies on PCP contamination in the Yangtze River. A low level of PCP in sediment of the Yangtze River was reported. However, the risks from independent and joint effects of PCP and associated PCDDs/DFs impurities have to be considered. Because of limited equipment and limited trained personnel, PCDDs/DFs have not been thoroughly investigated in the Yangtze River, unlike other pollutants such as PAHs and OCPs. PCDDs/DFs levels in water at TGR are relatively low. PCDDs/DFs levels in sediments of the Yangtze Estuary were lower than those in the Pearl River, but were marginally higher than the concentrations in Yellow Estuary and Mondego Estuary (Spain). The most abundant congeners of PCDD in water and sediments were OCDD, indicating that the residues of PCDDs/DFs in the Yangtze River were derived from the usage of Na-PCP in 1960s. Due to the combustion of petroleum and coal, and the incineration of solid wastes in China (Zheng et al., 2008), a continuous monitoring of PCDDs/DFs concentrations in water and sediments in the Yangtze River are urgently needed. Large amounts of imported e-waste have become the most important input sources of PBDEs in China. No studies reported on PBDEs in water of the Yangtze River. Lower concentrations of PBDEs in sediments were detected at the Lower Yangtze Reaches. This was attributed to dilution effects, due to huge amounts of water and suspended matter from the Upper Reaches (Chen et al., 2006). BDE-209 is the predominant PBDE in the Yangtze River. The PBDE contamination of

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Chapter 2 Solution by dilution? ______coastal sediments of the East China Sea was partly attributed to the Yangtze River. Stronger contaminated sites were deposition areas for PBDE containing fine-grained sediments discharged by the river. PFCs have been listed as POPs in Stockholm Convention 2009. The production of PFCs in China has increased 4 times from 2003 to 2006 and reached to more than 200 tons. Two of eight PFC factories in China are located at the Yangtze River (Wuhan and Shanghai). High PFOA levels were reported at TGR (Xituo section and Fengdu section), indicating that input sources were located around these areas. The Yangtze River at Wuhan section and Huangpu River in Shanghai also showed high PFOA values, which stood in accordance with locations of PFC factories in both cities. The highest PFOS levels could be detected in water of the Yangtze Estuary’s South Branch, at a site that served as freight terminal and passenger wharf. However, at Chongming Island high levels of PFOS were detected in sediments. Pan and You (2010) reported a high correlation between salinity and the affinity of PFOS to sediment at the Yangtze Estuary. Thus, it is to be expected that PFOS from upstream sources will accumulate in sediments of the estuary due to the dramatic change in salinity. Furthermore, also available information about PAEs, NP and BPA in the Yangtze River were reviewed, indicating a potential risk to some areas. The maximum concentrations of DBP and DEHP in water of the main stream at Chongqing and Wuhan section, as well as the Yangchenghu Lake in the Yangtze Delta have exceeded the surface water quality standard of China. Endocrine disrupting chemicals like BPA were reported in some drinking water samples and barreled water samples in Shanghai. Thus attention should be paid to the water quality surveillance. Emerging pollutants are not necessary new chemicals, but also substances that may have been present in the environment already for a long time while their presence and significance has just been realized within the past two decades. Data about the presence of emerging pollutants in the Yangtze River are still scarce. There is a long way to monitor and regulate emerging pollutants in the Yangtze River, considering that the analytical methods are still at an early stage of development and regulatory criteria for some emerging pollutants in water/sediment are still under discussion. Though China has built the organic pollutants management framework in the past two decades, it still lags behind developed countries with regard to the control of certain POPs and emerging pollutants in water and sediments (Wang et al., 2005). Two guideline values (ERL and ERM) can provide a basis for estimating potential biological impacts associated with

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Chapter 2 Solution by dilution? ______chemical analysis of pollutants in the sediment (Long et al., 1995). However, limitations exist between the incidence of adverse biological effects and the level of selected pollutants. Some pollutants can even induce responses although they are below the limit of quantification. In addition, chemical contaminants in sediments rarely affect organisms as single substances, but instead cause adverse effects as diverse mixtures (Ahlf et al., 2002). Integrated evaluation methods, such as the triad approach (Chapman, 1990; Chapman and Hollert, 2006), effect- directed analysis (Hecker and Hollert, 2009) and the hierarchical assessment on contaminated sediment method (Ahlf et al., 2002) can be employed in the ecotoxicological assessment of Yangtze River.

2.6 Effect assessment of water and sediment (in vitro and in vivo)

The chemical analysis of pollutants is often not able to explain ecotoxicological effects of complex environmental samples. Risk assessment based on concentrations, e.g., of priority pollutants in sediments or water, cannot reflect the risk of the actual mixture of contaminants, but only the risk of those pre-selected toxicants. Thus in vitro and in vivo test systems with endpoints like genotoxicity, mutagenicity and endocrine disruption are taken into account. The application of bioassays indicating effects on cellular, organism or population level in laboratory test systems and the availability of concepts for linking measurable effects of complex environmental samples to distinct toxicants are required to bridge the gap between the chemical contamination and ecological status (Brack et al., 2007). The bioassays applied in the Yangtze River were summarized in Annex I, Table I.5.

2.6.1 Genotoxicity and mutagenicity

Several substances, like PAHs, PCBs and PFCs, are known to possess mutagenic or genotoxic properties, which influence the genome of an organism and thereby can lead to severe impacts on health, e.g., cancer formation (ATSDR, 2000; 2009; Stahl et al., 2011). Mutagenicity describes permanent changes in the structure and/or amount of the genetic material of an organism that can lead to heritable changes in its function. It includes gene mutations as well as structural and numerical chromosome alterations (Eastmond et al., 2009). Genotoxicity refers to the capacity to give rise to mutations, but is not necessarily associated with it. It includes all directly or indirectly mediated adverse effects on genetic information, e.g., damage to DNA and/or cellular components regulating the fidelity of the genome, like spindle apparatus, topoisomerases, DNA ______57

Chapter 2 Solution by dilution? ______repair systems and DNA polymerases. Genotoxic events are reversible due to cellular repair processes. Thus not all genotoxic events become evident as mutations (Eastmond et al., 2009; UKCOM, 2011). Sediments, contaminated with mutagenic substances, pose a hazard to indigenous biota (Chen and White, 2004). Yet only limited research investigating genotoxic hazards of aquatic sediments using standardized bioassays (e.g., Ames assay with Salmonella typhimurium (Reifferscheid et al., 2012), micronucleus assay (Reifferscheid et al., 2008) and Single Cell Gel Electrophoreses (SCGE/comet) assay has been employed in the river (Annex I, Table I.5). Upper Reaches: Shu et al. (2002) and Qiu et al. (2003) both investigated the mutagenicity of source water in Chongqing section of the Jialing and the Yangtze River, using the Ames and SCGE assay, respectively. In the study by Shu et al. (2002), the mutation rate of the Salmonella typhimurium strains TA98 (frameshift mutagen indicator) and TA100 (baseshift mutagen indicator) were used as parameters to assess surface water quality. They concluded that most organic extracts from the two rivers possessed mutagenic potential. The source water of most sites exhibited the highest mutagenicity during spring and Jialing River extracts showed comparably higher mutagenicity than those from the Yangtze River. The latter observation matched the SCGE assay results by Qiu et al. (2003). The authors attributed the more serious pollution in Jialing River to agricultural and industrial discharges, domestic sewages and a poor self-purification capacity compared with the Yangtze River. It was also detected that the chlorinated drinking water possessed much higher mutagenic potential than the source water, indicating that chlorination during water treatment may have produced disinfection byproducts, which enhanced the DNA damage in the bioassay. Middle Reaches: Yuan et al. (2005) tested genotoxic effects in human HepG2 cells in the comet assay with chlorinated drinking water extracts from the three main water bodies Dong Lake, Han River and the Yangtze River at Wuhan section. The results suggested that genotoxicants and/or genotoxic disinfection byproducts were present among the three water bodies. The DNA damage in Han River was more serious than in Dong Lake and the Yangtze River. Lu et al. (2004) also studied genotoxic effects of chlorinated drinking water processed from raw water from Dong Lake and Yangtze River at Wuhan section, applying the comet assay (HepG2 cells) and the micronucleus assay. Results revealed that drinking water produced from polluted raw water using chlorination caused a significant and dose-dependent increase of DNA damage in the human cell

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Chapter 2 Solution by dilution? ______lines. Also, chlorinated water samples collected in the cold season (March) showed higher micronuclei frequencies than samples in the high summertime (August). However, the lowest concentration (lowest observed effect level) that caused a significant increase in DNA migration in the comet assay was ten times lower in Dong Lake and a hundred times lower in Yangtze River for samples taken at the same location in August than in March. Dong et al. (2010) compared the mutation rate of nonvolatile organic compounds (NVOCs) of source water and processed water extracts, from the Yangtze River and Han River in the Ames assay during normal (March), low (December) and high (July) flow period. The study revealed that the tap water was stronger mutagenic than the source water, and that the mutagenicity of Han River was higher than of Yangtze River. During normal flow period all samples exhibited mutagenicity in strain TA98 with and without S9-mix (a rat liver homogenate as an exogenous metabolic activation system), except finished water sourcing from Yangtze River was negative to TA98 with S9. During low flow period only the finished water and the terminal tap water of Yangtze River were positive to TA98 without S9. In high flow period, all samples were positive to TA98 with or without S9, except the source water of Yangtze River. None of the samples from the different periods was positive to TA100 with or without S9. The results showed that the NVOCs mainly contained frameshift mutagens. Lower Reaches: Shen et al. (2003a) collected tap water and source water samples in different areas of Shanghai, sourcing from Yangtze River and Tai Lake. Testing the samples in the Ames, Arabinose resistance test (Ara test) and SOS/umu test proved that tap water samples (sampled in south and middle Shanghai) originating from Tai Lake possessed mutagenic potentials, whereas two tap water samples (sampled in north Shanghai) originating from Yangtze River, were not mutagenic. Shen et al. (2003a) also found that the water displayed an even stronger mutagenic potential, compared to its original tap water after boiling. Alike Shu et al. (2002), Dong et al. (2010) and Wu (2005), the authors referred the molecular mechanism of mutagenicity to be associated with a frame-shifting potential. Chlorinated tap water extracts from city middle and their corresponding source water were compared with respect to their chemical composition. Gas chromatography-mass spectrometry (GC-MS) analysis revealed qualitatively similar spectra, except for the peaks of three chlorinated aromatic hydrocarbon compounds (3,4-dichloroaniline, 2,4-dichloroaniline, 2,6-dichloro-4-nitroaniline), which existed only in the tap water. The authors concluded that the chlorination step lead to the creation of more toxic compounds, including

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Chapter 2 Solution by dilution? ______mutagens. The authors stated that cancer risks may be elevated in areas where heavily chlorinated water is consumed. Li et al. (2006) investigated genotoxic effects in the comet assay with human peripheral blood lymphocytes and the micronucleus test with Vivia faba root tip cells after exposure to organic extracts from water of Yangtze River at Nanjing section. DNA damages in lymphocytes and root tip cells indicated mutagenic potentials at this section. Wu (2005) investigated the genotoxicity of surface water samples from Yangtze Estuary with the Ames assay. Genotoxicity was detected in several samples by the strain TA98, while no response could be detected in all samples with strain TA100. The mutation rate increased when S9-mix was added. This indicated that the mutagenic potential of the estuarine water samples was manifested in the bacteria’s genome by the mechanism of frame shifting (strain TA98). The water of the Yangtze Estuary’s South Branch and some samples from the seaward end of the estuary exhibited genotoxic potentials. The responsible toxicants were both direct (-S9 mix) and indirect (promutagenic) (+S9 mix) mutagens (Wu, 2005).

2.6.2 Endocrine disrupting activity

The groups of endocrine disruptors are composed of a large variety of molecules. Besides natural (e.g., 17β-estradiol) and synthetical hormones (e.g., 17α-ethinylestradiol) as well as natural components (e.g. phytoestrogens like genistein), this group compound include synthetic chemicals used as industrial solvents/lubricants and their byproducts (PCBs, polybrominated biphenyls [PBBs] and dioxins), plasticizers (BPA, phthalates), pesticides (methoxychlor, chlorpyrifos, DDT), fungicides (vinclozolin), and pharmaceutical agents (diethylstilbestrol [DES]) (Diamanti-Kandarakis et al., 2009). Due to their influence on the hormone (endocrine) system, endocrine disruptors are known to have an impact on wildlife health (e.g., feminization of fish and masculinization of snails) (Sumpter and Jobling, 1995; Jobling et al., 1996). Chronic exposure to low concentrations (5 - 6 ng/L) of 17α-ethinylestradiol even lead to the almost extinction of fathead minnow in one Canadian lake (Kidd et al., 2007). In addition, endocrine disruptors are also hypothesized to have effects on human health as well (e.g., male and female reproduction, breast development and cancer, prostate cancer, neuroendocrinology) (Diamanti- Kandarakis et al., 2009). Specifically, the USA, Japan, EU, and OECD have established testing approaches and regulatory frameworks with aim to assess the risks associated with chemicals that have endocrine disrupting properties (for review (Hecker and Hollert, 2011)). There is evidence that endocrine disruptors have been detected in the Yangtze River. Nonylphenol (NP) was ______60

Chapter 2 Solution by dilution? ______reported in river water, drinking water and fish tissues in Chongqing section (Shao et al., 2005) and natural estrogens estrone (E1) and 17β-estradiol (E2) were detected in municipal wastewater extracts at Nanjing section (Lu et al., 2010b). Furthermore, NP and BPA were also detectable in surface sediments of the Yangtze Estuary and the adjacent East China Sea (Bian et al., 2010). To estimate the ecotoxicological effects bioassay studies have been performed. Upper Reaches: Zhu et al. (2003) assessed the estrogenic activity of organic extracts from water of Chongqing section with the cell proliferation test with MCF-7 cells. The samples were taken at five water treatment plants sourcing from the Jialing River (upstream and central city section) and the Yangtze River (upstream, central and downstream city section after confluence). Estrogenic activity was detected in all samples, with higher activity at the central and downstream sections than in the upstream sections of Chongqing. The authors attributed the effects to the discharge of sewage and industry wastewater from Chongqing. The estrogenic activity was higher in summer than in winter, which was attributed to stronger runoff from agricultural fields during the rainy season in this time of the year (Zhu et al., 2003). It might also be possible that estrogenic active compounds were remobilized from agricultural fields on the riverbank or from waste that accumulated along the shore, both being dry during low and indundated during high water period. Di-iso-butyl-phthalate (DiBP) and DBP were identified as the main pollutants (Tian, Shu et al. 2003). Middle Reaches: The nonvolatile organic compounds (NVOCs) in samples of the source water and tap water from the Yangtze River and Han River in Wuhan section were investigated by Dong et al. (2010) with the Yeast Estrogen Screen (YES) assay in low, normal and high flow periods. Only the source water samples of the Yangtze River were estrogenic active in low flow period. During normal flow period both the source water samples of the Yangtze River and the Han River showed estrogenic activity, with a higher activity in the Yangtze River samples. No water sample showed estrogenic activity in high flow period. Because activity could only be found in the source water samples, the estrogenic activity of NVOCs was most likely erased by the routine process of water works. Lower Reaches: The estrogenic content of the Yangtze River in Nanjing section was assessed in an in vivo bioassay with adult male goldfish (Carassius auratus) (Lu et al., 2010a). The assay revealed significant serum vitellogenin (VTG) and 17ß-estradiol (E2) induction as well as gonad atrophy in the treated fish. The result was consistent with the levels of water estrogens

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Chapter 2 Solution by dilution? ______determined by chemical analysis. Steroidal estrogens were the major causal agents responsible for the estrogenic responses in the Jiangxinzhou and Daqiao sections, while phenolic estrogens were the main contributors in the Sanchahe section. Song et al. (2010) held polar contaminants responsible for the estrogenic activities in Jiaxingzhou and Daqiao section, while mid-polar and nonpolar contaminants led to the majority of estrogenic activities in Sanchahe section. The estrogenic activities in this area originated from the effluents of waste water treatment plants. In addition to the mentioned estrogenic activity, thyroid hormone (TH) agonist and antagonist activities of water sources along the Yangtze River between Nanjing and Nantong (near Shanghai) were examined by a green monkey kidney fibroblast (CV-1) cell-based TH reporter gene assay (Shi et al., 2011). Responsible thyroid-active compounds were determined by chemical analysis. To predict the TH agonist and antagonist activities instrumentally derived L-3,5,30-triiodothyronine (T3) equivalents (T3-EQs) and thyroid receptor (TR) antagonist activity equivalents referring to dibutyl phthalate (DBP-EQs) were calculated from the concentrations of individual congeners. It could be shown that only one water source from Nanjing section and two water sources from the section between Taizhou and Changzhou contained TR agonist activity equivalents (TR-EQs), ranging from 286 to 293 ng T3/L. On the other hand anti-thyroid hormone activities were found in all water sources with the TR antagonist activity equivalents referring to DBP (Ant-TR-EQs), ranging from 51.5 × 106 to 55.5 × 107 ng/L. The comparison of equivalents from instrumental and biological assays led to the conclusion that the TR antagonist activities might be attributed to high concentrations of dibutyl phthalate and di-2-ethylhexyl phthalate (DEHP) at some locations of the Yangtze River (Shi et al., 2011).

2.6.3 Other endpoints

This chapter summarizes the outcome of those studies, obtained from the available articles in the scope of this review, which investigated endpoints that did not belong to any of the former categories (Annex I, Table I.5). Upper Reaches: Organic extracts from surface water of the Yangtze River and Jialing River at Chongqing section, sampled during high (August) and low water period (January), have been examined by Cui et al. (2009) with respect to the endpoints ethoxyresorufin-O-deethylase (EROD) activity, cytochrome P-450 1A1 (CYP1A1) mRNA expression, aryl hydrocarbon receptor (AhR) binding capacity and activation of xenobiotic response element (XRE). The according methods were the EROD assay, reverse-transcription polymerase chain reaction (RT-PCR) and ______62

Chapter 2 Solution by dilution? ______electrophoretic mobility shift assay (EMSA) applied to H4IIE rat hepatoma cells. The sampling sites were both located in urban areas near water supply sites of Chongqing city without any significant industrial discharges nearby. The main pollutants originate from traffic emissions and domestic sewage. The EROD assay revealed toxic equivalency (TEQ) values from 1 × 10-4 to 13 × 10-4 pg 2,3,7,8-TCDD/L river water. The low water samples exhibited higher TEQ values than the high water samples from the same site, and Jialing River samples showed higher TEQ values than samples from the Yangtze River from the same sampling season. However, overall the TEQ values were categorized to be relatively low. The time-dependent induction of CYP1A1 also demonstrated a difference between the seasons. An induction could still be measured in low water period samples after 48 hours, whereas the high water samples already showed no response at this time. PAHs were considered to be the responsible contaminants for the CYP1A1 induction, which were quantified by GC/MS analysis in a range from 231 to 994 ng/L. The Yangtze River samples from low water period contained the highest total PAHs concentrations. The authors attributed the differences between the seasons to a lower flow rate in the low water period, which led to an enrichment of organic pollutants in the rivers. In a study by Li (2006), a subchronic toxicity test on rats was adopted to assess the risk of Chongqing drinking water samples - originating from the Jialing River - to humans. Pathological changes, hematological parameters, analysis of urine, biochemical serum parameters and an index related to free radical damages were observed at different doses. Genetic damage in body cells as well as reproductive cells could be shown. Although the organic extracts had potential toxicity on many organs the authors stated that with respect to the dosage of people’s daily intake the water might be safe for human usage (Li, 2006). Another toxicity test on the reproductive system of male mice (Mus musculus) with organic extracts from tap water originating from the Jialing River suggested that mid and high doses of organic extracts could disturb the male reproductive system in mice. The number of epididymal sperm in the high organic extracts group as well as serum testosterone and follicle-stimulating hormone levels in the treated groups decreased significantly (p < 0.05), whereas the frequency of sperm abnormalities in all treated groups increased significantly (p < 0.05). Besides significant decrease of acid phosphatase and increase of γ-glutamyl transpeptidase activity (p < 0.05), histological changes were observed in the mid- and high-dose organic extracts-treated groups (Zhao et al., 2011).

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Chapter 2 Solution by dilution? ______

Lower Reaches: Another toxicological study with male mice (Mus musculus), concerning the Yangtze River at Nanjing section as a source of drinking water, demonstrated that the source water had a toxic impact on the reproductive system of the tested animals. Alterations in different germ cell populations were observed as well as a significant increase of the percentage of abnormal sperms. Furthermore there were obvious testicular histopathology distinguishes noticed in expansion of interstitial space and reduction in the number and size of leydig cells (Zhao et al., 2009). Wang et al. (2010b) exposed goldfish (Carassius auratus) to organic water extracts taken near inlets of main branches or the outlets of wastewater treatment plants at three representative sections (Daqiao, Sanchahe and Jiangxinzhou) of the Yangtze River in Nanjing section. Acetylcholinesterase (AChE), glutathione-S-transferase (GST), 7-ethoxyresorufin-O-deethylase (EROD), glutathione peroxidase (GPx) and Na+/K+-ATPase activities were determined and alterations could be observed. EROD and GST activities appeared to be the more sensitive biomarkers. The levels of AChE, GST, EROD, Na+/K+-ATPase activities changed with the change of the extracts polarity. Most significant responses were detected for fractions with intermediate and weakly polar components. To evaluate the impact the integrated biomarker response index (IBR) was calculated. The authors concluded that wild fish at Nanjing section of the Yangtze River were at potential ecological risk. Water extracts from Jiangxinzhou exhibited greatest adverse biological effects, followed by Sanchahe. Samples from Daqiao possessed the comparably lowest adverse potencies (Wang et al., 2010b). Furthermore organic extracts of surface water of the Tai Lake section in Jiangsu Province of the Yangtze River was investigated by Wang et al. (2011), with in vitro cytotoxicity assays (MTT cell viability test, lactate dehydrogenases release and annexin V–PI staining combined with flow cytometry) applying the sertoli, leydig and spermatogenic cells from adult male Sprague–Dawley rats. In addition, selected pollutants including PCBs, OCPs and PAHs were quantified by instrumental analysis. Though the total concentration of PAHs did not exceed national drinking water source quality standards (Ministry of Health - China, 2006), the concentrated organic extract (286-fold) induced significant reproductive toxicity. PAHs were considered responsible to pose the greatest risk of the chemicals studied. The authors stated that the extract was enriched to determine if there were adverse effects due to exposure to interactions among measured residues or from unquantified residues that might have been in the mixture (Wang et al., 2011). As an

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Chapter 2 Solution by dilution? ______example enrichment factors for OCPs were 39 to 59 fold and for PCBs were 510 to 42 × 103 fold in fish of the Yangtze River in Jiangsu area (Hu et al., 2009b). The results indicated that chronic exposure to the water can cause adverse effects on the male reproductive system, e.g., by bioconcentration of the compounds present in the water (Wang et al., 2011). Wu et al. (2010) evaluated the sediment quality of the Yangtze Estuary using zebra fish (Danio rerio) embryos. The chemical analysis revealed that the concentrations of Zn and fluorene in the sediment samples were 2.4 × 105 ng/g and 46 ng/g, respectively. According to the sediment contact assay and fish embryo toxicity test, the survival rate and heart rate of zebra fish embryos were reduced, while abnormalities and delayed hatching were induced, indicating that the sediment from the Yangtze Estuary may have teratogenic effect on biota.

2.6.4 Discussion: Effect assessment of water and sediment (in vitro and in vivo)

The assessment of environmental samples focused mainly on mutagenicity/genotoxicity (Ames, comet and micronucleus assay) and endocrine effects (Cell proliferation, YES and in vivo assay with Carassius auratus) in the compartment water, which has to be differentiated into source/surface water, tap water and drinking water. The main sections of interest were the same that were predominantly investigated with respect to pollutant levels in water and sediment: the highly populated areas of Chongqing in the Upper, Wuhan in the Middle and Nanjing, as well as the Yangtze Estuary in the Lower Reaches. A large amount of citizens, who require also a large amount of unpolluted drinking water and food, as well as main polluters that discharge contaminants into the local rivers, like industry and domestic wastewater, are concentrated in these areas. Thus, human health is there at a particular risk. Based on the reviewed articles it can be stated that the inflowing Jialing River at Chongqing in the Upper Reaches, though both rivers possess mutagenic and endocrine potentials, seems to be more seriously polluted than the Yangtze River. This was mainly attributed to agricultural, industrial and domestic discharges along the Jialing River, as well as a poor self-purification capacity compared to the Yangtze River (Qiu et al., 2003). Water from the Yangtze River, Dong Freshwater Lake and the Yangtze feeding Han River at Wuhan section in the Middle Reaches showed mutagenic and endocrine activity as well. The tributary in this section possessed also a higher mutagenic potential than the mainstream (Dong et al., 2010). Alike the other sections the Lower Reaches revealed mutagenicity and endocrine activity in the Yangtze River water at Nanjing and the Yangtze

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Estuary. According to mutagenicity the South Branch of the estuary seems to be more seriously polluted than the North Branch (Wu, 2005). Another interesting observation was that all Ames mutagenic assays match in the outcome that especially frameshift mutagens were the responsible inducers (Shu et al., 2002; Shen et al., 2003b; Wu, 2005; Dong et al., 2010). Similar observations were made in the German Rhine River (Kosmehl et al., 2004). In addition, genotoxicity was increased when the metabolically active S9-mix was added, indicating a predominant presence of promutagenic substances (Wu, 2005). The pattern of genotoxicity was comparable to that previously described by Dobias et al. (1999, who applied the Ames assay in an effect-directed chemical analysis to measure the differences in mutagenicity of the main and subfractions of extractable organic material in the ambient air at workplaces of a coke oven. Based on these results they concluded (1) that most of the mutagenicity found in the main fractions required metabolic activation in vitro (+S9 mix) and (2) frameshift mutations were the predominant type of mutation observed. The authors attributed the most important role to the carcinogenic PAHs and genotoxic nitrocompounds (Dobias et al., 1999). The frameshift mutagenicity of nitro-PAHs has also been proven in other studies (McCoy et al., 1981; Xu et al., 1982) and they were considered to be among the main responsible components for mutagenicity in Danube River sediments in Germany (Higley et al., 2012). Nitro- PAHs emerge from the emission of combustion sources and form in the environment from - reactions of certain PAHs, e.g., phenanthrene, with the highly reactive nitrite (NO2 ). The latter is produced mainly naturally through biological processes, like nitrification (ammonia oxidation) and denitrification (nitrate reduction). Oxygen deficiency or pollution with nitrogenous waste, e.g., from fertilizers, promotes the creation of nitrite. Nitrite has already been proven to be toxic to freshwater fish, including carps and catfish (Lewis Jr and Morris, 1986). In addition to that can it enhance the toxicity of PAHs by causing severe hepatic damage in fish and impact the metabolism of PAHs (Shailaja and Rodrigues, 2003), as well as increasing the formation of mutagenic metabolites in fish, e.g., exposed to refinery effluent (Shailaja et al., 2006b; Shailaja et al., 2006a). As previously described is the water and sediment in many sections of the Yangtze River polluted by PAHs, which primarily originated from combustion sources. This supports the assumption that the mutagenic activity in the Yangtze River region might be attributed to a high degree to PAHs and nitro-PAHs from combustion sources, as well as nitro-PAHs that form from polycyclic hydrocarbons in the aquatic environment. Yet further effect-directed analysis should

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Chapter 2 Solution by dilution? ______be utilized to clarify what the responsible components are. Although mutagenic/genotoxic chemicals like heavy metals, PAHs, heterocyclic amines and pesticides were detected in surface waters all around the world in numerous studies, linking those to the measured mutagenicity has been difficult. Thus many major putative mutagenic/genotoxic compounds in most surface waters with high mutagenic/genotoxic activity in the world remain yet unknown (Ohe et al., 2004). Furthermore, it was observed that chlorination of polluted source water during drinking water treatment increased its mutagenic potential (Shu et al., 2002; Shen et al., 2003b; Wu, 2005; Dong et al., 2010), whereas the estrogenicity could be erased after the routine process of water works (Dong et al., 2010). Boorman (1999) pointed out that several mutagenic byproducts can originate from disinfection of drinking water, e.g., after chlorination. Chlorine gas (Cl2) and hypochlorite are commonly used for chlorination processes in water treatment. Hypochlorite and hypochlorous acid (HOCl) are the main chlorine species under typical water treatment conditions (pH 6-9), with HOCl being the major reactive form. Hypochlorous acid originates from the hydrolyzation of Cl2 in water. The main reactivity of chlorine results from electrophilic attacks of HOCl on organic and inorganic compounds (Deborde and Von Gunten, 2008). The main disinfection byproducts resulting from the reaction of chlorine in drinking water with naturally occurring organic matter in water are trihalomethanes (THM) and haloacetic acids (HAA). Further attention is drawn to the family of chlorinated furanones, because most of the mutagenicity found in chlorinated drinking water can be referred to 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)- furanone, a member of this family (WHO, 1997; Boorman, 1999). Variables that influence the formation of chlorine species and disinfection byproducts are chloride concentration, concentration of dissolved organic matter, pH, temperature and bromide concentration in the water (Deborde and Von Gunten, 2008; Platikanov et al., 2010). Chen et al. (2010) measured THM and HAA concentrations in raw water and processed water of Yangtze and Huangpu River in Shanghai section. In addition to that they examined fluctuations during transmission and distribution in the pipeline network. While THM concentrations of finished water from Yangtze River (21 – 31 µg/L) exceeded those from the Huangpu River (7 – 9 µg/L), a reverse picture could be shown for HAAs (Yangtze: 19 – 23 µg/L; Huangpu: 25 – 37 µg/L). For matters of comparison, neither exceeded the maximum allowable annual average level given by the USEPA (THM: 80 µg/L; HAA 60 µg/L). The fluctuations of THM and HAA levels in the distribution network were low during transmission and distribution process.

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The cancer risk originating from the formation of trihalomethanes in drinking water sourcing from the Yangtze River in Shanghai section was shown by Liu et al. (2011b). Based on an adopted human health risk assessment developed by the USEPA (2005) it could be found that the total cancer risk (oral, dermal and inhalation) was highest in spring (male: 8.23 × 10-5 meaning 82.3 cases/million persons; female: 8.86 × 10-5) and lowest in summer (male: 3.57 × 10-5; female: 3.84 × 10-5). Levels for autumn (male: 5.38 × 10-5; female: 5.79 × 10-5) and winter (male: 5.69 × 10-5; female: 6.15 × 10-5) lay in between. The lifetime cancer risk of THMs was the highest via oral ingestion. The average concentration of THMs was lowest in autumn (66 µg/L) followed by winter (78 µg/L), summer (98 µg/L) and the highest level in spring (101 µg/L). Because these values demonstrate only one variable of the cancer risk assessment, beside chronic daily intake, exposure frequency, duration and others, the high difference in cancer risk can be explained between spring and summer despite comparable THM concentrations. The high level in spring was related to a higher bromide ion concentration resulting from the intrusion of tidal saltwater into the Yangtze River during the dry season. In addition to that it was found that the presence of Fe(III) increased the levels of THMs and subsequently the cancer risk to humans (Liu et al., 2011b). According to the guidance of USEPA (1989), a cancer risk below 10-6 (1 case per million persons) is negligible and above 10-4 (100 cases per million persons) is considered to be sufficiently large that some sort of remediation is desirable. A cancer risk between 10−6 to 10−4 is classified as an acceptable risk (Environmental Protection Agency - USA, 2013). The background cancer risk over a lifetime is 1 case in 3 persons (Environmental Protection Agency - USA, 1997). Based upon this classification the cancer risk originating from THMs in the investigated drinking water is acceptable throughout all seasons (Liu et al., 2011b). Yet it has to be considered that the treated water bears a complex mixture of disinfection byproducts which add to the individual cancer risk of THMs. This includes, besides non-mutagenic dissolved organic matter as parent substances, also the chlorination of pollutants like PAHs (Deborde and Von Gunten, 2008). The primary generation of chlorinated PAHs (Cl-PAHs) is during pyrosynthesis and a secondary reaction process also occurs in the aquatic environment. Certain Cl-PAHs induce even higher mutagenicity and aryl hydrocarbon receptor activity compared to their corresponding parent PAHs (Ohura, 2007). Colmsjö et al. (1984) could show that mutagenicity of the weak mutagenic PAHs pyrene could be multiplied after chlorination. Some Cl-PAHs are reported to induce tumorigenicity and oncogene activation (Fu et al., 1999).

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Shiraishi et al. (1985) demonstrated the presence of Cl-PAHs in concentrations of 10-1 to 10-2 ng/L in chlorinated tap water of Tsukuba, Japan. Shen et al. (2003a) discovered the chlorinated monoaromatic hydrocarbons 3,4-dichloroaniline, 2,4-dichloroaniline and 2,6- dichloro-4-nitroaniline in processed tap water, which were not detected in the corresponding source water, indicating a formation of these substances associated with the disinfection step. Beyond was observed that boiling of the mutagenic tap water led to even stronger mutagenicity. The authors referred this phenomenon to an accelerated chlorination of non-volatile organic residues by excessive chlorine in the tap water during the heating process. This is especially critical as it is widely believed, as in Shanghai, that boiling reduces the risk originating from the water (Shen et al., 2003a). Furthermore several epidemiological studies have suggested that drinking chlorinated water may be associated with increased incidences of bladder, rectal, and colon cancer (King and Marrett, 1996; Koivusalo et al., 1997; Hildesheim et al., 1998) and adverse reproductive effects (Waller et al., 1998). To summarize, it should be considered that chlorination of non-mutagenic dissolved organic matter and other parent substances during water treatment adds to the total mutagenicity of components in the polluted source water. Another striking observation was that the greatest mutagenic/genotoxic activities were observed during spring (March) in the majority of all cases. The Yangtze River passes annual fluctuations in its water level, which is influenced by the rainfall throughout the year. There is no clear definition about the timeframe for each flow period. As a rough classification, the water level is lowest in the dry season in winter/spring (December to April), at a medium level in spring/summer (May to June) as well as autumn (October to November), and highest in the rainy season in summer (July to September) with occasionally occurring flood events (Sun et al., 2002). The seasonality of the water level in the Yangtze River in the TGR section is inverted due to the construction of the TGD (Cui et al., 2009). Since the impoundment, the water level is lowest in summer (June to September) and rapidly rising towards the highest level over winter (October to March) with middle levels in spring (April to May). The inverted water levels are attributed to the buffer capacity of the reservoir to prevent flood events and supply the Middle and Lower Reaches with water during the dry season. The observation that the highest mutagenicity/genotoxicity could often be observed in spring is most likely associated with higher pollutant concentrations in the water - due to lower dilution - during the low water period in this season. This assumption is supported by the observation that PAHs levels in the South Branch of the Yangtze Estuary during

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Chapter 2 Solution by dilution? ______dry season in spring were comparably higher than those in the rainy season in summer, as described before (Ou et al., 2009). Also CYP1A1 inductions of surface water extracts from Yangtze River and Jialing River at Chongqing section were greater in low water period (January) than in higher water period (August). The authors attributed the differences between the seasons to a lower flow rate in the low water period, which led to an enrichment of organic pollutants in the rivers (Cui et al., 2009). Due to the inverted water level situation in the TGR since impoundment, it is to be expected that the changes of hydrology manifest as changes in contamination conditions, with higher pollutant and effect levels in summer during rainy season than during dry season (Cui et al., 2009), caused by a lesser dilution and reduced velocity in the reservoir. Hydrophilic compounds may be carried with the discharged water past the dam, but hydrophobic substances will most likely remain in the sediments of the reservoir. It has also to be considered that the elevated precipitation in summer is associated with an increased amount of air-borne particles washed out from the air as well as a stronger runoff from fields, carrying pollutants into the TGR, which may also be trapped there and subsequently accumulate in this region. As PAHs seem to play a major role in the Yangtze River Region it is surprising that only limited research was performed on biomarkers or bioassays displaying the cytochrome P450 activity, a major enzyme family for the metabolism of lipophilic xenobiotics. PAHs, PCBs, dioxins and furans are metabolized by the CYP1A enzyme subfamily. Their activity can be measured, e.g., by the EROD assay, which is widely applied and accepted to test dioxin-like activity of water and sediment in vitro as well as to measure the biochemical response in organisms in vivo and in situ exposed to the previously mentioned xenobiotics (Hilscherova et al., 2000; Whyte et al., 2000; Brack et al., 2005). Also more specific in vitro assays, like the H4IIE assay, are widely accepted and able to measure either enzyme activity (i.e., EROD) or agonistic receptor binding (e.g., PCBs, PAHs, PCDDs/DFs) at the AhR, which is considered to be involved in the mediation of dioxin- like effects (Poland and Knutson, 1982; Tillitt et al., 1991; Villeneuve et al., 2001). As most of the studies applied organic extracts in varying concentrations to the bioassays, there is a certain degree of uncertainty regarding the actual risks associated with the native samples. Therefore it is crucial to also conduct in situ studies in the same area, to bridge the gap between laboratory and field (Chapman, 1990). Regardless, analyses of organic extracts provide useful

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Chapter 2 Solution by dilution? ______information with respect to the presence of compounds that may lead to effects after chronic exposure and/or bioaccumulation.

2.7 Pollutant levels and adverse effects in aquatic organisms (in situ studies)

The fish fauna of the Yangtze River basin is one of the richest world-wide, offering plentiful fish resources (Fu et al., 2003; Chen et al., 2009), but at present these fishery resources are seriously depleted and the fishery yield is significantly reduced (Chen et al., 2009). The fishery yield in the Yangtze River grew from 1949 to 1954 and peaked at about 427,000 tons in 1954. The annual yields during 1955 to 1971 were stable at about 261,000 t per year and began to decrease to 200,000 t from the 1980s. In 1990s, the fishery yield was even halved to about 100,000 t (Chen et al., 2002a) (More recent numbers were not available). The fish community has been altered, with a reduction in the quantity of rare, peculiar and economically important fish species populations, an increase in the number of exotic fish species, decrease in migratory fish species and a severe trend in fish stunting. Habitat fragmentation and shrinkage, resources overexploitation, invasion of exotic species and water pollution were held responsible for these changes (Chen et al., 2009). Industrial wastewater and communal sewage discharge carrying toxicants are considered to induce the destruction of spawning grounds, depletion of brood stocks, decrease in production and even high mortality in fish (Chen et al., 2004). Moreover, the pollutants dislocate from the water column to particulate matter, and finally accumulate in sediments which may play a role as secondary contamination sources (Brinkmann et al., 2010). The contaminants can accumulate in sediment-dwelling organisms, and be transferred to higher trophic levels through the food chain (Liu et al., 2003; Hu et al., 2009b). Bioaccumulation of toxicants may pose a potential health risk for local residents with regard to the consumption of contaminated food from the Yangtze River. The reported publications about in situ studies in the Yangtze River were summarized in Annex I, Table I.6.

2.7.1 Pollutant levels in aquatic organisms

Upper Reaches: A major study carried out by the Greenpeace (2010) evaluated the presence of PFCs (including PFOS) and alkylphenols (including NP and 4-tert-octylphenol [OP]) within commonly eaten wild fish (Cyprinus carpio carpio, Silurus soldatovi meridionalis) from the Upper (Chongqing), Middle (Wuhan) and Lower (Ma’anshan and Nanjing) Reaches. These fish contained detectable levels of alkylphenols and PFCs. Nonylphenols were the predominant ______71

Chapter 2 Solution by dilution? ______alkylphenols (present in over 95% of carps, and over 85% of catfish) and PFOS as dominating PFC. Whereas average NP levels were comparable in carp (23 ng/g ww) and catfish livers (24 ng/g ww) from Chongqing, OP (1 g/g ww; 3 ng/g ww) and PFOS (< 0.3 ng/g ww; 22 ng/g ww) levels differed considerably. The carp samples from Chongqing were the only samples that did not contain any detectable perfluorinated compounds. On the other hand catfish samples from the same area showed the highest OP concentrations (3 ng/g ww) (Greenpeace, 2010). Shao et al. (2005) also detected the bioaccumulation of NP and nonylphenol ethoxylates (NPOEs) in fish samples (Coreius guichenoti, Coreius heterodon, Leptobotia elongate, Rhinogobio typu, Rhinogobio ventralis) from Jialing River and Yangtze River at Chongqing section. The residual NP concentrations in muscle, gills, liver and stomach ranged from n.d. to 90 ng/g ww, 100 to 400 ng/g ww, 800 to 1.9 × 103 ng/g ww and 200 ng/g ww, respectively. While NPEO levels ranged from 400 to 1.3 × 103 ng/g ww for muscle, 2.1 × 103 to 5.8 × 103 ng/g ww for gills, 20.2 × 103 to 48.3 × 103 ng/g ww for liver and 2.2 × 103 ng/g ww in stomach. The authors compared these concentrations to levels of NP and NPEO in the surrounding water, gaining bio-concentration factors which indicated that NPEOs were more easily bio-concentrated in the fish than NP. Both chemicals concentrated especially in the fish livers. The authors concluded that under the assumption that one person takes in 200 g river fish tissues and 2 L drinking water, the maximum amount of NP (390 × 103 ng/person/day or 65 × 103 ng/kg bodyweight) was far below the concentration that can elicit sub-chronic toxicity on laboratory rats (Cunny et al., 1997). Whereas the risk of NP was low from the view of human consumption they pointed out that measured NP levels in the Yangtze River were close to the threshold concentration (10 × 103 ng/L) that affects fish reproduction (Jobling et al., 1996), thus posing a potential ecological risk. Middle Reaches: Carp and catfish liver samples from Wuhan contained even higher average levels of the dominant alkylphenols and perfluorinated compounds (carp: 85 ng NP/g ww; 2 ng OP/g ww; 42 ng PFOS/g ww; catfish: 32 ng NP/g ww; 40 ng PFOS/g ww), except for OP in catfish (3 ng/g ww). In comparison to the other sampling sites in this study highest NP (85 ng/g ww) concentration could be detected in carp from Wuhan (Greenpeace, 2010). Lower Reaches: Greenpeace (2010) could only investigate carp in Ma’anshan due to a scarcity of catfish during the sampling period. The carp samples contained lowest average levels of NP (6 ng/g ww) and OP (0.3 ng/g ww), except for PFOS (2 ng/g ww) which was higher than in

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Chongqing. The carp samples from Nanjing contained second highest average NP (34 ng/g ww), OP (2 ng/g ww) and PFOS (28 ng/g ww) concentrations compared to the carps from other study areas. Whereas the catfish showed the highest NP concentration (61 ng/g ww), they contained the lowest OP (2 ng/g ww) and PFOS (18 ng/g ww) average concentration among their species from the other sites (Greenpeace, 2010). Furthermore, PBDEs were determined in some species of aquatic biota from the lower reaches of the Yangtze River, including fish, crabs and shrimps (Xian et al., 2008; Gao et al., 2009; Su et al., 2010). The concentrations of PBDEs and hexabromocyclododecanes (HBCDs) in muscle of nine freshwater fish species (Hypophthalmichthys molitrix, Ctenopharyngodon idella, Aristichthys nobilis, Carassius auratus, Cyprinus carpio, Coreius heterodon, Parabramis pekinensis, Siniperca chuatsi, Channa argus) from the Yangtze River (near Nantong/Shanghai), reached to 1.1 × 103 ng/g and 330 ng/g (lipid weight), respectively (Xian et al., 2008). Findings of another study revealed that PBDEs and methoxylated PBDEs (MeO-PBDEs) were accumulated in four species of anchovy (Coilia nasus, Coilia mystus, Coilia nasus taihuensis, Coilia brachygnathus) from the Yangtze River (Nanjing to Yangtze Estuary), Tai Lake and Hongze Lake with concentrations up to 4 ng/g ww and up to 8 ng/g ww, respectively. The authors concluded that PBDEs in anchovy are primarily of synthetic origin and released by human activities, while MeO-PBDEs in anchovy primarily sourced as natural products from the sea, instead from metabolism of synthetic PBDEs in the animals itself (Su et al., 2010). Su et al. (2012) measured the concentrations of PBDEs and analogues in freshwater fish (Cyprinus carpio, Carassius auratus, Pelteobagrus fulvidraco, Hemicculter leuciclus, Coilia macrognathos Bleeker, Silurus spp.) from the Lower Yangtze Reaches and marine fish (Sinonovacula constrzcta, Drepane punctata, Ilisha elongate, Suggrundus meerdervoortii, Pseudosciaena polyactis) from the Yellow Sea. Finally, the potential risk of these PBDE analogues was assessed by measuring the dioxin-like activity in the H4IIE-luc rat hepatoma transactivation bioassay. Most of the PBDE analogues were detected in the marine organisms. Of the eleven detected PBDE analogues, six were found to have measurable dioxin-like potency. Some PBDE analogues exhibited significant dioxin-like potency. However, concentrations of 2,3,7,8-TCDD equivalents, indicating the strength of potency, were less than the tolerance limit proposed by the European Union (2006 and the oral reference dose (RfD) derived by Environmental Protection Agency - USA (2012), respectively (Hazard Quotients [HQ] < 0.005). Although the PBDE levels in aquatic biota in the

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Chapter 2 Solution by dilution? ______lower reaches of the Yangtze River and adjacent Yellow Sea did not exceed the safety levels, there should still be paid attention, because some analogues have been detected in other environmental media, as well as human blood (Su et al., 2012). Perfluorinated compounds (PFCs) were measured in water and wildlife (Carassius auratus, Ictalurus punctatus, Pseudorasbora parva) and farmed fish (Carassius auratus, Ictalurus punctatus) as well as home-fed chicken and ducks collected from Shenyang (northeast China) and the Yangtze River Delta (Lu et al., 2011). The study aimed at the evaluation of the human health risk growing from the intake of PFOS and PFOAs via fish and domestic poultry dietary. The water of the Yangtze River Delta (PFCs: 42 to 170 ng/L; average river waters 105 ng/L; average surface seawaters 77 ng/L), with highest concentrations in the Shanghai section of the Yangtze River, exceeded the concentrations in water samples from the rivers in Shenyang (PFCs: average 5 ng/L). PFOA were the dominating compounds in both sections, in addition to PFOS in Shenyang. PFOS seemed to play only a minor role in the Yangtze River Delta. Urban sewage was claimed to be the main source of PFOS, and perfluorohexane sulfonate (PFHxS) in the investigated surface waters of Shenyang. In all biota samples PFOS (Averages: Shenyang poultry 1 ng/mL serum and 1 ng/g dw liver; Shenyang fish 3 ng/g dw tissue; Chongming Island Fish 6 ng/g dw tissue) and perfluoroundecanoic acids (PFUnDA) in fish (Averages: Shenyang fish 1 ng/g dw tissue; Chongming Island fish 4 ng/g dw tissue) were the most abundant. The authors concluded that the acceptable daily intake of PFOS and PFOA through the diet of fish and poultry in the studied areas did not exceed the RfD for non-cancer health effects (Hazard ratio values [PFOS; PFOA] < 1.0). Wen et al. (2008) reported PCDDs/DFs in bivalves (Limnoperna lacutris, Cobicula fluminea) in the Yangtze Estuary. The concentrations of total PCDDs/DFs and the toxic equivalent (TEQs) in bivalves varied between 0.3 and 1 ng/g dw as well as 3 × 10-3 and 11 × 10-3 ng/g dw, respectively. The BSAF (biota-sediment accumulation factor) values were studied, examining the relationship between concentrations of PCDDs/DFs in animal and sediment in this area. While it was found that the BSAF had an inverse correlation with the number of chlorines in PCDDs/DFs. According to the authors the herbicide pentachlorophenol and sodium pentachlorophenol and the waste discharge from a local sinter plant were held responsible to be the main source of PCDDs/DFs in the area.

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A holistic approach to determine the accumulation of PCBs, PAHs, HCHs and DDTs in fish and benthos was performed by Hu et al. (2009b) at Jiangsu section. Fish (Cyprinus carpio, Hypophthalmichthys molitrix, Ctenopharyngodon idellus) and benthos (Bithynia fuchsiana, Bellamya aeruginosa) as well as water and sediment samples were taken between Jiangning (upstream Nanjing) and Haimen (Yangtze Estuary). The highest levels for PCBs, PAHs, HCHs and DDTs in fish were 23 ng/g, 7 × 10-3 ng/g, 28 × 10-3 ng/g and 76 × 10-3 ng/g, respectively. Analogous to that have been the highest levels in benthos bodies 14 ng/g, 21 × 10-3 ng/g, 26 × 10-3 ng/g and 82 × 10-3 ng/g. Four- to five- ringed chlorinated biphenyls were chiefly detected among all PCBs, with PCB 105 being the major representative. According to the authors, the PAH concentrations have been higher in benthos (21 × 10-3 ng/g) than in fish (7 × 10-3 ng/g) including a significant positive correlation (p < 0.05), with two- to four- ringed PAHs as the main components upon all PAHs measured. DDTs consisted entirely of p,p'-DDE isomers. Comparing the PCB burden in the fish to the environmental compartments sampled at the same sites, it was observed that fish from Jiangning, Jiangyin (halfway Nanjing and Shanghai) and Haimen accumulated PCBs with enrichment factors from 5.1 × 102 to 4.2 × 104 fold. Enrichment factors for OCPs ranged from 3.9 × 101 to 5.9 × 101 fold in fish and 6.8 × 101 to 1.46 × 102 fold in benthos at the same sites. The authors concluded that this demonstrated the remaining presence and accumulation of already banished hazardous compounds in biota (Hu et al., 2009b). Organochlorine pesticides HCHs and DDTs were detected in sediment-dwelling animals including mollusks (Corbicula fluminea, Sinonovacula constricta) and crabs (Sesarma dehaani) from the Yangtze Estuary and nearby coastal areas. Levels of HCHs ranged from 1 ng/g to 6 ng/g and averaged 4 ng/g in mollusks, while DDTs concentrations ranged from 26 to 69 ng/g, with a mean of 35 ng/g. In crabs HCHs concentrations varied from 2 to 26 ng/g and averaged 14 ng/g, whereas the concentrations of DDTs were in the range of 2 to 25 ng/g with a mean value of 6 ng/g (Yang et al., 2006). The concentrations of DDTs were far higher than those in sediments in this area (up to 1 ng/g, dw) (Liu et al., 2003). Liu et al. (2004) measured concentrations of OCPs in addition to PCBs in the same mollusks (Corbicula fluminea, Sinonovacula constricta, Bullacta exarata, Potamocorbula ustulata) and crab species (Sesarma dehaani), as well as shrimp (Exopalaemon carinicauda) and fish (Mugil cephalus) also in the Yangtze estuarine and coastal areas. The gas chromatography-electron capture detector (GC-ECD) analysis determined HCHs concentrations ranging from 1 to 77 ng/g with a mean of 13 ng/g and with DDTs

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Chapter 2 Solution by dilution? ______concentrations from 2 to 159 ng/g with a mean of 34 ng/g. The concentration distribution of PCBs ranged from 44 to 1.3 × 103 ng/g with a mean value of 343 ng/g. Animals of the same species were characterized by higher contamination levels for males than females, and small individuals than large ones. The authors evaluated the contamination status of the animals to be at a moderate level. Another study by Ma et al. (2008) measured HCHs and DDT concentrations in shellfish (Oyster, Mussel, Mactra veneriformis Reeva, Meretrix meretrix Linnaeus, Scapharca subcrenata) from the Yangtze Estuary and adjacent waters. Gas chromatography analysis determined a concentration range of n.d. to 12 ng/g ww for HCHs (mainly alpha- and delta-HCH) and averaged at 1 ng/g, conforming to the first level of criterion (20 ng/g) of the Marine Biology Quality Criterion by the State Oceanic Administration - China (2001). For DDTs (mainly o,p'- and p,p’-DDT) the concentrations ranged from 4 to 282 ng/g with a mean of 58 ng/g exceeding the first level (10 ng/g), but conforming the second level (100 ng/g) of the Marine Biology Quality (State Oceanic Administration - China, 2001). The highest concentration could be found at Shengsi (Island southeast of Shanghai), followed by Yangkougang (coast north of estuary - CNE), Lvsi (CNE), Dongyuan (CNE) and Beibayao (Chongming Island). Low concentrations were observed at Changsha (CNE), Beidaodi (Southeast of Chongming Island) and Gouqi (Island southeast of Shanghai). Comparing the samples from 2006 and 2007 the concentration of HCHs and DDTs in most sites decreased, except for Yangkougang, Dongyuan, Beidaodi, Lvsi, and Shengsi. Of all studied animals oyster had the highest bioaccumulation ability. Overall it could be stated that the studied areas were slightly affected by organochlorine pesticides, particularly by DDTs. Chen et al. (2002c) measured the accumulation of petroleum hydrocarbons and volatile phenols in two migratory fish species (Coilia ectenes - CE, Eriocheir sinensis - ES, Anguilla japonica - AJ), three semi-migratory fish species (Leiocassis longirostris - LL, Pelteobagrus vachellii - PV) and five sedentary fish species (Silurus asotus - SA, Siniperca chuatsi - SC, Cyprinus carpio - CC, Megalobrama amblycephala - MA, Coreius heterodon - CN) from the river sections of Anqing (halfway Wuhan and Nanjing) (CC, PV, MA, CN), Nanjing (CE, ES, AJ, LL, SA, SC), Zhenjiang (downstream Nanjing) (CE, ES, AJ, LL, CC, PV) and the Yangtze Estuary (ES). Mean levels of petroleum hydrocarbons ranged in Anqing from 1.10 × 103 to 6.83 × 103 ng/g ww, in Nanjing from 3.21 × 103 to 8.72 × 103 ng/g ww in Zhenjiang from 3.25 × 103 to 9.23 × 103 ng/g ww and in the Yangtze Estuary 2.83 × 103 ng/g ww could be detected. Mean levels of volatile

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Chapter 2 Solution by dilution? ______phenol ranged in Anqing from 240 to 620 ng/g ww, in Nanjing from 520 to 3.18 × 103 ng/g ww in Zhenjiang from 390 to 3.27 × 103 ng/g ww and in the Yangtze Estuary 250 ng/g ww could be measured. The accumulation of these substances indicated that the area has been polluted by industrial effluents.

2.7.2 Adverse effects in aquatic organisms

Lower Reaches: Li and Shen (2010) measured the peripheral blood erythrocyte micronucleus rate of the wild Carassius auratus catched in the Yangtze River in Jiangsu province (upstream Shanghai) as an in situ biomarker for genotoxicity. It was found that the genotoxic effects caused by the water of the cities Changshu (0.212 ‰ to 0.219 ‰; p < 0.01) and Jiangyin (0.199 ‰ to 0.200 ‰; p < 0.01) showed significant effects, while those of Haimen (0.144 ‰ to 0.152 ‰; p < 0.05) were comparably lower, yet still significant. Those from Jiangpu (0.120 ‰ to 0.133 ‰; p > 0.05), Jiangning (0.120 ‰ to 0.133 ‰; p > 0.05), Jingjiang (0.112 ‰ to 0.119 ‰; p > 0.05) and Zhengjiang (0.120 ‰ to 0.126 ‰; p > 0.05) were the lowest and revealed no significance compared to the control group (0.093 ‰). Chen et al. (2002c) also tested the formation of micronuclei in Cyprinus carpio from the river sections at Anqing (3.56 ‰), Nanjing (4.25 ‰), Zhenjiang (4.50 ‰) and the Yangtze Estuary (3.65 ‰). A high frequency of micronuclei (control group 0.82 ‰) indicated that the Yangtze Rivers lower reaches possess high potential for adverse effects in the area. Micronucleus rates were also measured for Ctenopharyngodon idellus (3.55 ‰) and Leiocassis longirostris (4.68 ‰) from Nanjing and Zhenjiang, respectively, but no control was applied for those species. Chen et al. (2002c) pointed out that the observed formation of micronuclei might be associated with accumulated volatile phenol and petroleum hydrocarbons in the fish.

2.7.3 Discussion: Pollutant levels and adverse effects in aquatic organisms (in situ studies)

Diverging from the research on the pollutant levels and effects in vitro and in vivo of water and sediment, the assessment of pollutant levels and adverse effects in wildlife organisms primarily focused on the lower reaches of the Yangtze River: from Nanjing to the estuary and the adjacent coastal area. The Upper and Middle Reaches were hardly investigated in this context. Upon the studies on biota those quantifying organic pollutants in the organism were higher in number than those investigating effects. Taking into account that cities like Chongqing and Wuhan have a major impact on the surrounding area, and as the bioassay studies already indicated

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Chapter 2 Solution by dilution? ______ecotoxicological impacts in this area, further in situ studies are highly recommended. The reviewed articles chiefly focused on perfluorinated compounds (mainly PFOS) and PBDEs in fish (mainly carp and catfish), as well as organochlorine pollutants (mainly HCHs and DDTs) in mollusks and crabs. Yet there is only little knowledge available about the predominant priority pollutants that could be measured along the Yangtze River - like PCBs and PAHs - present in and their influence on aquatic organisms in this area. They have been demonstrated to potentially induce toxicopathic hepatic lesions in fish after long-term low exposure or short-term high exposure (Myers et al., 1998; Ortiz-Delgado et al., 2007). With respect to toxicological endpoints the only response measured were genotoxic effects manifested in the formation of micronuclei in carp and catfish from the Lower Reaches. A broader spectrum of endpoints - like histopathology, immunotoxicology, biochemical alterations and endocrine disruption - should also be taken into consideration along the whole river, especially in areas with elevated agricultural, industrial and domestic discharge levels. These information will help to further elucidate the reasons for the depletion of fishery resources and can be utilized to initiate counteractive measures (cf. (Boettcher et al., 2010; Grund et al., 2010)). As wild fish and other aquatic organisms still remain an important dietary source in China (Su et al., 2012), bioaccumulation of pollutants in the aquatic organisms has to be considered. The accumulation poses a risk to the organisms on the one hand and on further consumers on the other, as shown by Scholz-Starke et al. (2013). The sustenance with polluted food, e.g., fish, muscles and crabs, might lead to secondary intoxication. This can either result in acute toxicity or to effects like higher risk for cancerogenicity after chronic exposure to pollutant levels that exceed the acceptable daily intake (ADI). However, the reviewed articles revealed only a low risk of secondary intoxication with organic pollutants by consuming aquatic organisms from the Yangtze River. To gain a comprehensive view on the potential risk it would yet be necessary to extend the variety of investigated organic pollutants in biota for other important bioaccumulative toxicants, e.g., PCBs. They have been considered only in two studies, though they could be widely measured in water and sediment along the river. Moreover, besides organic components also heavy metals are known to accumulate in biota and therefore need to be integrated in an overall risk assessment. To summarize, prior to a risk of secondary intoxication for humans the detected organic contaminants pose a higher risk for adverse effects in the aquatic organisms themselves.

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2.8 Overall conclusion

Concerning the question if there is a solution of the pollution problem by dilution, we agree with the findings of Mueller et al. (2008) that the Yangtze River’s immense amounts of water and sediment indeed reduce the ecotoxicological risk growing from pollution all along the river, but do not eliminate it. Though the largest share of measured pollutant levels in water was below Chinese national standards (Ministry of Environmental Protection - China, 2002), ERL/ERM values (Long et al., 1995) suggested ecotoxicological impacts in certain areas, which could also be detected in vitro, in vivo and in situ. Hotspot pollution of highly populated areas, as economic centers like Wuhan, revealed an immediate local impact. Further, as it can be seen from the example of the Yangtze Estuary, the particulate-bound pollution carried away from upstream sources can manifest in the downstream course and influence distant areas. The intrusion of saltwater at the river’s mouth causes a reduction in flow rate, which elevates the sedimentation rate and leads to an accumulation of particle bound pollutants. In addition, the increase in salinity can cause a shift in the pollutants affinity from water to sediment, as shown for PFOS. The consequence is that hydrophobic organic pollutants are trapped in the estuary. With respect to the high persistence of the detected compounds like PAHs, PCBs and OCPs, and the enduring production of emerging pollutants it is to be expected that the Yangtze Estuary will continue to suffer the pollution discharge of the river and that the contamination in this area will still increase in the future. Prevention including both direct and diffuse pollution control should therefore be the first choice, remediation the second. Counter measures like the improvement of domestic and industrial wastewater treatment plants are recommended. The water level’s influence on the concentrations and effects of pollutants in the investigated rivers could be shown in various studies. Those were generally higher during low water period in the dry season in winter/spring than during the rainy season in summer. This observation has been attributed to different hydrologic factors: on the one hand to the difference in amounts of discharged water and particulate matter, causing a greater or lesser dilution, and the reduced velocity during low water level on the other hand. The lower flow rate causes an enrichment of pollutants in the water as well as higher sedimentation rates, which lead to the accumulation of particle bound pollutants in sediments. Due to the inverted water level situation in the TGR since impoundment, with high water level in winter and low water level in summer, it is to be expected that the changes in hydrologic conditions manifest as changes in the contamination status, with ______79

Chapter 2 Solution by dilution? ______greater pollutant and effect levels during rainy season than during dry season. Hydrophilic compounds may be carried with the discharged water past the dam, but hydrophobic substances will most likely remain in the sediments of the reservoir. It has also to be considered that the elevated precipitation in summer is associated with an increased amount of air-borne particles washed out from the air as well as a stronger runoff from fields, carrying pollutants into the TGR, which may also be trapped there and subsequently accumulate in this region. To verify this assumption it is recommended to continuously monitor these changes. Moreover it has to be considered that solely organic pollutants were in the scope of this review. Other impacts and risks on the Yangtze River’s biota and residents, e.g., growing from heavy metals or eutrophication due to high input of nitrates and phosphates from sewage and fertilizers as well as land use changes and the construction of dams that influence the river’s ecosystem, have to be kept in mind and included into an overall assessment. The main research reflected by the number of reviewed publications was performed between Chongqing and the Yangtze Rivers mouth at the East China Sea, with particular focus on the areas of Chongqing, Wuhan, Nanjing, Shanghai and the Yangtze Estuary. Whereas the reaches upstream Chongqing were only little investigated. In comparison the areas of main interest were also the main contaminated sites. This can be associated with an increasing population, industrialization and intensified agricultural usage from the Upper Reaches along the river’s downstream course to the Middle and Lower Reaches, with certain hotspots in highly populated and industrial areas. Furthermore, despite its increasing importance only a small amount of published studies were available on the Three Gorges Reservoir after impoundment. A striking observation was that many tributaries presented higher pollution levels and toxicity than the main stream. This was in particular the case at some economic centers such as Chongqing (Jialing River) and Wuhan (Han River). The comparably lower levels in the main stream can most likely be attributed to the vast amounts of water carried by it. The main stream incorporates also less polluted tributaries and thereby dilutes the input and toxicity from higher polluted sources. This demonstrates the need to also monitor and initiates counter measures at tributaries of the entire Yangtze River network, which are home to numerous people and habitat of a large variety of biota. The largest part of the reviewed studies focused on pollutant levels in water and sediment, especially on PAHs, PCBs, OCPs, PCDDs/DFs, PBDEs, and PFCs. PAHs were the dominant

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Chapter 2 Solution by dilution? ______pollutant class upon all contaminants detected in the river, predominantly sourcing from combustion of coal, wood and petroleum as well as from vehicle emission. The combustion of coal in addition to wastewater discharges seemed to be the main contributors to PAHs contamination in Chongqing section. The PAHs in sediments at Wuhan section were mainly caused by coal burning and petroleum combustion. Whereas PAHs found in Yangtze Estuary in the near-shore area mainly derived from petroleum and biomass (mainly coal) combustion, as well as vehicle emission; those detected in the farther shore zone originated mainly from petroleum combustion of shipping processes and shore side discharge. Chongming Island in Yangtze Estuary revealed PAHs from sewage as well as petroleum and coal combustion on the island itself. As an alluvial island, meaning formed by redeposited suspended matter, it is also influenced by upstream sources, because particle-bound PAHs carried by the water accumulate around the island. Furthermore, due to its special climate the south wind in summer carried PAHs in suspended particles from Shanghai City to Chongming Island. Environmental quality standards for total PAHs are not applicable due to the complexity of this substance class (EWFD Directive 2000/60/EC, 2000; European Union, 2008). However, BaP can be used as an indicator for PAH contamination (ICPR, 2009). The available studies revealed that BaP levels in water at Wuhan section, Jiangsu section exceeded the relevant standards given by the Ministry of Environmental Protection - China (2002 and also those of the European Water Framework Directive (EWFD Directive 2000/60/EC), an integrated river basin management to improve the quality of European water bodies. However, the concentrations of BaP in sediments along the River were all below the “relevant contamination” (> fourfold ICPR target value) set forth by the ICPR (2009), which is based on the EWFD (EWFD Directive 2000/60/EC, 2000; European Union, 2008). Based on the ERL and ERM guideline values (Long et al., 1995) maximum concentrations of total PAHs at Wuhan section and Yangtze Estuary rise concern for ecotoxicological impacts on aquatic life. Measured PCB levels along the Yangtze River were also below a “relevant contamination” according to the ICPR classification (ICPR, 2009). Yet the exceeded ERL guideline value (Long et al., 1995) indicates that the PCB levels in sediments at Wuhan section and the Yangtze Estuary may induce adverse effects. Attention should be paid to the release of PCBs as well as PBDEs into the environment from improper importation and disposal of electronic waste along the river.

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DDTs, HCHs and PCDDs/DFs residues in the Yangtze River have originated from the usage of pesticides in the past decades. DDT concentrations in all the sampling sections exceeded the ERL value (Long et al., 1995) suggesting a potential ecotoxicological risk in those areas. Attention should be paid to new input sources of DDTs and HCHs, e.g., by the usage of lindane and dicofol in some agricultural areas of the middle and lower Yangtze Reaches. Biological and chemical degradation can contribute to the reduction of organic pollutant levels in the environment in addition to dilution, and have to be considered as additional diminution factors for a broad variety of organic compounds. But most of the dominating substance classes that were reported by the reviewed articles shared the critical property persistence, which means that they are hardly degradable. Thus can degradation only play a minor role for decreased concentrations of these compounds. Against the background of an immensely fast developing economy and considering the critical properties persistence and toxicity of pollutants like PAHs and PFCs it is to be expected that the pollution levels will raise in the future. Besides, though most of the contaminants are represented only in low levels at the TGR, except partially high PFOA concentrations, the organic pollutant distribution in sediments along the river has been influenced by the construction of TGD (Li et al., 2012a; Wang et al., 2012b) , and require a long- term monitoring. Among measurable ecotoxicological endpoints especially mutagenicity and endocrine activity were of predominant interest and widely detectable along the river. The mutagenicity in several sections of the Yangtze River and its tributaries can most likely be attributed to the widely distributed PAHs and nitro-PAHs, predominantly originating from combustion sources. Counter measures to reduce the emission of PAHs into the environment are necessary, e.g., catalysts for cars, pollutant filter for factories and chiefly alternatives to fossil energy sources. In several studies an increase in mutagenicity of tap water after drinking water treatment was shown and was most likely induced by disinfection byproducts, like trihalomethanes, emerging from the chlorination of dissolved organic matter and other substances during the disinfection process. Thus it is highly recommended to substitute the chlorination of polluted source water by less harmful water treatment methods for the preparation of drinking water. Though some information, mainly on OCPs, PBDEs and PFCs in carp, catfish, mollusks and crabs were available, hardly any toxicological endpoints, except the formation of micronuclei in fish, were examined in situ. These studies, focusing on genotoxic effects, revealed that DNA

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Chapter 2 Solution by dilution? ______damages occurred in fish from the Yangtze River, proving environmentally relevant concentrations of pollutants in the water. Wild fish and other aquatic organisms are an important dietary source in China. Marine algae and plants are considered to be nutritionally rich and wild fish are thought to be beneficial to human health. Therefore relatively large quantities of them are still consumed in China (Su et al., 2012). This means that contaminated fish, crabs, muscles and other aquatic organisms as well as contaminated drinking water bear a risk to residents benefiting from the river as a source for sustenance with respect to acute and chronic intoxication. However, the available articles revealed only a low risk of secondary intoxication with organic pollutants by consuming aquatic organisms from the Yangtze River. Prior to a risk of secondary intoxication for humans the detected organic contaminants posed a higher risk for adverse effects in the aquatic organisms themselves. To estimate the entire risk growing from bioaccumulation to the organisms as well as the consumers it is recommended to intensify the research on especially hazardous and well known bioaccumulative contaminants, like the in water and sediment ubiquitously detected PCBs as well as heavy metals. Europe faced and still faces comparable issues as the Yangtze River basin. The Rhine, which is one of the economically, socially and environmentally most important rivers in Europe, and also an intensely examined and monitored water body, can serve as an example for future approaches concerning the Yangtze River. Though concentrations of organic pollutants may be lower in Yangtze River than in Rhine River, due to a comparably higher mass transport of water and particulate matter, it still results in comparably larger amounts of organic pollutants that end up in the Yangtze River’s receiving water body. The Yangtze River’s mean water discharge (30,200 m3/s) (National conditions - China, 2003) is about fourteen times greater compared to the Rhine River (2,200 m3/s) (Huisman et al., 2000). This implicates that at same contamination levels in both rivers the fourteen fold amount of toxic substances would still enter the East China Sea. Therefore, the impact of the Yangtze River on the East China Sea needs to be taken into account. Several approaches can help to preserve and improve the water quality of the Yangtze River: (1) The Yangtze should be seen in context of the River Continuum Concept (Vannote et al., 1980), an approach to describe and evaluate a river from its source to its estuary, considering it as an open and holistic ecosystem. It integrates biotic parameters, physical and hydrological factors, energy and nutrient input as well as output. This has also been applied for European river systems

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Chapter 2 Solution by dilution? ______in the European Union Water Framework Directive (EWFD Directive 2000/60/EC). (2) Chemical/ecotoxicological and hydraulic approaches should be combined, e.g., to estimate the risk growing from remobilization of pollutants during flood events (Woelz et al., 2009; Brinkmann et al., 2010). This appears to be even more important compared to the Rhine River due to higher mass transport of water and sediment in the Yangtze River. (3) Bioassay-directed analysis should be applied to identify relevant toxic components (Hecker and Hollert, 2009). (4) In situ investigations of mechanism-specific toxicity, like genotoxicity in blood cells from fish caught in the river, should serve as additional lines of evidence in Weight of Evidence studies, in order to prove the relevance of lab-based assays for the situation in the field (Chapman and Hollert, 2006; Boettcher et al., 2010). (5) Integrated assessments, like the triad approach (Chapman, 1990), should be deployed to evaluate the relevance of pollutants for organisms and to gain a comprehensive view on the pollution status. At least, each effect assessment studied in vitro, in vivo or in situ should be accompanied by chemical analysis to identify possible inducers. (6) Toxicity reduction evaluation should be applied to areas with elevated toxicity levels. Advanced methods of wastewater treatment and integrated strategies to minimize the impact of point and non-point sources should be further developed - e.g., by transfer of knowledge from national research programs and applications (Huckele and Track, 2013; Triebskorn et al., 2013) to bilateral and international joint projects in China (e.g. (Bergmann et al., 2012) and Clean Water Initiative, China).

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2.9 Acknowledgements

This review has been carried out as part of the MICROTOX project (“Transformation, Bioaccumulation and Toxicity of Organic Micropollutants in the Yangtze Three Gorges Reservoir”), which is integrated into the joint environmental research program “Yangtze-Hydro - Sustainable Management of the Newly Created Ecosystem at the Three Gorges Dam” (Bergmann et al. 2012, www.yangtzeproject.de). The project has been financed by the Federal Ministry of Education and Research, Germany (BMBF) as part of the research cluster “Pollutants/Water/Sediment - Impacts of Transformation and Transportation Processes on the Yangtze Water Quality”. The review was also supported by a personal grant to Hongxia Xiao by the scholarship program of the Chinese Scholarship Council. The authors want to express their gratitude for the great and helpful effort that was put into the manuscript by the two anonymous reviewers.

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Chapter 3 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China

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Parts of this chapter published in peer-reviewed journals as:

Floehr, T., Scholz-Starke, B., Xiao, H., Koch, J., Wu, L., Hou, J., Wolf, A., Bergmann, A., Bluhm, K., Yuan, X., Roß-Nickoll, M., Schäffer, A., Hollert, H. (2015): Yangtze Three Gorges Reservoir, China: A holistic assessment of organic pollution, mutagenic effects of sediments and genotoxic impacts on fish. Journal of Environmental Sciences 38: 63-82.

Floehr, T., Scholz-Starke, B., Xiao, H., Hercht, H., Wu, L., Hou, J., Schmidt-Posthaus, H., Segner H., Kammann, U., Yuan, X., Roß-Nickoll, M., Schäffer, A., Hollert H. (2015): Linking Ah receptor mediated effects of sediments and impacts on fish to key pollutants in the Yangtze Three Gorges Reservoir, China – A comprehensive perspective. Science of the Total Environment 538: 191-211.

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3.1 Abstract

The Three Gorges Reservoir (TGR), created in consequence of the Yangtze River’s impoundment by the Three Gorges Dam (TGD), faces numerous anthropogenic impacts that challenge its unique ecosystem. In order to record organic contamination, to find links to ecotoxicological impacts and to serve as reference for ensuing monitoring, several sites in the TGR area were screened applying the triad approach with additional lines-of-evidence as a holistic assessment method. Sediments and the benthic fish species Pelteobagrus vachellii were sampled in 2011 and 2012, respectively, to identify relevant endpoints. In the chapter, (a) sediment was analyzed for 54 relevant organic compounds based on the European Water Framework Directive, and (b) investigated in vitro assay with the Ames fluctuation assay for mutagenicity, as well as (c) the ethoxyresorufin-O-deethylase (EROD) induction assay for AhR-mediated activity. Further, (d) the sediment was investigated in vivo with the fish embryotoxicity test (extractable fraction) and (e) sediment contact assay (bioavailable fraction) with Danio rerio to both test for embryotoxicity/teratogenicity. In situ studies with Pelteobagrus vachellii comprised (f) the quantification of biliary pollutant metabolites and (g) micronucleus formation in erythrocytes to assess genotoxic impacts. In addition, activities of hepatic (h) phase I (EROD) and (i) phase II (glutathione-S-transferase) biotransformation enzymes were measured in situ to both determine AhR-mediated activities. Further, (j) histopathological alterations in liver and excretory kidney of Pelteobagrus vachellii were evaluated, inter alia to assess immunotoxic impacts. EROD induction was tested in vitro and in situ to evaluate possible relationships between the activity of sediments and effects determined in the fish. According to the study, only polycyclic aromatic hydrocarbons (PAHs) could be detected in sediments from 2011 (165 – 1,653 ng/g), emphasizing their role as key pollutants of the area. Their ubiquity was confirmed at Chongqing (150 – 433 ng/g) and Kaixian (127 – 590 ng/g) in 2013. Concentrations were comparable to other major Chinese and German rivers. However, the immense sediment influx suggests a deposition of 216 – 636 kg PAH/day (0.2 – 0.6mg PAH/m2/day), indicating an ecotoxicological risk. PAH source analysis highlighted the primary impact of combustion sources on the more industrialized upper TGR section, whereas petrogenic sources dominated the mid-low section. Sediment extracts from several sites exhibited significant impacts of frameshift promutagens in the Ames fluctuation assay. Additionality, the

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Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______sediments induced in the in vitro/in vivo bioassays AhR-mediated activities and embryotoxic/teratogenic effects – particularly on the cardiovascular system. These endpoints could be significantly correlated to each other and respective chemical data. However, particle- bound pollutants showed only low bioavailability. The in situ investigations suggested a rather poor condition of Pelteobagrus vachellii, with histopathological alterations in liver and excretory kidney. Significant genotoxic impairments in erythrocytes of Pelteobagrus vachellii were detected (Chongqing/Kaixian), demonstrating the relevance of genotoxicity as an important mode of action in the TGR’s fish. In addition, fish from Chongqing city exhibited significant hepatic EROD induction and obvious parasitic infestations. The PAH metabolite 1-hydroxypyrene was detected in bile of fish from all sites. By accounting all endpoints in combination with the chemical data, it is concluded that adverse effects might be induced to the fish on the long term, and thus with an influence on the overall fitness of the fish and other aquatic organisms in TGR. Two regional “hot-spots” near the cities of Chongqing and Kaixian were identified and need to be further investigated.

Key words: Three Gorges Reservoir, Mutugenicity, AhR-mediated Activity, Embryotoxity, PAHs

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3.2 Introduction

Contaminated sediments may exhibit severe hazardous effects. Toxic chemicals may accumulate in sediments and show many times higher concentrations than that in the free water column concentration (Hollert et al., 2002), which may cause an increase of (eco)toxicologically relevant effects through bioaccumulation to humans. In addition, sediments may functions as the final receptor of persistent pollutants and sinks for lipophilic pollutants. It becomes sources of pollutants through suspensions of particulate matter, e.g. as a consequence of flood events (Hollert et al., 2003; Gerbersdorf et al., 2005; Hilscherova et al., 2007). Thereafter, sediment contaminants with toxicity, e.g. carcinogenic, mutagenic, reproductive and endocrine disruptive potencies under low-dose and long term exposure are of great concern. A common way to monitor the pollution status of a water body is to determine the chemical concentrations in water and sediment with chemical-analytical methods. This approach however is insufficient. Those way only selected compounds are in the scope of the quality assessment, and an even larger variety of toxic non-target parent compounds and metabolites is not considered, their synergistic and antagonistic effects, as well as the metabolism in the organisms are not taken into account (Altenburger et al., 2015). Thus, in order to know the complex situation in the field, a holistic assessment in environmental monitoring is demanded, that considers not only the concentrations of selected compounds, but integrates them with the toxic effects the environmental sample may induce (Heugens et al., 2001; SedNet, 2004; Brils, 2008; Hollert et al., 2007; 2009; Malaj et al., 2014; Wernersson et al., 2015). The Yangtze River’s fish fauna is described as one of the richest worldwide, providing plentiful fish resources, but they showed a serious decline with a significantly reduced fishery yield in the past years (Fu et al., 2003; Chen et al., 2009). Further, the species composition shifted in its quantitative relationships and decreased in diversity (Chen et al., 2002a; 2002b; 2009). The impoundment of the Three Gorges Reservoir (TGR) further threatened endemic and rare fish species, particularly those adaptable to the prior running water conditions, with demersal fishes like the darkbarbel catfish (Pelteobagrus vachellii) becoming more important. However, only few quantitative studies on Yangtze fishes area available. A number of qualitative research demonstrates that aside habitat fragmentation and shrinkage, invasion of exotic species and resources overexploitation, water pollution was ranked among the responsible factors for the registered depletion and structural changes of the fish resources (Chen et al., 2009).

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Moreover, sediments that are contaminated with mutagenic substances pose a hazard to indigenous biota (Chen and White, 2004). It has been shown that fish populations in rivers and lakes in industrial areas in the US and Europe displayed an elevated prevalence for tumours (Balch et al., 1995). The endpoint mutagenicity was already for particular interest in several studies of the Yangtze River (as reviewed in Chapter 2). As a possible result of genotoxic effects on parental cells, the formation of micronuclei can be observed in the daughter cells after cell division (Leme and Marin-Morales, 2009). For convenient application, blood has been proven to be a sensitive compartment for genotoxicity measurement in fish (Kilemade et al., 2004; Rocha, 2009; Boettcher et al., 2010). Due to their impact on the genetic material of the organism, the mutagenic and genotoxic effects on cellular level can have not only impairments on the organism itself, but also multigenerational impacts, thus effecting the population and ecosystem level (White et al., 1999; Diekmann et al., 2004). Organic pollutants, particularly aryl hydrocarbon receptor (AhR) agonists, appeared to play a crucial role in the TGR (Wang et al., 2009; Wolf et al., 2013b; a; Deyerling et al., 2014; Floehr et al., 2015a). Such compounds are known to induce cytochrome P450 1A (CYP1A) by ligand- activation of the aryl hydrocarbon receptor (AhR) (Van den Berg et al., 2006; Sorg, 2014). It has been reported that the binding of xenobiotics to the AhR can trigger a broad spectrum of biochemistry, physiology and reproduction in many organisms (Grund, 2011; Sorg, 2014). As fish are in particular sensitive state at early life stages, the in vivo Fish Embryo Toxicity Test (FET) (Braunbeck et al., 2005) and Sediment Contact Assay (SCA) with eggs of Danio rerio (Hollert et al., 2003) have proved to be suitable methods in ecotoxicological assessment. In particular, as the FET can serve as a substitute for the acute toxicity test with adult fish (OECD guideline 203, 1992) registering mortality, it also allows to record sublethal impacts that might affect the overall fitness of the fish (Nagel, 2002; Braunbeck et al., 2005; Lammer et al., 2009). The main objective of the present study was to evaluate the ecotoxicological status of the TGR, with focus on evaluate the ecotoxicological relevance of potentially contaminated sediment from several sites of the TGR in order to support further management strategies of the TGR and other Chinese water bodies. To address potential environmental risk in the sediment of TGR, the specific aim to this study were (a) chemical analyzed organic compounds selected on basis of the European Water Framework Directive (EWFD Directive 2000/60/EC, 2000) and (b) tested in vitro with the Ames fluctuation assay for mutagenicity, the ethoxyresorufin-O-deethylase (EROD) induction assay with RTL-W1 cells for AhR-mediated activities, and (c) in vivo with the FET.

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Further, the bioavailable fraction of particle bound pollutants was tested with native (freeze-dried) sediments in vivo with the SCA. Both in vivo assays were performed with eggs of Danio rerio to screen for embryotoxic and teratogenic impacts for embryo toxicity.To complement these studies, hepatic (d) EROD and (e) GST activities of the darkbarbel catfish (Pelteobagrus vachellii, Richardson 1846) were investigated. Further, (f) bile of the fish was tested to identify a potential exposure to PAHs, and (g) histopathological alterations in liver and kidney were recorded in parallel. By accounting all the enpoints and chemical burden, a holistic perspective of the ecotoxicological risk in TGR was summarized.

3.3 Material and methods

3.3.1 Sampling campaign

Sampling campaign May 2013 - Sediments

In a first sampling campaign in September 2011 in TGR area, at a water level of 160 - 166 m altitude above sea level (a.s.l.), the surface layers (~5 cm) of sediments were collected at four major tributaries using a Van-Veen sampler – Jialing River at Chongqing (CNG), Long River at Fengdu (FEN), Pengxi River/Xiao River at Yunyang (YUN), Daning River at Wushan (WU)- along the Yangtze River mainstream of the TGR, as well as the artificial Hanfeng Lake at Kaixian (HF-L) and the Baijiaxi River in the Pengxi River Wetland Nature Reserve (BJX-R) in the TGR watershed (Fig. 3.1). The Pengxi River Wetland Nature Reserve was selected as reference site, assuming minimum pollution in the poorly industrialized area. At each site along the mainstream three locations were sampled - upstream (U) and downstream (D) the tributary’s inlet, as well as in the tributaries (T). At the artificial lake site and the reference site only one location was sampled. The samples from each location were pooled samples. Each pooled sample consisted of sediment from at least three individual sampling spots at this location, which were combined to reach a total volume of 500 mL. The individual samples were taken in a radius of 50 m from the first sampling spot to cover intrinsic heterogeneity of each sampling location. The pooled samples were stored in inert PTFE-bottles at 4°C for transportation. The samples were freeze dried, sieved with a 2 mm sieve to remove sticks and stones, and kept in amber glass bottles at 4°C for further analysis.

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Fig. 3.1 Overview map of sampling locations in the Three Gorges Reservoir area. The Three Gorges Reservoir covers the Yangtze River section between Jiangjin district (Chongqing) and the Three Gorges Dam. Names in grey depict geographical markers: water bodies (italicized), cities and the dam. Fish were sampled in the TGR close to the cities Chongqing (CNG), Fengdu (FEN), Yunyang (YUN) and Wushan (WU), as well as in the TGR watershed in the Hanfeng Lake (HF) and Baijiaxi River (BJX). Labels in black signify sediment sampling locations from campaign September 2011 (with asterisks; CNG-U,-T,-D; FEN-U,-T,-D; YUN-U,-T,-D; WU-U,-T,-D; HF-L; BJX-R) and campaign May 2013 (without asterisks; YAN-A,-B,-C; JIA-A,-B,-C; TGR-A,-B; HAN-A,-B,-C,-D); U = upstream, T = tributary, D = downstream, L = lake, R = reference; A,B,C,D = order in flow direction.

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Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______

Sampling campaign May 2012 – Fish

In a second sampling campaign in May 2012, at a water level of 160-164 m altitude a.s.l., fish samples were taken at the sampling sites Chongqing (CNG 2012), Fengdu (FEN 2012), Yunyang (YUN 2012) and Wushan (WU 2012) in the TGR, as well as the Hanfeng Lake (HF 2012) and Baijiaxi River (BJX 2012) in the TGR watershed (Fig. 3.1). The Baijiaxi River in the Pengxi River Wetland Nature Reserve was again selected as reference site. The darkbarbel catfish (Pelteobagrus vachellii, Richardson 1846) was chosen as monitoring fish species due to its demersal lifestyle and low to medium migration, thus being suitable to indicate local contamination over longer periods of time, as well as its distribution all along the TGR and other sections of the Yangtze River. Furthermore, it frequently occurred in catches from all sampled sites and has a major economic importance (Ministry of Environmental Protection - China, 2007). At each site 10 fish of comparable size (mean ± SD: total length 165 ± 15 mm; standard length 142 ± 14 mm; weight of 40 ± 11 g; indifferent sex) (Table 3.1) were sampled with help from local fishermen. The fish were kept in water of the local water body in an aerated tank till each fish was euthanized with clove oil, followed by an additional blow to the head. Blood samples were obtained from a caudal section with a heparinized syringe and transferred to two microscope slides per fish according to the methods given by Rocha et al. (2009) and Boettcher et al. (2010). Each blood smear was fixed in methanol for 5 min. Bile samples were obtained from the gall bladder with a syringe, transferred to 2 mL cryovials and stored at 4°C. Excised liver samples were divided, with one half immediately transferred to a 2 mL cryovial and stored in liquid nitrogen at -80°C. The other half was fixed in 10% buffered formalin and stored at room temperature.

Sample campaign May 2013 - Sediment & fish

In a third sampling campaign in May 2013, at a water level of 160 m altitude a.s.l., sediment and fish samples were taken in parallel at two identified regional hot-spots – near the cities of Chongqing (CNG 2013) and Kaixian (HF 2013). To ensure the same biological status of the fish according to their seasonal rhythm, in order to prevent major physiological oscillations that could influence the biomarkers, and because May and September have shown comparable temperature, precipitation and TGR water level, May was chosen for the combined sampling of sediment and fish. For sediment, at Chongqing three samples were taken in the Jialing River (JIA), three samples in the Yangtze River (YAN) upstream of the conversion zone of both rivers, and two

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Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______samples downstream of the conversion zone in the reservoir (TGR). Samples were labelled in order of flow direction (A, B, C) (Fig. 3.1). In addition four samples were taken in the Hanfeng artificial lake (HF) at Kaixian. Samples were labelled in order of flow direction (A, B, C, D) (Fig. 3.1). For fish, 20 individuals of comparable size (mean ± SD: total length 189 ± 23 mm; standard length 163 ± 21 mm; weight 61 ± 21 g; indifferent sex) (Table 3.1) were sampled at both sites (CNG; HF) (Fig. 3.1). Sampling procedures for sediment and fish, as well as further treatment were as described before.

Table 3.1 Main morphological parameters of Pelteobagrus vachellii from sampling campaign May 2012 and 2013. The Fulton condition factor (k-value) was calculated according to Barnham and Baxter (1998) based on Fulton (1902) and Ricker (1975) with total length (tot) and standard length (std); Values are stated as means with standard deviation; 2012 samples n=10; 2013 samples n=20.

Sites Total length Standard length Weight k-value k-value [mm] [mm] [g] (tot) (std) CNG 2012 154 ± 17 132 ± 15 37 ± 12 1.01 ± 0.08 1.59 ± 0.13 FEN 2012 172 ± 12 148 ± 12 39 ± 9 0.75 ± 0.08 1.20 ± 0.11 YUN 2012 174 ± 7 149 ± 6 42 ± 6 0.81 ± 0.05 1.27 ± 0.09 WU 2012 156 ± 11 134 ± 8 34 ± 7 0.90 ± 0.13 1.41 ± 0.17 HF 2012 167 ± 13 145 ± 11 45 ± 12 0.95 ± 0.09 1.46 ± 0.14 BJX 2012 166 ± 19 144 ± 20 45 ± 15 0.96 ± 0.12 1.48 ± 0.15 CNG 2013 177 ± 15 152 ± 13 54 ± 15 0.96 ± 0.12 1.54 ± 0.23 HF 2013 201 ± 24 175 ± 21 69 ± 24 0.83 ± 0.10 1.27 ± 0.18 Mean 2012 165 ± 15 142 ± 14 40 ± 11 0.90 ± 0.13 1.40 ± 0.18 Mean 2013 189 ± 23 163 ± 21 61 ± 21 0.90 ± 0.13 1.40 ± 0.25

3.3.2 Chemicals and materials

Acetone (≥99.5%, p.a.), n-hexane (≥99%, p.a.), dichloromethane (≥99.8%, p.a.), isooctane (≥99%, p.a.), cyclohexane (≥99.5%, p.a.), hydrochloric acid (37%, ACS reagent), ethanol (≥99.8%), 3,4-dichloroaniline (DCA), 7-ethoxresorufin, fluorescamine, reduced ß-nicotinamide adenine dinucleotide phosphate (NADPH), reduced L-glutathione, 1-chloro-2,4-dinitrobenzene (CDNB) and acridine were purchased from Sigma Aldrich GmbH (Deisenhofen, Germany) and methanol (99%) from Sigma Aldrich Inc. (Shanghai, China). 2,3,7,8-tetrachlorodibenzo-p-dioxin was purchased from LGC Promochem GmbH (Wesel, Germany). Dimethylsulfoxide (DMSO), aluminium oxide (for column chromatography) and sodium sulfate (≥99%, p.a.) were supplied by Carl Roth GmbH & Co. KG (Karlsruhe, Germany), silica gel 60 (size: 0.063-0.200 mm) and

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Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______acetonitrile (gradient grade, p.a.) by Merck group (Darmstadt, Germany), S9 fraction (from phenobarbital/ß-naphthoflavon treated rats) by Harlan Cytotest Research GmbH (Rossdorf, Germany), heparine from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China), cleaned silica sand by Büchi Labortechnik AG (Flawil, Switzerland) and Quartz sand (grain size W4) by Quarzwerke GmbH (Frechen, Germany). Amber glass bottles and vials, PTFE bottles, syringes, cryovials and microscope slides were purchased from VWR International GmbH (Darmstadt, Germany), 24-, 96- and 384-well microplates from TPP Techno Plastic Products AG (Trasadingen, Switzerland) and gas permeable foil from Renner GmbH (Dannstadt, Germany).

3.3.3 Sediment extraction

Each freeze dried sediment sample was extracted with acetone-hexane (1:1) (p.a.) in a pressurized liquid extractor (Speed Extractor E-916, Büchi Labortechnik AG, Flawil, Switzerland) at 100°C and 120 bar in two cycles (heat up 1 min; hold 10 min; discharge 2 min; flush with solvent 1 min; flush with gas 4 min). For process controls, extraction tubes were filled with cleaned silica sand and further processed in parallel. After the reduction in volume with a rotary evaporator and under a gentle nitrogen stream, the extracts were redissolved in dimethylsulfoxide (DMSO) to a final concentration of 20 g sediment equivalents (SEQ) per mL DMSO and stored at 4°C in amber glass vials for further testing in biotest. The whole process was performed under protection from light to prevent photodegradation of toxic compounds. In the following, sediment concentrations in gram SEQ per milliliter solvent/medium will be given as gram per milliliter (g/mL).

3.3.4 Triad A: Chemical analysis

Every extract has been screened for 54 organic compounds, including 17 pesticides, 16 priority polycyclic aromatic hydrocarbons (PAH), 8 polybrominateddiphenyl ether (PBDE), 6 polychlorinated biphenyls (PCB) and 8 other relevant intermediate and by-products of chemical production (Table 3.2). Per sample 20 g freeze dried sediment has been extracted as described before, with process controls in parallel. After reduction in volume the samples have been cleaned up chromatographically on columns filled with 2 g silica gel and 2 g aluminium oxide using 15 mL n-hexane, 5 mL n-hexane: dichloromethane (9:1) and 20 mL n-hexane: dichloromethane (4:1) as elute. After adjustment to 200 µl each sample has been provided for gas chromatography–mass spectrometry (GC-MS) analysis using an Agilent GC-MS system (6890

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Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______

GC and 5973 mass selective detector (MSD) single quadrupole mass analyzer) equipped with an automatic sampler (MPS 2) and Cooled Injection System (CIS 4, Gerstel).

Table 3.2 Target compounds in sediment extracts of campaign September 2011 (a) and May 2013 (b). Pesticidesa Chlorpyrifos, Chlorfenvinphos, Aldrin, Isodrin, Dieldrin, Endrin, alpha-Endosulfan, beta-Endosulfan, p,p’-DDE, p,p’-TDE, o,p’-DDT, p,p’-DDT, Trifluralin, Simazine, Atrazine, Lindane (Gamma-HCH), Alachlor, Hexachlorobenzene PAHsa,b,c Naphthalene, Acenaphthylene, Acenaphthene, Fluorene, Phenanthrene, Anthracene, Fluoranthene, Pyrene, Benzo[a]anthracene, Chrysene, Benzo[b]fluoranthene, Benzo[k]fluoranthene, Benzo[a]pyrene, Indeno[1,2,3-c,d]pyrene, Dibenzo[a,h]anthracene, Benzo[g,h,i]perylene PBDEsa BDE 28, BDE 47, BDE 99, BDE 100, BDE 153, BDE 154 PCBsa PCB 28, PCB 52, PCB 101, PCB 138, PCB 153, PCB 180 Other intermediate and 1,2,3-Trichlorobenzene, 1,2,4-Trichlorbenzene, 1,3,5-Trichlorbenzene, Alpha-HCH, by-productsa Beta-HCH, Delta-HCH, Hexachlorobutadiene, Pentachlorobenzene c priority PAHs (Environmental Protection Agency & Office of the Federal Registration - USA, 1982)

3.3.5 Triad B: In vitro/In vivo bioassays

Ames fluctuation assay with Salmonella typhimurium

The Ames fluctuation assay is a modified version of the traditional Ames test (Ames et al., 1975; McCann et al., 1975). While the traditional test is performed using agar plates (plate incorporation technique), the Ames fluctuation assay uses a liquid medium to expose and incubate bacteria in 24- and 384-well microplates, respectively. The Ames fluctuation assay including cytotoxicity determination was performed according to the method described by the (ISO 11350 2012) as detailed by Reifferscheid et al. (2012) and Higley et al. (2012). Each sample was tested in three individual replicates. Two tester strains were used to examine the samples’ mutagenic potential. Both strains are auxotrophic mutants of the bacterium Salmonella typhimurium. While TA98 carries a frameshift mutation (hisD3052), TA100 carries a base pair substitution (hisG46). Both mutations disable the bacteria from growing in histidine-free medium unless they are reverted to a prototrophic state by back mutation (Ames et al., 1975). To determine metabolic activation of promutagenic substances, each sample was tested additionally with a liver homogenate S9-fraction from phenobarbital/ß-naphthoflavon treated rats, further supplemented for application in the assay (S9 supplement). Tests were valid when mean values of revertants were > 0 and ≤ 10 per 48 wells in negative controls and ≥ 25 per 48 wells in positive controls. If data showed normal distribution and ______98

Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______variance homogeneity William’s multiple sequential t-test (α=0.05) was used to determine No Observed Effect Concentration (NOEC) values. The results of the above tests allow for the use of the parametric William’s test without further transformation. If data showed normal distribution but variances were heterogeneous, Welch-t test with Bonferroni-Holm adjustment was performed. In the following, test concentrations in milligram SEQ per milliliter solvent will be given as milligram per milliliter (mg/mL).

EROD induction assay with RTL-W1 cells

To determine the dioxin-like activity of the sediment extracts the EROD induction assay was conducted according to Woelz et al. (2008) based on the procedure described by Behrens et al. (1998). In order to avoid cytotoxic effects in the EROD assay, the neutral red retention assay (NR) was performed beforehand to assess the initial concentrations of the sediment extracts set by the

NR80 value (80% viability). The NR assay was based on the method described by Babich and Borenfreund (1992). Both assays were performed with the CYP1A expressing fibroblast-like permanent cell line RTL-W1 from primary hepatocytes of rainbow trout (Oncorhynchus mykiss) (Lee et al., 1993). The cells-kindly provided by Drs. Niels Bols and Lucy Lee from the University of Waterloo, Canada were cultured according to the procedure described by Klee et al. (2004). Samples were tested in three independent replicates, each with three internal replicates serially diluted with medium in seven 1:2 steps. As positive control 2,3,7,8-tetrachlorodibenzo-p- dioxin was used in a test concentration range from 3.13 to 100 pM. To avoid cytotoxicity, the maximum DMSO concentration per well was restricted to 1%. The protein amount was measured fluorometrically applying the fluorescamine method by Kennedy and Jones (1994) at excitation 360 nm and emission 465 nm. The enzyme activity was computed based on the quantity of produced resorufin per total amount protein and reaction time. The concluding concentration response curves for EROD induction were calculated by nonlinear regression (Prism 5.0, GraphPad Software Inc., San Diego, USA) applying the classic sigmoid curve or Boltzmann curve as model equations.

To provide comparability, EC25 values of the EROD induction of each sample replicate (EC25 sample) were related to the EC25 of the maximum response in the corresponding TCDD standard curve (EC25 TCDD) to compute the bioassay-derived toxic equivalents (bio-TEQs) (Equation 3.1), according to the fixed effect level quantification method (Engwall et al., 1996).

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Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______

푝푔 푇퐶퐷퐷 푝푔 푇퐶퐷퐷 퐸퐶25 푇퐶퐷퐷 [ ] 퐵𝑖표 − 푇퐸푄 [ ] = 푚푙 (Equation 3.1) 푔 푆퐸푄 푔 푆퐸푄 퐸퐶25 푠푎푚푝푙푒 [ ] 푚푙 The EC25 was used for means of calculation, because the EC50 was not well defined by the dose- response curves of several samples. Hence, the EC25 was considered more reliable, as suggested by Engwall et al. (1996). In the following, bio-TEQ values in picogram TCDD per gram SEQ will be given as picogram per gram (pg/g). Given are mean values, with standard deviation (± SD) if applicable. Moreover, the measured BEQs in fractions were compared to the 2,3,7,8-TCDD equivalents (TEQs), which were obtained by multiplying the concentration of each compound in each sample by its relative potency (REP) in RTL-W1 cells, and summing up these values (Equation 3.2). This comparison enables mass-balance estimation of the contribution of the analyzed chemical contaminants to the measured AhR-mediated activity. 푛 푝푔 푃퐴퐻 푝푔 퐶ℎ푒푚 − 푇퐸푄 [ 훴16] = ∑ 푐표푛푐. 푃퐴퐻 [ ] 푥 푅퐸푃 푔 푆퐸푄 푖 푔 푆퐸푄 푖 (Equation3.2) 푖=1

Fish Embryo Toxicity Test with Danio rerio

Maintenance of Danio rerio was conducted according to Braunbeck et al. (2005) with modifications previously described by Peddinghaus et al. (2012). The Fish Embryo Toxicity Test (FET) was performed according to the German Standard guideline DIN 38415-6 (2009) and the corresponding draft OECD guideline (OECD draft, 2006) with Danio rerio over a prolonged testing period of 96 h. The aim was to determine the embryotoxic and teratogenic potential of the sediment extracts. The FET was started no later than 3 h past fertilization (128-cell stage). Fertilized freshly spawned eggs were identified with a stereo microscope (SMZ 1500; Nikon GmbH, Düsseldorf, Germany), at a minimum magnification of 25×, on basis of the following criteria: the chorion surrounded the perivitelline space, which contained the yolk, and the blastodisc was located at the animal pole of the yolk. Exclusively normally developed eggs at minimum 8-cell stage were chosen for application in the FET. Till further testing eggs were transferred into artificial water (ISO7346-3, 1996) at 26 ± 1°C to guarantee homogenous water quality and ion concentration in each test series. Sediment extracts were diluted with DMSO in five steps and added to the artificial water. Thereby, the highest tested concentration for all samples was 100 mg SEQ/mL and all dilution steps were adjusted to a DMSO concentration of 0.5%. Preliminary experiments with DMSO and artificial water displayed no differences in the results. Thus, artificial water in

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Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______the absence of the extracts was applied as negative control (validity criteria: mortality < 10%). As solvent control, DMSO was tested in a concentration of 0.5% in artificial water. Further, 3.7 mg/L DCA dissolved in ultrapure water (ZFMQ 23004, Millipore, Molsheim, France) in absence of the extracts was used as standard positive control (validity criteria: mortality > 10%). Subsequently, eggs were transferred to 24-well plates with 2 mL test solution and controls per well. Lethal and sublethal effects were evaluated with an inverted microscope (Eclipse TS 100, Nikon GmbH, Düsseldorf, Germany) at magnifications of 40× and 100×. The following criteria were assessed as lethal endpoints according to DIN 38415-6 (2009): (a) coagulation of the embryo, (b) no heartbeat (c) non-detachment of the tail. In addition to that, sublethal endpoints were: (d) weak heartbeat, (e) weak or (f) no blood circulation, (g) edema, an (h) underdeveloped or (i) malformed embryo, (j) missing eye primordia, (k) weak or (l) no pigmentation of the body, (m) missing eye pigmentation, (n) curved or (o) deformed spine and (p) malformed or (q) missing fins. Each test series was scored valid when the negative control did not exceed 10% mortality and the positive control showed more than 10% mortality. Each extract from sampling campaign was tested in three independent replicates with ten eggs per test concentration. Results were analyzed according to lethality and overall effects (lethal plus sublethal). The concluding concentration response curves were plotted by nonlinear regression (Prism 5.0, GraphPad Software Inc., San Diego, USA) applying the classic sigmoid curve or Boltzmann curve as model equations to calculate LC50 and EC50 values with 95% confidence intervals (CI). In the following,

LC50 and EC50 values in milligram SEQ per milliliter water will be given as milligram per milliliter (mg/mL).

Sediment Contact Assay with Danio rerio

The Sediment Contact Assay (SCA) was performed on basis of the German standard guideline DIN 38415-6 (2009), adapted for sediment testing as described by Hollert et al. (2003) and detailed by Zielke et al. (2011). The aim was to examine the embryotoxic and teratogenic potential of bioavailable fractions of particle-bound pollutants in native (freeze-dried) sediment cf. (Kosmehl et al., 2007). Initially, samples were prescreened at the highest sediment concentration (100%) to identify possible inducing samples. Each sample was tested in three independent replicates, each with three parallel replicates, with five fish eggs per parallel replicate. Controls were tested in four parallel replicates with each five fish eggs. Lethal and sublethal criteria were evaluated according to the FET.

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Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______

Freeze-dried sediment from campaign May 2013 was not investigated in detail in the SCA as it was decided that - based on prescreening of the corresponding sediment extracts in the FET and with regard to results from other bioassays on the same samples - no significant effects could be expected. Thus, to avoid unnecessary usage of fish embryos the samples were not investigated any further.

3.3.6 Triad C: In situ biomarkers

Fulton condition factor

The Fulton condition factor (k-value) can be consulted to judge the long-term condition of fish. It was calculated according to Barnham and Baxter (1998) based on Fulton (1902) and Ricker (1975) (Equation 3.3). The weight (W) was given in gram and the standard/total length (L) was given in mm. Given are mean values, with standard deviation ( ± SD) if applicable. 105W k = (Equation 3.3) L3 Micronucleus assay with erythrocytes

The micronucleus assay was performed with minor modifications according to the methods given in Rocha et al. (2009) and Boettcher et al. (2010). To determine micronucleus formation rate acridine orange was used for staining of the fixated blood smears on the slides. Per fish 4000 erythrocytes (2000 per slide) were examined using an epifluorescence microscope (BIOREVO BZ-9000E, Keyence, Neu-Isenburg, Germany) equipped with an oil-immersion lens at a 600× magnification. The cells were evaluated according to the scoring criteria of the ISO guideline 21427-2 (2006): (a) only erythrocytes with intact cell membrane were scored. The micronuclei should have (b) a maximum size of about 30% of and (c) the same staining intensity as the main nucleus, as well as (d) a clear separation from it. Further, induction factors (IF) were computed, which give the induction of the mean micronucleus frequency of each sample referred to the reference site.

Hepatic biotransformation enzyme activities of Pelteobagrus vachellii

Determination of EROD activity: Homogenization and centrifugation of excised liver samples was performed according to Bonacci et al. (2003) to prepare enzymatic active liver fraction (S9) and the activity of 7-ethoxy-resorufin-O-deethylase was measured as described by the fluorometric method of Maria et al. (2005) as described by Kammann et al. (2014). All samples

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Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______were kept on ice during the complete procedure till measurement. Each sample was measured in duplicate. The specific activity was expressed as pmol resorufin produced per mg total protein and minute. Further, induction factors (IF) were calculated, which give the induction of the mean EROD activity of each sample referred to the reference (BJX 2012). Determination of GST activity: Homogenization and centrifugation of excised liver samples was performed as described above. The activity of glutathione-S-transferase (GST) was measured according to Habig et al. (1974) as described by Kammann et al. (2014). Each sample was measured in duplicate. The specific enzyme activity was expressed as nmol conjugated 1-chloro-2,4-dinitrobenzene per mg total protein and minute. Further, induction factors (IF) were calculated, which give the induction of the mean GST activity of each sample referred to the reference. Determination of protein concentrations: Protein concentrations of the S9 fractions were determined photometrically with a bicinchoninic acid protein assay kit (Sigma Aldrich GmbH, Deisenhofen, Germany). The assay is based on the Lowry procedure. Histopathological evaluation: Fixed samples of excised liver and kidney with surrounding tissue of Pelteobagrus vachellii from sampling campaign May 2012 were paraffin-embedded and routinely processed for histological examination, sections of 4 μm thickness were cut. Sections were stained with haematoxylin-eosin (H&E) and examined by light microscopy. Histopathological changes of liver and kidney were graded as 0 (no), 1 (scattered), 2 (mild), 3 (mild to moderate), 4 (moderate), 5 (moderate to severe) or 6 (severe). Analysis of PAH metabolites in bile: Fish bile of Pelteobagrus vachellii from campaign May 2012 was analyzed for PAH metabolites 1-hydroxypyrene (1-OHPyr) and 1-hydroxyphenanthrene (1-OHPhe) according to Kammann (2007). Due to low bile volumes available samples had to be combined into 3 pooled samples per site to reach the minimum required volume (25 µL), with exception of WU where bile was only sufficient for 2 pooled samples. Available bile samples were n=7 at WU, n=9 at FEN and the reference site BJX, and n=10 at CNG, YUN and HF. Absorption of bile at 380 nm was applied to normalize the 1-hydroxypyrene level. Fish of campaign 2013 did not supply sufficient bile volumes, thus required minimum amounts for analysis were not achieved.

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Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______

3.3.7 Statistical analysis

Statistical analysis were generally conducted with SigmaPlot 12.5 (Systat Software Inc., San Jose, USA), with exception of mutagenicity determination by the rate of revertants (see below), which was computed with ToxRat 2.10 (ToxRat Solutions GmbH, Alsdorf, Germany). ANOVA on ranks (multiple comparison) followed by Dunn’s post hoc test (α=0.05) was used to determine significant differences in the in vitro EROD induction by the sediment extracts, and the graded histopathological changes of liver and kidney, in comparison to the reference (BJX-R and BJX 2012, respectively). The same was applied for pairwise comparison of the Fulton condition factors for fish from all sites. Mann-Whitney Rank Sum Test (α=0.05) was conducted to compute significant differences in hepatic EROD and GST activities, as well as micronucleus formation of the samples in comparison to the reference (BJX 2012). Deviations of the sample values from control are classified as significant with a p-value smaller than a significance level of 5 % (* Fig.3.3 and Fig.3.5 ) or 1% (**) and as highly significant with p-value smaller than a significance level of 0.1 % (*** Fig.3.5 ).

3.4 Results and discussion

3.4.1 Chemical analysis

PAH pollution

In the extracts of sediment samples, among all 54 analyzed compounds (Table 3.2) only the 16 priority PAHS could be quantified above a LOD of 10 ng/g. Concentrations below the LOD have been considered as not-detected. The total PAH (PAHΣ16) concentrations ranged from 165 ng/g (BJX River/reference site) to 1,653 ng/g (CNG-U) (Table 3.3). In the sampling campaign during September 2011, with less than 600 ng/g, the samples FENT-T at Long River (310 ng/g ), CNG-T at Jialing River (450 ng/g SEQ) and WU-T at Daning River (552 ng/g) possessed a lower levels. Concentrations more than 1,000 ng/g were the samples CNG-U (1,653 ng/g), YUN-D (1,563 ng/g), WU-U (1,198 ng/g), FEN-U(1,122 ng/g) and YUN-U (1,121 ng/g), which presented a higher concentration. The spectrum ranged from 750 ng/g (FEN-D) to 1,653 ng/g (CNG-U) at the mainstream of the Yangtze River and 310 ng/g (FEN-T) to 824 ng/g (YUN-T) in the tributaries (Table 3.3).

Each upstream sample of the four sites along the mainstream displayed the highest PAHΣ16 content – with exception of Yunyang – and the tributaries the lowest, giving the dominating

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Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______pattern for PAHΣ16 content per site: upstream > downstream > tributary. Inter-site comparison along the mainstream, integrating each three samples per site, showed the following order for

PAHΣ16 content: Yunyang (mean: 1,169 ± 37 ng/g) > Chongqing (mean: 973 ± 617 ng/g) >Wushan (mean: 876 ± 323 ng/g) > Fengdu (mean: 727 ± 406 ng/g) (Table 3.3).

Further, it could be observed that the PAHΣ16 content from the downstream location of one site typically increased to the upstream location of the next site in flow direction – with exception of YUN-D/WU-U (Table 3.3), which could hint to an accumulation of PAH contamination between the tributaries, due to lesser dilution.

In sampling campaign May 2013, PAHΣ16 concentrations ranged from 150 ng/g (TGR-B) to 433 ng/g (YAN-C) at Chongqing, and 127 ng/g (HAN-B) to 590 ng/g (HAN-C) at Kaixian

(Table 3.4). In general, a decline in the PAHΣ16 concentration could be measured in the sediments along the flow direction with exception of YAN-C. The concluding pattern was YAN-C > YAN-A > YAN-B > TGR-A > TGR-B for the Yangtze River, and JIA-A > JIA-B > JIA-C > TGR-A for the Jialing River.

In comparsion, the Yangtze River displayed a higher PAHΣ16 content than the Jialing River and the TGR (Table 3.3 and Table 3.4). These values correspond to a study by Tang et al. (2011), who found comparable PAH Σ16 in sediments of the Yangtze River (257 – 723 ng/g) and Jialing River (132 – 349 ng/g) sampled at Chongqing in October 2009.

In Kaixian area, HAN-D and HAN-C possessed a three to four times higher PAHΣ16 concentration than HAN-B and HAN-A (Table 3.4). This indicates a pollutant influx between HAN-B and HAN-C, which could originate from the city of Kaixian on the south side and/or the Dong or Toudao River, which enter the lake on the north side. This assumption is underlined by the observation that benzo[b]fluoranthene was alike benzo[k]fluoranthene below the LOD in the upper part of the Hanfeng Lake (HAN-A, HAN-B), but both could be measured in the lower part of the Hanfeng Lake (HAN-C, HAN-D) (Table 3.4).

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Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______

R

-

8

50 24 22 12 15 30 11 81 57 92

165

<10 <10 <10 <10 <10 <10 <10 <10 <10

BJX

+ o WHO

L

+

-

Kaixian

7

21 60 53 24 81 17 27 36 11 57 93

60

140 827 395 335

<10 <10 <10

240

HF

U

+

-

13 61 44 76 51 13 81 13 87

130 130 110 210 694 564

<10 <10 <10 <10

280

1198

WU

T

-

61 62 23 55 19 32 21 39 11 89

140 100 552 311 250

<10 <10 <10 <10 <10 <10

WU

Wushan

D

-

11 41 70 30 56 35 10 54 12 78

190 120 110 150 877 512 392

<10 <10 <10 <10

WU

-

9); + = concentration exceeded „effect

8

U

62 41 71 4 15 75 11 89

250 130 120 110 200 676 546

<10 <10 <10 <10 <10

1121

YUN

-

+

8

T

73 76 34 81 22 43 33 10 61 92

130 824 427 353

<10 <10 <10 <10 <10

260

YUN

Yunyang

-

D

64 23 56 35 54 22 78

180 110 100 780 160

<10 <10 <10 <10 <10 <10

1563 1228 1118

YUN

-

+ +

U

29 62 79 28 52 44 10 55 10 90

16 36

140 230 120 100 120 514 394

<10

1122

FEN

W1 cells (Bols et al., 199

-

T

-

7

14 82 38 34 15 29 36 11 15 16 20 93

310 160 122

<10 <10 <10 <10 <10

FEN

Fengdu

D

-

+

12 20 95 81 52 60 24 43 36 41 13 87

28

150 110 750 419 324

<10 <10 <10

FEN

U

+ +

-

+ +

12 41 73 38 76 57 12 76 12 88

33 64

170 150 120 170 717 547

220 340

1653

CNG

T

-

+

6

15 55 47 19 41 55 12 21 21 25 94

21

120 450 223 168

<10 <10 <10 <10

CNG

Chongqing

D

-

PAH content in sediment extracts of sampling campaign September 2011(ng/g)

+

3

14 27 99 64 65 24 57 41 44 25 75

.

20

130 120 110 816 482 362

<10 <10 <10

3

CNG

Table

a,b,c

a,b,c

a,b,c

a,b,c

a,b,c

TEQ (%)

-

TEQ (%)

]pyrene

a,b,c

-

c,d

a

-

]anthracene

]perylene

hene

a,b,c

a,h

TEQ/Bio

]anthracene ]fluoranthene ]pyrene

]fluoranthene

-

a b k a g,h,i

TEQ/Bio

-

Chem

PAHs /wPAHs Ames activity

a = Substance has been shown to be mutagenic in the Ames assay (Pérez et al. 2003); b = Substance is cancerogenic according t (2010); c = Substance has been shown to induce EROD activity in RTL range valuelow“ according Long to (1995). et al.

-

Substance Naphthalene Acenaphthylene Acenapht Fluorene Phenanthrene Anthracene Fluoranthene Pyrene Benzo[ Chrysene Benzo[ Benzo[ Benzo[ Indeno[1,2,3 Dibenzo[ Benzo[ PAHs Σ Σ /wPAHs Σ EROD activity Chem 1

______106

Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______

The temporal variation between the campaigns in September 2011 and May 2013 showed that PAH concentrations were generally higher in September 2011. These observations stand in agreement with Wang et al. (2013), who referred the temporal differences in pollution to the drawdown period of the TGR (around May), where water quality is better than during the other seasons.

With regard to PAHΣ16 concentrations in the water phase, Wang et al. (2009) could measure overall 14 to 97 ng/L at sites along the reservoir applying passive samplers in surface water in May 2008. The highest concentrations were detected at the upper part of the TGR at Changshou (97 ng/L) – located between Chongqing and Fengdu, and Chongqing (43 ng/L), but also in the lower part of the TGR at Maoping (45 ng/L) near the dam. They could also find other organic pollutants in the surface water of the reservoir. PCBs (0.08 to 0.51 ng/L) were highest in the area near the dam, and organochlorine pesticides (2.33 to 3.60 ng/L) were homogenously distributed along the TGR. Particularly HCB and its metabolite PeCB, as well as HCH and DDT dominated the organochlorinepestice spectrum. Overall, they stated that obvious regional variations of PAHs, PCBs and organochlorine pesticide levels appeared along the reservoir and that its water could be classified as being polluted by HCB and PAH based on water quality criteria (Wang et al., 2009). In another study Wolf et al. (2013; b) could determine PAH concentrations – napthalene excluded – between 5 to 101 ng/L (mean: 22 ± 33 ng/L) along the TGR between Chongqing and Yunyang, and also at the Hanfeng Lake in September 2011. The detected concentrations were designated to be in comparable ranges or even lower than measured in surface waters in western industrialized countries. Furthermore, concentrations of perfluorinated compounds, polychlorinated biphenyls and polybrominateddiphenyl ethers, were below the respective detection limits, which were referred to their low solubility (Wolf et al., 2013a). The analyzed organic pollutants, with exception of the two pesticides – picloram and clopyralid, met the standards for the Chinese National Drinking Water Quality Standard GB5749 (Ministry of Health - China, 2006) and the European Union (EU) Council Directive 98/83/EC on the quality of water intended for human consumption (The Council of the European Union, 1998) and the EU Directive 2008/105/EC on environmental quality standards in the field of water policy (European Union, 2008; Wolf et al., 2013a). In comparison to other sections of the Yangtze River, where the same 16 priority PAHs were also measured, the sampled locations in this study exhibited a similar PAHΣ16 content, but were mostly rather in the lower part of the ranges detected elsewhere. Feng et al. (2007b) measured a

______107

Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______

PAHΣ16 burden between 303 and 3,995 ng/g in Yangtze River mainstream sediments at the city of Wuhan further downstream of the TGD in June 2005, and between 72 and 1,206 ng/g in December of the same year. However, the sediments from tributaries of Wuhan section sampled in June (4,121 to 4,262 ng/g) and December (31 to 4,813 ng/g) displayed generally a much higher

PAHΣ16 content than those investigated at the TGR in the scope of this study (Feng et al., 2007b). It demonstrated a more critical role of the tributaries as pollutant influx sources to the mainstream at Wuhan than in the TGR. The Yangtze Estuary, where the river empties into the East China Sea near the city of Shanghai, an important industrial region for the country, has been investigated by

Wang et al. (2012a), who detected PAHΣ16 contents between 77 to 2,937 ng/g (average: 450 ng/g) in sediments of the estuary in 2010. However, Liu et al. (2014) determined only concentrations between 22 and 191 ng/g in the estuary in 2012. Other Chinese rivers exhibited also comparable and mainly higher PAHΣ16 concentrations. Mai et al. (2002) detected 323 – 14,812 ng/g PAHΣ16 in the Pearl River in 1997, and Yu et al. (2009) measured 1,415 ng/g in the Yellow River in 2005.

Also in international comparison the total PAHΣ16 content of TGR surface sediments were below or at the lower limit of, e.g. German main water bodies. Keiter et al. (2008) measured PAHΣ16 concentrations between 240 and 26,320 ng/g (average: 4,899 ± 8,791 ng/g) along the Danube Rivers course. Furthermore, the Rhine River was investigated by Kosmehl et al. (2004), who detected PAHΣ16 – benzo[e]pyrene was analyzed instead of benzo[a]pyrene – in a range of about 1,300 to 4,300 ng/g (2,056 ± 971 ng/g) in surface sediments.

______108

Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______

D

-

+

HAN 63 16 <10 37 68 17 34 30 21 47 87 47 <10 <10 <10 20 484 234 201 9 91

C

-

+

HAN 86 21 <10 42 73 19 40 34 24 53 116 59 <10 <10 <10 26 590 291 251 10 90

B

-

HAN 35 <10 <10 13 18 <10 17 16 14 15 <10 <10 <10 <10 <10 <10 128 46 29 1 99

A

-

Kaixian Hanfeng Lake HAN 33 <10 <10 14 20 <10 17 16 14 16 <10 <10 <10 <10 <10 <10 127 47 30 1 99

B

-

Substance cancerogenicis according to

TGR 24 <10 <10 12 17 16 25 21 20 17 <10 <10 19 <10 <10 <10 129 61 36 2 98

A

-

W1 cells (Bols et al. 1999); + = concentration

-

TGR TGR 37 <10 <10 14 22 17 39 29 24 25 25 25 <10 <10 <10 <10 150 137 98 14 86

C

-

JIA 38 16 <10 15 22 17 35 29 26 22 16 25 19 <10 <10 <10 254 141 107 13 87

B

-

+

JIA 62 18 <10 19 30 17 35 31 23 23 13 22 <10 <10 <10 <10 278 115 80 11 89

A

-

Jialing JIA 58 22 <10 18 35 18 40 33 23 22 13 20 <10 <10 <10 <10 299 117 77 6 94

C

-

YAN 45 17 <10 18 42 19 57 45 31 41 44 36 25 <10 <10 18 433 232 176 14 86

B

-

YAN 35 16 <10 16 29 18 43 35 25 29 27 27 <10 <10 <10 <10 297 150 107 11 89

A

-

Chongqing Yangtze YAN 27 16 <10 14 30 18 63 55 28 46 31 34 24 <10 <10 <10 383 226 163 23 77

a,b,c

a,b,c

(%)

a,b,c

a,b,c

a,b,c

TEQ (%)

-

TEQ

]pyrene

a,b,c

-

c,d

a

-

]anthracene

PAH content in sediment extracts of sampling campaign 2013May (ng/g)

]perylene

4

a,b,c

.

a,h

TEQ/Bio

]anthracene ]fluoranthene ]pyrene

]fluoranthene

3

-

a b k a g,h,i

TEQ/Bio

-

Chem

Table

-

Substance (ng/g) Naphthalene Acenaphthylene Acenaphthene Fluorene Phenanthrene Anthracene Fluoranthene Pyrene Benzo[ Chrysene Benzo[ Benzo[ Benzo[ Indeno[1,2,3 Dibenzo[ Benzo[ PAHs Σ /wPAHs Σ Ames activity /wPAHs Σ EROD activity Chem 1 a = Substance has been shown to be mutagenic inAmes the assay (Pérez et al. 2003); b = WHO (2010); c = beenSubstance has shown to EROD activityinduce in RTL exceeded „effect range low“value according to et Long al. (1995).

______109

Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______

Considering the mass balance for PAHs, under assumption that annually 151 – 172 Mt of sediment remain in the TGR (2003 – 2008) (Yang et al., 2007; Hu et al., 2009a) and that the average PAHΣ16 burden in sediment along the reservoir is 936 ± 414 ng/g (September 2011 campaign), an amount of about 79 – 232 t PAHΣ16 per year or 216 – 636 kg PAHΣ16 per day are deposited in the reservoir. Further, adopting the reservoirs surface area of 1080 km2 as potential bottom deposition area of the reservoir (water body pictured as a cuboid) this results into about 0.2 – 0.6 kg PAH/km2/day (0.2 – 0.6 mg PAH/m2/day). This assumption neglects seasonal differences and less industrialized regions between the major tributaries, but also that an even larger portion of PAHs enters the reservoir, remains in the water phase and is discharged – solved in water or bound to particles – behind the dam. These numbers are in agreement with the mass balance estimation of Mueller et al. (2008) that about 500 to 3,500 kg of phenols, chlorinated compounds, aromatic hydrocarbons and PAHs are discharged by the Yangtze River per day.

Zhang and Tao (2009) estimated an annual atmospheric emission of 114,000 t PAHΣ16 for China in 2004 – accounting for 22% of the global emission, followed by India (17%) and the United States (6%).

PAH Source analysis

Anthropogenic sources are the main PAH contributors in urbanized and industrialized regions, and can be divided into two groups. Petrogenic sources relate to crude oil, coal and their products, mainly released by spillage, while pyrogenic sources relate to incomplete combustion of biomass, like wood burning, and fossil fuels (Neff, 1979; Mostert et al., 2010). Because PAHs are always emitted as a mixture and different emission sources are considered to have characteristic relative PAH ratios, it is possible to trace the contamination in environmental samples (sediment, soil, water, air and tissue) – with some restrictions – back to their sources (Yunker et al., 2002; Tobiszewski and Namieśnik, 2012). As emitted PAHs undergo various fate processes, which depend on factors like solubility and adsorption, typically substances of the same molecular mass and similar physicochemical properties are compared to minimize differences due to diverging transformations (Readman et al., 1987; McVeety and Hites, 1988).

______110

Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______

-

f

e

0.5 0.5 0.5

R

- -

-

0.35 0.35 = 0.35 =

0.52 0.44

- -

<0.47

<0.17

BJX

L

-

0.08 0.53 0.23 0.38

HF

0.5 0.5 = liquid fossil 0.5 = liquid fossil

- -

Kaixian D

-

e

D

-

0.4 = petroleum, 0.4

0.52 0.37 0.40

HAN 0.20 0.53 0.31 <0.34

<0.05

WU

e

Concentration Concentration of Indeno[1,2,3

T

-

C

f

Source: Source: petroleum, <0.4 = 0.4

Source: Source: <

-

0.49 0.29 0.35

WU <0.07

Kaixian

HAN 0.20 0.54 0.31 <0.28

ng/g;

Hanfeng Lake

e

U

B

-

-

Source: Source: <0.2 = petroleum, 0.2 Source: <0.2 = petroleum, 0.2

0.50 0.36 0.39

<0.03

WU

HAN <0.36 0.52 0.48 n.d.

Wushan

e

D

]perylene, ]perylene,

-

A

-

0.52 0.08 0.39

g,h,i g,h,i

<0.05

YUN

HAN <0.33 0.52 0.47 n.d.

r 2011 with r related sources. 2011 with

enzo[

]anthracene, ]anthracene, CHR = Chrysene, Source: <0.2 = petroleum, 0.2 ]anthracene, CHR = Chrysene, Source: <0.2 = petroleum, 0.2

FLA FLA Fluoranthene, = PYR = Pyrene,

a a

FLA = Fluoranthene, PYR = Pyrene,

e

T

b

b

-

B

entration of anthracene was <10

-

0.49 0.30 0.35

<0.04

YUN

Conc

TGR 0.49 0.55 0.54 n.d.

e

TGR

e

U

BaA BaA = Benzo[ BaA = Benzo[

-

A

-

c c

; n.d. = no diagnostic ratio could be determined. beno diagnostic could ratio = ; n.d.

]pyrene, ]pyrene, BghiP = Benzo[ ]pyrene, BghiP = B

0.52 0.36 0.39

<0.04

YUN

TGR 0.44 0.58 0.49 n.d.

c,d c,d

Yunyang

- -

D

- C

-

0.12 0.54 0.46 0.47

JIA 0.44 0.54 0.54 <0.59

FEN

Indeno[1,2,3 Indeno[1,2,3

e

T

-

Roche Roche et al., 2009); Roche et al., 2009);

B

- -

0.1 combustion (Pies = et al., 2008);

-

0.53 0.34 0.44

<0.11

Jialing

FEN

IcdP IcdP = IcdP =

JIA 0.36 0.53 0.51 n.d.

d d

U

-

A

-

Chongqing

0.11 0.54 0.44 0.44

JIA 0.34 0.54 0.51 n.d.

FEN

Fengdu

Diagnostic ratios for PAHs in sediment from campaign May inrelated sources. campaign2013 with ratios sediment PAHs from for Diagnostic

D

-

C

6

-

.

Diagnostic ratios for PAHs in sediment from campaign Septembe in campaign ratios sediment PAHs from for Diagnostic

3

0.17 0.55 0.49 0.48

5

.

CNG

3

YAN 0.31 0.56 0.43 <0.36

Source: Source: <0.1 petroleum, = >

Source: Source: <0.1 = petroleum, >0.1 = combustion (Pies et al., 2008);

Table

0.5 = grass/wood/coal combustion (Yunker et al., 2002);

T

B

Table -

-

0.11 0.54 0.31 0.45

Yangtze

CNG

YAN 0.38 0.55 0.47 n.d.

U

-

-

0.35 0.35 = combustion (Yunker et al., 2002); 0.35 = combustion (Yunker et al., 2002);

0.5 = grass/wood/coal combustion (De la Torre 0.5 = grass/wood/coal combustion (De la Torre

0.11 0.53 0.38 0.43

YAN A 0.37 0.53 0.37 <0.53

CNG

Chongqing

ng/g.

d

d

a

b c a

b

c

was <10was

ratios

]pyrene ]pyrene

PAH PAH diagnostic

BaA/BaA+CHR

FLA/FLA+PYR

ANT/ANT+PHE

IcdP/IcdP+BghiP

ANT/ANT+PHE FLA/FLA+PYR BaA/BaA+CHR IcdP/IcdP+BghiP

ANT ANT = PHE Anthracene, = Phenanthrene,

ANT ANT = Anthracene, PHE = Phenanthrene,

PAH PAH diagnostic

ratios a = vehicular emission, > petroleum/combustion, > fuel combustion (vehicle/crude oil), > c,d a = vehicular emission, > petroleum/combustion, > 2002) combustionet al., oil), grass/wood/coal >0.5 = (Yunker fuel (vehicle/crude combustion ______111

Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______

The important role of liquid fossil fuel combustion all along the reservoir (Table 3.5) stands in agreement with results from the TGR water column by Wang et al. (2009; 2013). Urban traffic emissions and runoff, as well as intensified shipping activities since the impoundment of the reservoir, can be accounted to be the main contributors. Moreover, combustion sources had a major impact on the upper – highly urbanized and industrialized – part of the TGR (Table 3.5, Table 3.6), which is also verified by the water analysis by Wang et al. (2009; 2013). Particularly the cities of Chongqing and Changshou are important industrial centers at the TGR’s shore, where industrial emission – e.g., power generation from coal combustion, and urban air pollution, may have a serious influence on the contamination status of the reservoir in this region. On the other hand, the origin of PAHs in the middle (YUN) and lower part (WU) of the TGR rather could be traced back to petrogenic sources – in accordance with Wang et al. (2013), and upon combustion particularly to vehicular emission from traffic (Table 3.5). Alike all tributaries – with exception of CNG-T, which were also affected by petrogenic sources, this may be caused by oil and fuel spillage, e.g., from ships (Table 3.5). As the downstream location at Yunyang (YUN-D) stood out to be seriously influenced by a contamination source, the origin appears to be petrogenic (Table 3.5). The contamination at the Hanfeng Lake exhibited a mixture of petrogenic sources and combustion, especially from liquid fossil fuels as well as coal, grass and wood. This may be caused on the one hand side from urban air pollution, and on the other hand from biomass burning in homes as the city is located in a rather rural area. The lower locations at the Hanfeng Lake, that showed an increased contamination, were defined by petrogenic sources, which may originate from shipping activities and wastewater (Table 3.5 and Table 3.6). The reference site (BJX-R) showed a dominating pyrogenic impact, with a mixture of liquid fossil fuel and also coal, grass and wood combustion. This can also be referred to biomass burning in homes of the rural area and minor shipping activities in the area (Table 3.5). With regard to overall China, particularly biomass burning, domestic coal combustion and coke ovens have been identified as significant sources for the atmospheric emission of PAHs (Zhang et al., 2007b; Zhang and Tao, 2009).

Assessment of biological adverse effects according to sediment quality guidelines

PAHs have been classified as persistent organic pollutants, due to their (semi-)persistent, bioaccumulative and toxic properties. Upon those are the 16 analyzed PAHs listed as priority pollutants by the U.S. Environmental Protection Agency (1982). PAHs can be found throughout

______112

Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______all environmental media – air, soil, water, sediment, tissue – and exhibit impairing properties, for example cancerogenicity and mutagenicity (Pérez et al., 2003; WHO, 2010). Due to the complexity of PAH mixtures and their individual effects no official standards exist so far – only for indicator compounds like BaP – to evaluate the biological effects of total PAHs in sediment. However, one attempt to set evaluation values in order to anticipate biological effects is the ERL/ERM concept (“effect range low”/“effect range median”) by Long et al. (1995). These values delineate three ranges in substance concentrations that are associated with (a) rarely (

3.4.2 In vitro bioassay

Sediment mutagenicity in TGR

Mutagenicity of all sediments samples from campaign September 2011 were examined with Ames fluctuation assay using the tester strain TA100 and TA98 with and without metabolic activation (S9) at the highest concentration of 400 mg SEQ/mL. For the samples exposure to TA100 with and without S9 supplement with the sediment extracts, it resulted in no significant mutagenic effects (p > 0.05), with exception of YUN-D with S9 supplement (NOEC: 200 mg SEQ/mL). The same procedure with the tester strain TA98 without S9 supplement also led to no significant results, with exception of CNG-D (NOEC: 200 mg SEQ/mL). However, the exposure of tester strain TA98 with S9 supplement had in consequence that several sediment extracts showed significant mutagenic activity (Fig. 3.2). The strongest inducing samples were JL River (CNG-T), FEN Down (FEN-D), WU Up (WU-U) and WU Down (WU-D) with a NOEC of 25 mg SEQ/mL (p < 0.05), while FEN Up, DN River, HF Lake and the reference site BJX River exhibited no significant impact at all (NOEC: > 400 mg SEQ/mL; p > 0.05) (Fig. 3.2).

______113

Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______

Testing of the sediment extracts from the May 2013 sampling with bacteria tester strain TA98 in absence of metabolic activation revealed no significant mutagenicity in any of the samples (p > 0.05) (Data not shown). Also, adding of S9 supplement resulted only in a significant effect in the samples HAN-D (NOEC: 100 mg/mL) and HAN-A (NOEC: 200 mg/mL) (p < 0.05). However, cytotoxicity was observed for the extracts of JIA-A, JIA-B and HAN-C (400 mg/mL), which might explain the absence of mutagenicity in these samples.

250

200

)

L

m

/

Q 150

E

S

g

m

(

C 100

E

O

N

50

d

d

.

.

d

.

n

d

n

.

n

0 n T T -U - -D -U -T -D -U - -D -U -T -D -L -R G N N U F X G G N N N N U U H N N N E E E U U U W W W J C C C F F F Y Y Y B Sample location

Fig. 3.2 Mutagenic activity of sediment extracts determined in the Ames fluctuation assay with Salmonella typhimurium tester strain TA98 with exogenous enzymatic S9 supplement for metabolic activation of promutagenic compounds. Values given are No Observed Effect Concentrations (NOEC) in mg/mL, n.d: NOEC above the maximum tested concentration (400 mg/mL).

Potential sources for the mutagenic compounds in the strongest inducing samples (25 mg/mL) may be referred to local industries. The mutagenicity at CNG-T and the cytotoxicity found in JIA-A and JIA-B emphasize an industrial impact on the Jialing River, where several industrial sites have settled. At Fengdu, the downstream location FEN-D was clearly affected, while the upstream location (FEN-U) and the Long River (FEN-T) showed no or less impact, respectively. This suggests an input source between these points, like industry or domestic sewage disposal from Fengdu. The similar mutagenic potentials of WU-U and WU-D at Wushan suggest a drift of mutagenic compounds from upstream to downstream with no obvious influence from the Daning River (WU-T), with the source located upstream of WU-U. At the Hanfeng Lake, the cytotoxicity ______114

Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______in sample HAN-C and the mutagenicity in sample HAN-D further corroborate a considerable input source between HAN-B and HAN-C. Almost all mutagenic effects could be related to frameshift mutations after metabolic activation. Comparing this to the chemical results PAHs appear to be the obvious causative agents. Depending on the individual PAH, they can either induce frameshift (TA98) or base pair substitution (TA100) (Madill et al., 1999), but generally require metabolic activation (WHO, 2010). Chrysene for example, is most probably the inducer for base pair substitution in sample

YUN-D, because it constitutes 50% of the PAHΣ16 (780 ng/g) in the extract and reacts positively in tester strain TA100 with S9 (Madill et al., 1999). Mutagenicity could also be detected in surface water of the Yangtze River. Shu et al. (2002) could also detect base pair substitution and frame shift mutagenic potentials in surface water of the Yangtze and Jialing River at Chongqing before the impoundment of the reservoir, from which the latter appeared to be more seriously polluted. Metabolic activation led to a slight decrease in mutagenicity. Qiu et al. (2003) also found higher mutagenicity in surface water of the Jialing River and mainly referred this to agricultural, industrial and domestic discharges, in addition to a poor self-purification capacity compared to the Yangtze River. Further, direct- and indirect-acting frameshift inducers were found to be the responsible compounds in surface water of the Yangtze River at Wuhan section (Dong et al., 2010), Shanghai section (Shen et al., 2003a) and the Yangtze Estuary (Wu, 2005), as well as in sediments of the German Rhine River (Kosmehl et al., 2004) and Danube River (Higley et al., 2012). Generally, surface waters appear to be dominated by direct- and indirect-acting frame shift mutagens worldwide (Ohe et al., 2004).

AhR-mediated Activity in the sediment of TGR

The EROD induction assay with RTL-W1 cells was used to determine the Ah receptor mediated activity of sediment extracts. The mean bio-TEQ EC25 ranged from 93 ± 13 pg/g (BJX-R) to 1,161 ± 326 pg/g (YUN-U) (Fig. 3.3). The strongest inducing samples could be found at Yunyang (YUN-U), Chongqing (CNG-U), Kaixian (HF-L) and Wushan (WU-U), which also showed the only significant inductions compared to the reference site (BJX-R) (p < 0.05). The pattern of EROD induction per site at the mainstream – with exception of Wushan - was: upstream > tributary > downstream. Inter-site comparison of the mean and total induction per site along the mainstream each showed the following order: Yunyang > Chongqing > Wushan > Fengdu (Fig. 3.3).

______115

Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______

Fig. 3.3 Mean detected EROD inductions (Bio-TEQs) in comparison to calculated inductions of detected PAHs (Chem-TEQs) in sediment extracts from sampling campaigns September 2011 (A) and May 2013 (B). For each location the left bar relates to the Bio-TEQs on the left y-axis and the right bars to the Chem-TEQs on the right y- axis. Error bars represent standard deviations; n = 3; Percentages give differences between Bio-TEQs and Chem- TEQs (cf. Chapter 3.4.1); dotted lines indicate different sites, double dotted lines separate sites along the Yangtze River mainstream and the TGR watershed; Asterisks mark significant differences between samples and reference (BJX-R); Data was statistically analyzed with ANOVA on ranks (multiple comparison) with Dunn’s post hoc test, * = p < 0.05.

In campaign May 2013 the mean bio-TEQ EC25 range was between 87 ± 13 pg/g (TGR-B) and 883 ± 100 pg/g (HAN-C), with the highest induction potentials at Kaixian (HAN-C; HAN-D), ______116

Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______which also displayed the only significant induction compared to the reference site BJX-R (p<0.05) (Fig. 3.3). At Chongqing, the change of induction potentials in the Yangtze River (YAN) increased with the flow direction, whereas they overall decreased in the Jialing River (JIA) – with a light drop in the middle section – and continued to decline in the TGR after the conversion zone. Inter-site comparison of the Yangtze (316 ± 99 pg/g) and Jialing River (309 ± 84 pg/g) showed comparable inductions, with lower activities in the TGR (164 ± 93 pg/g). At Kaixian, a clear increase could be observed between the upper (HAN-A; HAN-B; 90 ± 12 pg/g) and the lower (HAN-C; HAN-D; 824 ± 94 pg/g) section of the Hanfeng Lake (Fig. 3.3). This indicates a pollution influx between those sites, as also observed by the chemical analysis, mutagenicity and cytotoxicity of the respective samples as described above. The potential origin was referred to the city of Kaixian on the south side and/or the Dong or Toudao River, which enter the lake on the north side. In national and international comparison AhR-induced activity appeared to be in the medium to upper range. The obtained bio-TEQ EC25 values mainly exceeded those of crude extracts measured with RTL-W1 cells from the Yangtze Estuary (39 – 324 pg/g ) (Liu et al., 2014), the German Elbe River Estuary (16 – 322 pg/g ) (Otte et al., 2013) and European Danube River (20- 123 pg/g ) (Keiter et al., 2008), whereas compared to the Tietê River in Brazil (n.d. – 24,170 pg/g) (Suares Rocha et al., 2010) and to the European Rhine River (2,387 – 4,553 pg/g; 3,620 and 7,920 pg/g) ((Heimann et al., 2011; Schulze et al., 2014), respectively) activities ranged in the lower to medium range for most of the sediments investigated there.

3.4.3 In vivo bioassay

Fish Embryo Toxicity and Sediment Contact Assay with Danio rerio

The SCA and FET were performed to record the lethality and overall embryotoxic/teratogenic effects of bioavailable (SCA) and extracted (FET) compounds on embryos and larvae of Danio rerio after 96 h. In the SCA all samples showed lethality and overall effects (lethal plus sublethal) on the fish only in range of the negative control (0 – 10%) (Data not shown). The screening of the extracted samples of campaign of May 2013 in the highest concentratin (100 mg/mL) showed no effects, thus niether SCA nor FET were conducted in detail. In the FET for campaign 2011, lethal impacts could only be registered for the samples from Long 2 River (FEN-T; LC50: 50 mg/mL; 95% CI: 46 – 54 mg/mL; r =0.95) and Hanfeng Lake (HF-L; 2 LC50: 30 mg/mL; 95% CI: 26-34 mg/mL; r =0.96). For all other samples the LC50 was larger

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Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______than the highest tested concentration of 100 mg/mL. In comparison, Wu et al. (2010) determined mortalities between 3 to 16% for sediment extracts (concentration: 60 mg/mL) from the Yangtze Estuary after 96 h exposure, whereas in this study mean mortalities ( ± SD) ranged from <10% in most of the samples up to 80 ± 10% (concentration: 60 mg/mL) in the Long River (FEN-T) and 80 ± 17% (concentration: 50 mg/mL) in the Hanfeng Lake. Schiwy et al. (2015a) determined

LC50 values of 160 mg/mL and 355 mg/mL for sediment extracts from Altrip and Ehrenbreitstein, respectively, in the FET after 96 h exposure. Both are sampling sites at the German Rhine River and have been classified previously to be moderately and low contaminated. Whereas they could determine a LC50 value of 1 mg/mL for sediment extracts from the Vering Canal at Hamburg, Germany, known as to be highly burdened with old environmental load (Feiler et al., 2009; Hoess et al., 2010).

Mainstream Tributary Reference Site 150 Chongqing Fengdu Yunyang Wushan Kaixian

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Fig. 3.4 Overall effects on Danio rerio exposed for 96h to sediment extracts given as half maximal effective concentration (EC50). Overall effects are constituted by the sum of lethal endpoints and sublethal endpoints; Symbols represent mean values and error bars 95% confidence intervals; n=3; dashed line indicates highest tested concentration 100 mg/mL; dotted lines indicate different sites, double dotted lines separate sites along the Yangtze River mainstream and the TGR watershed.

The overall effects (lethal plus sublethal) of the sediment extracts in the FET ranged from EC50 16 mg/mL (HF-L; 95% CI: 14 – 18 mg/mL) to 121 mg/mL (BJX-R; 95% CI: 108 – 135 mg/mL). The goodness of fit was in all cases r2 ≥ 0.89. None of the samples had an overlapping 95% ______118

Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______confidence interval with the reference site (BJX-R), thus all sites can be considered to display significant effects (Brandstätter, 1999) (Fig. 3.4). The most toxic samples could be found at Kaixian (HF-L), Chongqing (CNG-U), Yunyang (YUN-U) and Fengdu (FEN-U). The pattern of overall effects (lethal plus sublethal) at the mainstream – with exception of Wushan – was: upstream > tributary > downstream, analogous to the observations made with the EROD assay. Overall can be stated that most samples did not induce significant mortality in the fish eggs after 96 h. Aside the acute lethal effects numerous effects could be recorded that might result into severe consequences on the long run. The main set of effects were coagulation, yolk sac and pericardial edema, as well as disruptions of the cardiac function, manifested as diminished heartbeat and blood-circulation, or a total lack of both. A number of PAHs, PCBs and related dioxin-like compounds are known inducers of developmental disorders, among them the observed edema of the pericardium (Cantrell et al., 1998; Sundberg et al., 2005) and decreased blood circulation (Cantrell et al., 1998; Teraoka et al., 2010). Further, Incardona et al. (2004) described cardiac dysfunction, edema, spinal curvature and reduction in the size of the jaw and other craniofacial structures as part of a characteristic suite of abnormalities after exposure to complex PAH mixtures from petrogenic sources. They further highlighted secondary consequences of defects in the cardiac function on later stages of cardiac, renal, nervous and craniofacial morphology. Brette et al. (2014) demonstrated a cardiotoxic mechanism, by which water accommodated fractions of PAHs containing crude oil had a serious impact on the regulation of cellular excitability via direct effects on ion channels, with consequences for life- threatening arrhythmias in fish and other vertebrates. The detrimental impacts of PAHs on teleost, avian, or mammalian systems could be shown in a number of studies (Barron et al., 2004).

3.4.4 In situ biomarkers

Micronucleus assay

In the erythrocytes of Pelteobagrus vachellii sampled along the Yangtze River mainstream in May 2012 no significant differences (p > 0.05) in the micronucleus frequency could be detected at Chongqing (CNG 2012: 0.175 ± 0.121 ‰; IF 0.9; n=10), Fengdu (FEN 2012: 0.150 ± 0.129 ‰; IF 0.8; n=10), Yunyang (YUN 2012: 0.150 ± 0.129 ‰; IF 0.8; n=10) and Wushan (WU 2012: 0.175 ± 0.169 ‰; IF 0.9; n=10) compared to the reference site Baijiaxi River (BJX 2012: 0.200 ± 0.158 ‰; n=10). However, the samples taken from the Hanfeng Lake in 2012 displayed a significant effect (HF 2012: 0.400 ± 0.175 ‰; IF 2.0; n=10; p<0.05) compared to the Baijiaxi

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River. Furthermore, the erythrocytes sampled in Chongqing (CNG 2013: 2.250 ± 2.073 ‰; IF 11.3; n=20; p<0.001) and Hanfeng Lake (HF 2013: 1.450 ± 2.040 ‰; IF 7.3; n = 20; p<0.01) in May 2013 showed even stronger effects. Comparing the change between the years at Hanfeng Lake (HF 2012; HF 2013; p > 0.05) no significant difference could be measured, whereas at Chongqing (CNG 2012; CNG 2013; p < 0.001) the status changed significantly (Fig. 3.5).

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Fig. 3.5 Micronucleus frequency in erythrocytes of Pelteobagrus vachellii from sampling campaign May 2012 and May 2013. Symbols represent individual animals, bars the mean value, and error bars the standard deviation; circles (°)= May 2012; dotted (•)= May 2013; dotted lines indicate different sites, double dotted lines separate sites along the Yangtze River mainstream and the TGR watershed; 2012 samples n = 10; 2013 samples n = 20; Asterisks mark significant differences between samples and reference (BJX 2012); Data was statistically analyzed with Mann- Whitney Rank Sum Test, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, a = significant difference to reference site, b = significant difference to same site in May 2012.

The biological impairments may be attributed to PAHs, as detected at Chongqing and Kaixian in May 2013 (Table 3.4). Genotoxic properties of PAHs could be demonstrated before (White, 2002). Further, environmental sediments contaminated with PAHs have shown genotoxic effects in catfish (Di Giulio et al., 1993) and turbot (Kilemade et al., 2004), barbell (Boettcher et al., 2010), tilapia (Rocha et al., 2009), as well as re-suspended PAH-spiked sediments in trout (Brinkmann et al., 2013; Hudjetz et al., 2014). The harbor sediments that caused DNA damage in turbot blood cells contained PAH concentrations of about 1,000 ng/g after 7 days of exposure, and the reference site – containing about 500 ng/g PAH – also induced significant effects after 14 days. Likewise, other organs – liver, epidermis, spleen and gill – were also affected significantly

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(Kilemade et al., 2004). Thus, although PAHΣ16 concentrations were at 302 ± 87 ng/g at Chongqing and 332 ± 240 ng/g at Kaixian, the exposure time has to be taken into account, as the formation of micronuclei is dependent on contamination level and exposure time (Das and Nanda, 1986). PAHs generally persist in the environment with half-lives from days to years – depending on the individual structure and processes like photo- and biodegradation (Shuttleworth and Cerniglia, 1995; Kanaly and Harayama, 2000; Fasnacht and Blough, 2002), and can accumulate in organisms without the right metabolic detoxification systems due to their lipophilicity, and consequently biomagnify through trophic transfer (Coates et al., 1997; Kanaly and Harayama, 2000). However, they are easily biodegradable in organisms with the right metabolic mechanisms (Van der Oost et al., 2003; Möller et al., 2014), unfolding their toxic potentials through biotransformation to reactive intermediates (WHO, 2010).

PAH metabolites in bile of P. vachellii

In order to determine if PAHs could be detected in fish of the species P. vachellii, exemplarily for other species, the indicator metabolite 1-OHPyr – which contributes up to 76% to the sum of biliary PAH metabolites (Ruddock et al., 2003) – was analyzed in bile of the fish collected in campaign 2012. The presence of PAHs in the fish from campaign 2012 could be verified. The mean contents of 1-OHPyr ranged from 211 ± 88 ng/mL at Yunyang (YUN 2012) to 2,428 ± 871 ng/mL at Wushan (WU 2012) (Table 3.7). A repeated determination of 1-OHPyr in samples from campaign 2013 was not possible due to very low available amounts of bile in the gall bladder.

Table 3.7 Parameters for PAH metabolites in bile of Pelteobagrus vachellii from sampling campaign May 2012. Parameters were concentrations of 1-hydroxypyrene (1-OHPyr) given as ng/mL, absorption of bile at 380 nm in dimensionless absorption units (A380) given as 1/mL, the concentration of 1-OHPyr normalized by A380 given as ng/A380, and the ratio of 1-OHPyr to 1-hydroxyphenanthrene (1-OHPhe) in dimensionless units; Values are stated as means with standard deviation of pooled samples (n); n = 3, with exception of WU 2012 (n=2). Sites 1-OHPyr [ng/mL] A380 [1/mL] 1-OHPyr per A380 1-OHPyr/1-OHPhe ratio [ng/A380] CNG 2012 1120 ± 296 32 ± 21 47 ± 25 7 ± 2 FEN 2012 1211 ± 460 61 ± 6 19 ± 6 11 ± 3 YUN 2012 211 ± 88 10 ± 3 20 ± 3 7 ± 3 WU 2012 2428 ± 871 5 ± 0 499 ± 146 19 ± 7 HF 2012 1562 ± 212 43 ± 8 37 ± 6 5 ± 1 BJX 2012 328 ± 201 22 ± 8 22 ± 22 7 ± 2

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Based on the normalized 1-OHPyr contents and the 1-OHPyr/1-OHPhe ratio (Table 3.7) , fish from Wushan clearly displayed the strongest contamination. This can either be referred to a higher pollution at Wushan, as Blahova et al. (2014) determined a statistically positive correlation between biliary 1-OHPyr and the total PAH content in the water, or to an extreme situation, like a contaminated food source that was taken up by the fish not long before they were caught. Most individuals from Wushan had an amply filled stomach and displayed the lowest absorption of bile at 380 nm (Table 3.7), meaning that bile was secreted for digestion of the food and freshly produced. Hence, less concentrated bile was measured. Overall, in comparison fish from the sites Baijiaxi and Yunyang displayed the lowest contamination levels (Table 3.7). The results of the present study were compared to concentrations of PAH metabolites in fish caught in freshwater and coastal habitats from other countries. The mean PAH metabolite concentrations in eel from European countries (range of means; 1-OHPyr: 56 – 3,200 ng/mL; 1-OHPhe: 70-495 ng/mL) are in the same order of magnitude than those found in Pelteobagrus vachellii obtained in this study (means: 1-OHPyr: 1,087 ng/mL; 1-OHPhe: 136 ng/mL) (Ruddock et al., 2003; Nagel et al., 2012; Kammann et al., 2014; Szlinder-Richert et al., 2014). To evaluate the measured PAH metabolite concentrations with respect to a possible harm of PAH contamination for the organisms, the results were compared to internationally agreed threshold values for fish. 12 of 17 investigated pool samples exceeded the threshold value for 1-OHPyr for cod (Gadus morhua) of 438 ng/mL bile, and 9 of 17 pool samples exceeded the threshold value for turbot (Scophthalmus maximus) of 909 ng/mL (ICES, 2012). Even if this threshold has been calculated for another fish species, this comparison indicates that the PAH contamination in fish from China is of significance and may rise concern for the fish health in some cases. To the authors knowledge no such thresholds exist for freshwater fish. However, this comparison gives only an impression on single contaminants and might not reflect the overall situation of organic pollution. Although, a good relationship between PAH content of resuspended sediment, uptake and biliary PAH metabolites could be demonstrated in rainbow trout by Brinkmann et al. (2013). Further, the cited threshold values provide only a rough guidance because they have been calculated for marine fish. Beyond, due to the low number of available replicates these results should only be considered as indications.

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Nutrition status and condition of P. vachellii

The determined bile color (A380) also allows an interpretation of the short term nutrition status of the fish catched in campaign 2012, as an increase in concentration of bile pigments can be expected during starvation periods of fish (Richardson et al., 2004; Kammann et al., 2014). Fish from Wushan (WU 2012) and Yunyang (YUN 2012) seemed to have the best status, while fish from Kaixian (HF 2012) and Fengdu (FEN 2012) appeared to have not eaten for the longest time in comparison (Table 3.7). Significant differences could be found between WU 2012 with FEN 2012, HF 2012 and CNG 2012, as well as between YUN 2012 with FEN 2012 and HF 2012, in addition to FEN 2012 compared to BJX 2012 (p < 0.05). However, the long-term condition of the fish can be judged based on the Fulton condition factor (k-value; here: total length) (Table 3.7). It ranged from 0.75±0.08 (FEN 2012) to 1.01 ± 0.08 (CNG 2012) in 2012 and from 0.83 ± 0.10 (HF 2013) to 0.96 ± 0.12 (CNG 2013) in 2013. Significant differences could be determined between FEN 2012 with CNG 2012, HF 2012 and BJX 2012, further between CNG 2012 with YUN 2012 for samples from campaign 2012. For samples from campaign 2013 significant differences could be registered between CNG 2013 and HF 2013, as well as between HF 2013 and the reference BJX 2012 (p < 0.05). Both campaigns showed almost matching k-values, which underlines the consistency of this parameter over the years. Apparently fish from Chongqing seemed to be in the comparably best fed condition, whereas fish from Fengdu exhibited the comparably less fed status. The better nutrition status at Chongqing may be regarded to a higher supply of food the catfish can prey on, due to a higher supply of organic matter with origin in elevated levels of wastewater in the highly urbanized area. To the authors knowledge no k-values are available for wildlife Pelteobagrus vachellii from other studies so far. Zeng et al. (2010) computed condition factors (total length) between 0.92 ± 0.06 and 1.00 ± 0.10 for stocked juvenile Pelteobagrus vachellii under various stocking density conditions. The weight varied between 26.7 ± 7.9 g and 41.6 ± 12.8 g, the total length was in a range of 137.8 ± 13.1 mm to 159.5 ± 15.9 mm and the age was about five months (May to October). The common length of Pelteobagrus vachellii is given as 210 mm (Nichols, 1943), and the maximum body length as 300 mm (Chan and Ho, 2011). Based on this, the fish catched in this study can be classified to be in a juvenile to adult state, as average size and weight slightly exceeded the values by Zeng et al. (2010) (Table 3.1). It is further supported by the observation that the reproductive organs – testes and ovaries – in most of the fish of this study showed only ______123

Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______incomplete maturation. A reduced abundance of larger fishes could be observed during the sampling, which stands in accordance with Chen et al. (2009), who described a shift in the age structure of the catch from the upper Yangtze River – including the TGR region – from proportional decrease of older fish and an increase of juveniles and younger adult fish. Although k-values consider allometry of growth, and thus differ with age, a comparison with the k-values obtained in this study (k = 0.90 ± 0.13; total length) hints at a rather less good condition of the fish from the TGR (Table 3.1).

Table 3.8 Fulton condition factors (k-values) of Pelteobagrus vachellii and other catfish species catched from fish stocks or as wildlife. Condition factors are given as mean values with standard deviation; sexes are given as female (f), male (m) or combined (c; male plus female); Condition factors were calculated with total length (tot) or standard length (std). Country Site Month Species (family) Sex k-value Reference China River, May Pelteobagrus vachellii C 0.90 ± 0.13 (tot) This study lake (2012) (Bagridae) 1.40 ± 0.18 (std) China River, May Pelteobagrus vachellii C 0.90 ± 0.13 (tot) This study lake (2013) (Bagridae) 1.40 ± 0.25 (std) China Stock October Pelteobagrus vachellii C 0.92 ± 0.06 up (Zeng et al., 2010) (Bagridae) to 1.00 ± 0.10 (tot) Bangladesh River Year Mystus vittatus (Bagridae) F 2.32 (std) (Hossain et al., 2006)

Bangladesh River Year Mystus vittatus (Bagridae) M 2.20 (std) (Hossain et al., 2006)

Bangladesh River May Gagata cenia (Sisoridae) C 1.88 ± 0.22 (std) (Chaki et al., 2013)

Nigeria Lagoon Year Chrysichthys nigrodigitatus C 0.81 ± 0.20 (tot) (Fafioye and Oluajo, (Bagridae) 2005) Nigeria Lagoon Year Chrysichthys walker C 1.15 ± 0.96 (tot) (Fafioye and Oluajo, (Bagridae) 2005) Nigeria Lagoon Year Clarias gariepinus C 0.79 ± 0.15 (tot) (Fafioye and Oluajo, (Clariidae) 2005) For means of further interpretation, k-values were compared to other catfish species catched in rivers and lagoons. Overall, k-values (standard length) obtained in this study were clearly lower compared to those of M. vittatus and G. cenia, as well as the k-value (total length) of C. walkeri. Comparable k-values (total length) could be shown for C. nigrodigitatus and C. gariepinus (Table 3.8). Variations between species, gender, age and season have to be considered. Hossain et al. (2006) could register a significant difference (p < 0.05) between sampled males and females,

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Induction of hepatic EROD and GST activities in P. vachellii

As bile analysis showed a presence of PAHs in the fish, activities of Ah receptor mediated phase I (EROD) and phase II (GST) biotransformation enzymes were tested in excised liver samples from P. vachellii. The mean EROD activities for samples from campaign 2012 (n=10) and 2013 (n=20) ranged – with exception of Chongqing – from 2 ± 2 pmol/mg/min (HF 2013; IF 0.6) to 4 ± 3 pmol/mg/min (HF 2012; IF 1.2) and showed no significant differences (p > 0.05) compared to the reference site Baijiaxi River (BJX 2012; 4 ± 3 pmol/mg/min). However, samples from Chongqing stood out significantly both from campaign 2012 (CNG 2012; 13 ± 9 pmol/mg/min; IF 3.6; p < 0.05) and campaign 2013 (CNG 2013; 19 ± 10 pmol/mg/min; IF 5.1; p < 0.001) in comparison to the reference site. The difference between the years comparing the samples from Chongqing are not significant (p > 0.05) (Fig. 3.6). Further, the mean GST activities varied between 133 ± 56 nmol/mg/min at Fengdu (FEN 2012) to 168 ± 32 nmol/mg/min at Wushan (WU 2012) from samples of campaign 2012 (n=10), and between 143 ± 48 nmol/mg/min at Chongqing (CNG 2013) to 173 ± 49 nmol/mg/min at Kaixian (HF 2013) from samples of campaign 2013 (n=20). However, none of the samples exhibited a significant difference compared to the reference site (p > 0.05) (Fig. 3.6). The results claim no significant correlation between the biliary 1-OHPyr content and the hepatic EROD or GST activities, as well as between the EROD and GST activities themselves (campaign 2012: p > 0.05). A reasonable explanation for a lack of correlation despite present PAH metabolites could be that pyrene – the precursor to 1-OHPyr – is not regarded as an EROD inducer as described by Bols et al. (1999) in RTL-W1 cells. In contrast, Hudjetz et al. (2014) could show a similar but less pronounced gradient for EROD induction compared to 1-OHPyr increase in bile of rainbow trout exposed to sediment spiked with 16 PAHs in various concentrations in a laboratory experiment. 1-OHPyr is therefore considered as an indicator for PAH presence, but not as a guarantor for AhR inducers.

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Fig. 3.6 Hepatic EROD (A) and GST (B) activities in excised livers from Pelteobagrus vachellii determined for sampling campaign May 2012 and May 2013. Symbols represent individual animals, bars the mean value, and error bars the standard deviation; (°) = May 2012; dotted (•) = May 2013; dotted lines indicate different sites, double dotted lines separate sites along the Yangtze River mainstream and the TGR watershed; 2012 samples n = 10; 2013 samples n =20; Asterisks mark significant differences between samples and reference (BJX 2012); Data was statistically analyzed with Mann-Whitney Rank Sum Test, * = p < 0.05, *** = p < 0.001, a = significant difference to reference site; significant difference to same site in May 2012 were calculated but no significances could be determined.

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However, the biliary content of hydroxylated PAH metabolites at all sites demonstrate that PAH parent compounds must have been present and undergone metabolism. Brinkmann et al. (2013) proposed a biomarker cascade for rainbow trout exposed to resuspended PAH-spiked sediment, in which the peak in EROD induction preceded the peak of biliary PAH metabolites 1-hydroxypyrene, 1-hydroxyphenanthrene and 3-hydroxybenzo[a]pyrene. This is consequential, because CYP1A belong to the phase I enzymes that catalyze the reaction from the parent compounds to the metabolites, which then accumulate in the bile. Thus, a temporal variable may also be included into the situation: a peak exposure might have passed, followed by EROD induction in the liver. While an elevated enzymatic activity could not be observed anymore at the time point of sampling, the products still could be detected in the bile. A loss of EROD activities as a result from inconsistent cooling can be excluded due to the registered GST activities obtained from the same S9-mix. Furthermore, although it has been shown that several PAHs induce EROD and GST activity in liver tissues, basal activities could be determined for both systems under absence of PAHs. Moreover, GST induction appeared to be very modest compared to CYP1 expression (Pushparajah et al., 2008a; 2008b). The background activity of the enzymes may as well be regarded to a chronic exposure to low levels of xenobiotics. However, it can be concluded that the CYP1A system was not activated to a significantly higher degree at most of the sites with regard to the Pengxi River Nature Reserve as reference site (BJX-R) at the time point of sampling. In contrast to that, the highly industrialized area of Chongqing city triggered a significant up- regulation of AhR-mediated CYP1 enzymes in fish from campaign 2012 and 2013. This elevated level of activity in phase I of the hepatic detoxification system hints at environmental stressors at this site, including AhR agonists. Moreover, as a consequence the catalytic activity of CYP1A enzymes can lead to a bioactivation of present PAHs to reactive intermediates, which in their turn can induce mutagenicity (Penning et al., 1999). Significant genotoxic impacts could be observed on erythrocytes of Pelteobagrus vachellii from Chongqing (campaign 2013), as well as from Hanfeng (campaign 2012 and 2013) determined with the micronucleus assay, although samples from Chongqing from campaign 2012 turned out to be not affected significantly. Overall, whereas the in vitro EROD induction assay with RTL-W1 cells could be linked to the PAH content in the sediment extracts, in situ hepatic EROD and GST activities could not be directly correlated to the results from the bioassay and chemical analysis obtained in parallel in campaign 2013. At the site Chongqing city, the in vitro EROD induction by sediment extracts

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Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______ranged between 0.9 (TGR-B) to 4.2 fold (YAN-C) – with a mean of 3.0 fold –, and at Kaixian from 1.0 (HAN-B) to 9.5 fold (HAN-C) – with a mean of 4.9 fold – when set in relation to the reference (BJX-R). The hepatic EROD activity of the benthic P. vachellii, which should be in close contact with the sediment, showed a 5.1 fold induction at Chongqing (CNG 2013) and 0.6 fold at Kaixian (HF 2013) compared to the reference (BJX 2012). Moreover, integrating the temporal and spatial variations, by creating a mean of all in vitro EROD inductions (sediment), as well as a mean of all in situ hepatic EROD inductions (fish) of samples from the site Chongqing (campaigns 2011, 2012 and 2013), showed a 4.1 fold in vitro EROD and 4.6 fold hepatic EROD induction relative to the respective reference. At Kaixian, the same approach resulted in a 6.2 fold in vitro EROD and 0.9 fold hepatic EROD induction. So, a relationship may apply for the situation at Chongqing, but not for Kaixian. The discrepancy at Kaixian may be regarded to the strong spatial variation of pollution between the upper and lower part of the Hanfeng Lake as registered in 2013. As the catch was obtained from the fishermen at the middle of the lake, the main origin of the fish may as well be the less polluted upper section, where the in vitro EROD induction was found to be 1.0 fold (HAN-A, HAN-B) relative to the reference, which is similar to the EROD induction found in the fish. Additional monitoring would be required to verify this observation. In general, increase and decrease of EROD and GST activities are transient (Pesonen et al., 1987; Brinkmann et al., 2013) and the complex situation including bioavailability of the compounds, exposition and uptake pathways of the fish in combination with metabolization and secretion processes aggravate a direct correlation.

Histopathological evaluation of P. vachellii

Liver and kidney play a crucial role in the biotransformation and secretion of xenobiotics (Pesonen et al., 1987), hence they are frequently exposed to these compounds under contamination conditions. In their turn xenobiotics may leave their mark on the organs, which are therefore well suited for histopathological investigations, as applied for fish from campaign 2012. In the kidney, infiltration with macrophages and giant cells (Chongqing, Fengdu, Wushan), multifocal accumulation of macrophages (Yunyang, Wushan), degeneration of melanomacrophage aggregates (Chongqing), as well as deposition of foreign material – probably degenerated parasitic structures (Chongqing) could be observed (Fig. 3.7). However, integrated over the whole catch of fish none of the mentioned criteria was significant in its grade of

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Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______manifestation compared to the reference site (BJX 2012; p > 0.05). Still, these criteria can be related to inflammatory reactions as a response to foreign material in the tissues, e.g. parasites. At Chongqing, 30%, as well as Fengdu, 10%, and Wushan, 10% of the catch, were afflicted by the trematode Isoparochis hypselobagri (Billet, 1898), which infested the swim bladder next to the kidney, and the digestive tract. The abundance of parasites at Chongqing could be verified in campaign 2013 (CNG 2013; 20%). The appearance of the trematodes also closely correlated with the occurrence of infiltration with macrophages, degeneration of melanomacrophage aggregates and deposition of foreign material in the livers of fish from Chongqing. Furthermore, lymphoid tissue around biliary tracts (Fengdu, Yunyang, Hanfeng Lake, Baijiaxi River) and bile duct proliferation (Yunyang, Hanfeng Lake, Baijiaxi River), as well as degeneration of hepatocytes and bile duct epithelial cells, expressed by karyopyknosis (Baijiaxi River) could be observed in the liver (Fig. 3.7). These primarily account for more chronic degenerative and inflammatory reactions. Here also, integrated over the whole catch of fish, none of the mentioned criteria was significant in its grade of manifestation compared to the reference site (BJX 2012; p > 0.05). Although not significant, the observed effects can indicate negative environmental impacts. The recorded macrophage aggregates are reported to increase in size and frequency in conditions of environmental stress and have been proposed as biomarkers for water quality for pollution and deoxygenation as reviewed by Agius and Roberts (2003). Camargo and Martinez (2007) as well as Belicheva and Sharova (2011) ranked macrophage aggregates among other structural changes to responses to pollution obtained from in situ studies at a Brazilian urban stream and a Russian reservoir under anthropogenic influence.

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Fig. 3.7 Plate of histological alterations of liver and kidney with surrounding tissue from Pelteobagrus vachellii from sampling campaign May 2012. A. Chongqing, degenerated macrophage center (star), intermixed multiple structures with a cyst-like wall and a homogenous degenerated center (open and closed arrowheads), interpreted as foreign material, probably degenerated parasitic structures, macrophages, lymphocytes and plasma cells infiltrating the surrounding fibrous tissue (arrow), H&E, bar=50m; B. Fengdu, multiple structures resembling parasitic eggs with a yellowish wall and an eosinophilic homogenous center (open arrowheads), surrounded by a moderate amount of macrophages (closed arrowhead), embedded in a macrophage center (star), H&E, bar=25m; C. Wushan, liver, unaffected liver structure with bile duct (arrow), H&E, bar=25m; D. Baijiaxi, liver, bile duct proliferation with degeneration of bile duct epithelial cells expressed by karyopyknosis (arrowheads), surrounded by fibroblasts (fibrosis) and lymphocytes, plasma cells and fewer macrophages (arrows), H&E, bar=25m; E. Baijiaxi, kidney, unaffected kidney structure, H&E, bar=25m; F. Wushan, kidney, infiltration with mainly macrophages in the interstitial tissue (open stars), surrounding a vessel, multiple inflammatory cells in the vessel lumen, H&E, bar=25m.

Further, pollution can suppress the immune system of fish, thus increasing the chance for parasites to enter and settle in the host, if the host is more susceptible to the pollution than the parasite (Khan and Thulin, 1991; MacKenzie et al., 1995). PAHs and phenols have been shown ______130

Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______to suppress non-specific cytotoxic cells in teleosts, which are relevant in the immunologic defense from xenogenic targets, like certain fish parasites (Graves et al., 1985; Faisal et al., 1991; Seeley and Weeks-Perkins, 1997; Taysse et al., 1998; Reynaud and Deschaux, 2006). Beyond, Möller et al. (2014) demonstrated that PAHs are distributed into the immune organs head kidney and spleen in fish, and that those possess the capability to metabolize PAHs to potentially immunotoxic metabolites, which in their turn may diminish immunological responses (Khan and Thulin, 1991). Further, an increased pollution of the water with organic material can cause an increased reproduction of macroinvertebrates that can serve as intermediate hosts. According to Manna and Das (2003) the freshwater snail Indoplanorbis exustus (Deshayes, 1834) is a first intermediate host of I. hypselobagri in India. An increased amount of intermediate hosts allow a propagated reproduction of the parasites, consequently causing increased infection rates in the fish. The comparably higher frequency of parasites found at the strongly industrialized site of Chongqing, hence may be used as an indicator for environmental degradation (Khan and Thulin, 1991). The registered karyopyknosis at the Baijiaxi River, which indicated apoptosis of hepatic cells, further suggests effects of pollution also on the fish from the reference site. Hepatic lesions could be significantly correlated in other studies to total PAHs in bottom sediment, as well as metabolite concentrations in marine fish (Krahn et al., 1986; Landahl et al., 1990). However, the exposure must have been some time prior to the sampling, as all other analyses suggest only a minor influence of the site on the fish and the organ lesions show a more chronic character. One reason could be a local discharge, another an influence of the reservoirs backwater on the Baijiaxi River from downstream, due to the rise of the water level in the winter months prior to the sampling campaign in May 2012. Holbach et al. (2013; 2014) recorded a reversion of the flow direction at the Daning and Xiangxi River – both main TGR tributaries – forced by the reservoirs increasing water level. The Pengxi River, in which the Baijiaxi River discharges into further downstream of the reference site BJX-R, connects the city of Kaixian – as well as several industrial sites along its shore – with the TGR and exhibits frequent shipping activities (Fig. 3.1). As transitions in water level can be even noticed in the Pengxi-Baijiaxi River system, as consequence of the reservoirs water level fluctuation, it is suggested that a potential contamination in Pengxi River may be pushed upstream into the Baijiaxi River and affected the fish.

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Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______

3.4.5 Responsible compounds in the in vitro/in vivo assays

In order to identify the individual contribution of the analyzed compounds to the observed effects so called toxicity equivalents (TEQs) can be calculated in relation to a strong inducer. Although all 16 priority PAHs may induce adverse effects, not all of them are mutagenic. Particularly those with more than four rings possess adverse mutagenic properties, but often also require metabolic activation (Penning et al., 1999). Out of the selected 16 priority PAHs eight possess mutagenic activity in the Ames assay (Table 3.3 and Table 3.4). The proportion of those in the sediment samples from September 2011 range from 43% (CNG-U) to 79% (YUN-D), with a mean of 55 ± 9%. The proportion in the samples from May 2013 range from 39% (JIA-A; JIA- B) to 59% (YAN-A), with a mean of 48 ± 8% at Chongqing, and 36% (HAN-A; HAN-B) to 49% (HAN-C), with a mean of 42 ± 7% at Kaixian. As each PAH has a specific mutagenic activity, the individual inductions can be calculated in relation to a strong mutagen, e.g., benzo[a]pyrene. However, the application of benzo[a]pyrene toxicity equivalency factors (BEP or BaP-TEQ), according to Madill et al. (1999), also resulted in no clear trend between the activity of detected compounds and the triggered effects. Therefore, it is suggested that non-target compounds may play a significant role in the induction of mutagenicity (Brack et al., 2005). In other studies about 10 – 20% of the total mutagenicity was attributed to PAHs and the rest to non-target compounds (Chen and White, 2004; Aouadene et al.,

2008). The cytotoxicity in several samples, with comparably low PAHΣ16 content (JIA-A, JIA-B), and the direct mutagenicity (-S9) in sample CNG-D, corroborates the assumption of toxic non- target compounds with potential mutagenic properties. Only seven PAHs induce EROD activity in RTL-W1 cells. The proportion of those in the sediment samples from September 2011 range from 33% (CNG-U) to 72% (YUN-D), with a mean of 43 ± 9%. The proportion in the samples from May 2013 range from 24% (TGR-B) to 42% (YAN-A), with a mean of 34 ± 7% at Chongqing, and 23% (HAN-A; HAN-B) to 42% (HAN-C), with a mean of 32 ± 11% at Kaixian. To further identify the potential contribution of the detected PAHs to the registered EROD induction, their individual inductions were calculated as chem-TEQs in relation to the strong inducing dioxin TCDD. The chem-TEQs were compared to the bioassay derived bio-TEQ values from the EROD assay (Fig. 3.3). The detected PAHs in the sediments showed a contribution of 6-25% to the overall AhR-mediated activity in campaign September 2011 and of 1 - 23% in campaign May 2013 – assuming only additive effects. ______132

Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______

While Otte et al. (2013) could refer the majority of activity to priority PAHs in most of the samples from the Elbe Estuary, only a minority of the activity could be explained by PAHs in sediment studies about the Yangtze Estuary (max. 10%), the Tietê River (max. 7%) and most of the samples of the Danube River (max. 17%) (Keiter et al., 2008; Suares Rocha et al., 2010; Liu et al., 2014). A part of the missing activity in those studies could be explained by other persistent pollutants – like PCBs, polychlorinated dibenzodioxins (PCDDs) and -furans (PCDFs) – as isolated by multilayer fractionation (Keiter et al., 2008; Otte et al., 2013; Liu et al., 2014). However, PCBs as well as several pesticides, like atrazine, and several other compounds can be excluded as causative agents as they could not be detected in the sediment extracts of this study. Therefore, the discrepancies between bio-TEQs and chem-TEQs can be referred most likely to differences in specific induction potentials and undetected non-target compounds. Furthermore, additive, synergistic, antagonistic and masking interactions of detected and undetected pollutants impede explanations. In any case, a comparison of the high induction of effects in the FET with no particular effects in the SCA suggests only a low bioavailability of the particle-bound pollutants. This further underlines the contribution of rather lipophilic substances to the observed effects.

3.4.6 Toxicity assessment in TGR

Spatial and temporal variations of several endpoints were recorded for sediment and the darkbarbel catfish P. vachellii, from various sampling sites in the TGR mainstream, major tributaries and its watershed and are combined in Table 3.9. After the sediment assessment, more pronounced biomarker responses in the bottom-dwelling fish have been expected to find in May 2012, for example at Yunyang that was comparable to Chongqing and Kaixian in several endpoints (Table 3.9). It has to be considered that the situation in 2011 does not necessarily reflect the situation in 2012, although biliary PAH metabolites could be determined in fish from all sites. The only consistency was observed for the sites Chongqing and Kaixian, which were therefore further investigated in May 2013 for a more detailed and combined assessment of sediment and fish. Both sites revealed overall lower PAH concentrations in the sediments compared to 2011, but more pronounced biomarker responses (Table 3.9). Genotoxic impacts were particularly observable at Kaixian. EROD induction was particularly detectable at Chongqing. The identification of Chongqing city as site of particular significance in the TGR area makes sense,

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Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______due to its enormous population and industrial importance. The conspicuity of the artificial Hanfeng Lake at Kaixian may be regarded to a lesser dilution of discharged contamination compared to the TGR mainstream, and a potential accumulation of it in the lower section of the lake. Except those two “hot-spot sites” – that displayed a burden with contaminants in combination with inductions in vitro, in vivo and in situ – no clear pattern could be observed among the endpoints. Pollution patterns of PAHs at the different locations per site – that are also partly reflected by the correlated in vitro EROD induction and embryotoxic/teratogenic effects. Bioavailability of the pollutants, non-target compounds, as well as synergistic and antagonistic effects of the contaminants, in addition to uptake, metabolism and secretion pathways in the fish corroborate a direct correlation between the different endpoints. Since Pelteobagrus vachellii has proven to be a suitable monitoring species and the selected endpoints to be applicable, a continuous monitoring applying a comprehensive strategy – combining sediment, water and biota – is strongly suggested to verify the reported observations and clarify the open questions.

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Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______

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Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______

3.5 Conclusion

Under consideration of the implications the TGR faces since its impoundment – a progressive urbanization, industrialization, rising ship traffic and submersion of abandoned contaminated sites – the environmental situation during the sampling campaigns between September 2011 and May 2013 appeared to be less pronounced than expected. Although, the significant biological impairments at Chongqing and Kaixian suggest a toxicity reduction evaluation for these areas. However, most parts seem not yet to exhibit ecotoxicological effects or only to a minor degree. The TGR appears to be in a comparable or even less contaminated state than other national or international water bodies, based on detected pollution concentrations in sediment and water. As immense amounts of water and sediment are discharged into and carried by the Yangtze River, this observation may be primarily contributed to a strong dilution effect. This leads to lower concentrations and respectively less acute effects, but conceals the total chemical burden under consideration of a mass-balance approach. This means that a contamination problem of the Yangtze River is not solved, but simply relocated (Mueller et al., 2008; Floehr et al., 2013). The mass balance approach for PAHs referred to the immense sediment influx suggested a deposition of 216 – 636 kg PAH/day (0.2 – 0.6 mg PAH/m2/day) and the combustion of fossil fuels appeared to play a significant role in the emission of PAHs into the environment along the reservoir. Thus, it can be concluded that air pollution, which plays a considerable role in China (Zhang and Tao, 2009; Zhang et al., 2009b), also affects the quality of its water bodies. And as the PAHs pass several exposition pathways – air and water – they pose a risk to humans and wildlife already before they are deposited in the TGR. Compared to the external sources, like air pollution, it is suggested that the risk of internal pollution emission from the submerged sites plays only a minor role, due to the deposition of vast amounts of sediment in the TGR and the dilution effect. Further, it could be shown in this study that the bioavailability of particle bound pollutants was rather low. However, hydraulic effects like frequently occurring flood events in the Yangtze River can increase the bioavailability through remobilization of pollutants to peak situations (Stachel et al., 2005; Feng et al., 2007b; Brinkmann et al., 2010; Woelz et al., 2010b). In the same course particle bound pollutants can be relocated on agriculturally used areas of the TGR’s water fluctuation zone, which covers an elevation range of about 30 m from 145 m to 175 m altitude

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Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______

(a.s.l.). Therefore, the importance of sediments as sink and source for pollutants in the TGR needs to be considered. Especially with respect to mutagenic properties of pollutants low concentrations cannot directly be set equal to low risk, as threshold values are under discussion due to the mutagenic mode of action. The mutagenicity of sediment extracts and genotoxic impacts on the fish at particular sites along the reservoir and in its watershed have been demonstrated. This study supports the importance of mutagenic and genotoxic impacts for the quality of Chinese water bodies, e.g., as source for drinking water (Shu et al., 2002; Lv et al., 2015), and recommend their inclusion into routine monitoring programs as suggested before by Wu (2005) for marine systems. The individual outcomes of this study and the crosslinks between chemical analysis, in vitro, in vivo and in situ investigations support the important role of AhR-mediated adverse impacts. Further, it supports the role of PAHs as key pollutants in the TGR area, as also shown in other studies (Wang et al., 2009; Wang et al., 2013; Wolf et al., 2013a). As Hong et al. (2012) emphasized that urbanization effects on the environment can be recorded by PAHs in sediment, are PAHs hereby suggested as suitable marker compounds or indicators for monitoring of the reservoirs sediment pollution status. This is in accordance with Wang et al. (2013), who stated the same for the reservoirs water. However, only a certain amount of the recorded activities could be explained by the presence of PAHs. Thus, the focus should be extended further to non-target pollutants - like unsubstituted, nitro- and heterocyclic PAH derivatives and other important dioxin-like compounds - to identify more causative agents. In order to do so, bioassay-guided effect directed analysis has been proven to be a valuable tool (Brack and Schirmer, 2003; Brack et al., 2005; Hecker and Hollert, 2009). It has also to be considered that only organic pollutants were in the scope of this study. Inorganic contaminants, like heavy metals or high nitrogen levels due to excessive regional fertilization, as well as other biotic and abiotic factors, like pathogens or oxygen depletion due to eutrophication by repeatedly occurring algal blooms, may also play their part as detrimental factors. A complex variety of pathways exist that influence the reservoirs biota. The obtained results demonstrate that pollution has an effect on the fish of the TGR area. As a burden with PAH metabolites, in addition to a number of effects, like genotoxicity and Ah receptor mediated changes in metabolism, could be recorded in the fish from the identified hot- spot sites Chongqing and Kaixian. In this course, the chosen monitoring fish species Pelteobagrus vachellii, hence was proven to be well suited for biomonitoring, particularly due to

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Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______its wide distribution along the TGR and other sections of the Yangtze River. The biomarkers in combination with the chemical analysis from this study and water data from other studies however suggest rather a low-dose chronic exposure than an acute toxicity (Wang et al., 2009; Wang et al., 2013; Wolf et al., 2013a). Therefore, habitat destruction due to hydrological alterations of the TGR area and overfishing are considered to be the primary triggers of a fish decline in this region, as reported by Chen et al. (2009). With pollution currently rather playing a rather sublethal role, as an additional risk factor that promotes disorders, like cardiovascular diseases and cancer, and with an influence on the overall fitness of the fish. The shift in species composition and age structure with a decrease of adults towards younger fish, as described by Chen et al. (2009), can more likely be regarded to the altered hydrologic conditions, habitat fragmentation and shrinkage, as well as to resources overexploitation, respectively (Liu et al., 2005; Chen et al., 2009). Especially fish at early life stages are very sensitive to contamination, as demonstrated for AhR- mediated activities and PAHs (Walker and Peterson, 1991; Guiney et al., 1997; Le Bihanic et al., 2014). An interference of sublethal chronic exposure during this vulnerable stage may even lead to impairments that evolve only in the long-term (Hicken et al., 2011). Chronic exposure to lower doses of pollutants can evoke detrimental impacts on physiological, immunological and behavioral processes, consequently reducing the overall fitness and susceptibility to pathogens (Mason, 2001). The suggested rather poor condition of fish, may therefore also be regarded to an influence of sublethal pollution. The complex situation in the field and the difficulty to predict environmental hazards solely from chemical data or bioassay studies call for a holistic environmental assessment. This should include in situ approaches with higher organismic levels due to a greater ecological relevance than isolated in vitro/vivo laboratory studies, which yet in their turn allow a faster screening of environmental samples and help to identify modes of action. Those should be combined with chemical screening to deliver potential suspects for the role of the inducers. The combined application of these methods with a variety of endpoints and biomarkers was proven to be applicable and successful in this study. The recent study should represent a foundation for further monitoring programs of the area. And as an examination of the reservoirs status in the early days after its impoundment it should constitute a reference for the further development of the reservoirs environmental condition in course of proceeding urbanization and industrialization. It should help to initiate and enforce

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Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______necessary countermeasures, like improved wastewater treatment and emission standards, in time to prevent environmental degradation in the long-term and sustain this unique ecosystem.

3.6 Acknowledgements

The study has been carried out as part of the MICROTOX project (“Transformation, Bioaccumulation and Toxicity of Organic Micropollutants in the Yangtze Three Gorges Reservoir”), which is integrated into the joint environmental research program “Yangtze-Hydro - Sustainable Management of the Newly Created Ecosystem at the Three Gorges Dam” (Bergmann et al. (2012); www.yangtzeproject.de). The project has been financed by the Federal Ministry of Education and Research, Germany (BMBF) as part of the research cluster “Pollutants/Water/Sediment - Impacts of Transformation and Transportation Processes on the Yangtze Water Quality”.

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Chapter 3 The ecotoxicological impacts of Three Gorges Reservoir (TGR), China ______

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Chapter 4 4 Effect-directed analysis of aryl hydrocarbon receptor agonists in sediments from the Three Gorges Reservoir, China

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This chapter is based on a manuscript to be submitted to Environmental Science & Technology as:

Xiao, H., Krauss, Martin., Floehr T., Yan, Y., Bahlmann, A., Eichbaum, K,. Brinkmann, M.,Zhang, X,. Yuan, Y,. Brack, W,. Hollert, H. (2016) Effect-driected of aryl hydrocarbon receptor agonists in sediments from the Three Gorges Reservoir, China (in preparation)

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Chapter 4 Effect-directed analysis of aryl hydrocarbon receptor agonists ______

4.1 Abstract

The construction of the Three Gorges Dam (TGD) in the Yangtze River raises great concern in ecotoxicological research since large amounts of pollutants enter the Three Gorges Reservoir (TGR) water bodies after TGD impoundment. In this work, effect-directed analysis combining effect assessment, fractionation procedure, target and non-target analyses, was used to characterize aryl hydrocarbon receptor (AhR) agonists in sediments of the TGR. Priority polycyclic aromatic hydrocarbons (PAHs) containing four to five aromatic rings were found to contribute significantly to the overall observed effects in the area of Chongqing. The relatively high potency fractions in the Kaixian area were characterized by PAHs and methylated derivatives thereof and heterocyclic polycyclic aromatic compounds (PACs) such as dinaphthofurans. Benzothiazole and derivatives were identified as possible AhR agonists in the Kaixian area based on non-target liquid chromatography-high resolution mass spectrometry (LC- HRMS). To our knowledge, this study is the first one applying the EDA approach and identifying potential AhR agonists in TGR.

Keywords: AhR-mediated activity; EDA;target analysis; non-target analysis

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Chapter 4 Effect-directed analysis of aryl hydrocarbon receptor agonists ______

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Chapter 4 Effect-directed analysis of aryl hydrocarbon receptor agonists ______

4.2 Introduction

The Three Gorges Reservoir (TGR), created in consequence of the Yangtze River’s impoundment by the Three Gorges Dam (TGD), spreads over a distance of 663 km between the town of Sandouping, Hubei Province, and the Jiangjin district of Chongqing Municipality. Besides the obvious benefits of the TGD, such as hydroelectricity, navigation, and flood control, construction of the TGD also poses great challenges to the unique ecosystem (Shen and Xie, 2004), particularly when facing numerous anthropogenic impacts, e.g., overpopulation, industrialization, and intensified shipping activities (Floehr et al., 2015a; 2015b). Furthermore, construction of the dam reduced the river’s flow velocity (Chen et al., 2005; Wang et al., 2009), and consequently increased the sedimentation rate of suspend particles and adhering contaminants (Hu et al., 2009a). Sediments are considered as the final sink of persistent and lipophilic pollutants in the environment (Hollert et al., 2003; Hilscherova et al., 2007). They can become a potential source of pollutants through resuspension of particulate matter, e.g., during flood events (Gerbersdorf et al., 2007; Woelz et al., 2009; Woelz et al., 2010b). Frequently occurring floods in the water fluctuation zone of TGR may increase the pollutants bioavailability through remobilization and direct exposure to benthic organisms (Brinkmann et al., 2010; Woelz et al., 2010b). To ensure the environmental and public health, suitable and effective monitoring strategies of sediments in TGR are urgently demanded. Extensive chemical-analytical research has been performed on TGR, indicating that the sediments in TGR are polluted with a mixture of persistent organic pollutants (POPs), such as polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs) and polycyclic aromatic hydrocarbons (PAHs) (Floehr et al., 2013; 2015a; Deyerling et al., 2014). These compounds are known to induce cytochrome P450 1A (CYP1A) by ligand-activation of the aryl hydrocarbon receptor (AhR) (Van den Berg et al., 2006; Sorg, 2014). It has been reported that the binding of xenobiotics to the AhR can trigger a broad spectrum of adverse effects, such as on biochemistry, physiology and reproduction in many organisms (Eichbaum et al., 2014; Sorg, 2014; Schiwy et al., 2015a). Furthermore, exposure of early life stage of fish to AhR agonists may cause increased mortality in further developmental stages (Walker and Peterson, 1994; Andreasen et al., 2002; Brinkmann et al., 2013; Di Paolo et al., 2015a) and adverse outcomes in wild fish populations (Niimi, 1983; Gilbertson, 1992; Whyte et al., 2000; Van der Oost et al., 2003; Ankley et al., ______145

Chapter 4 Effect-directed analysis of aryl hydrocarbon receptor agonists ______

2010). This is of particular concern, as the Yangtze River plays an important role for fishery production in China (Liu and Cao, 1992; Liu et al., 2005). As recently shown in Chapter 2, only limited research was done on bioassays to determine AhR- mediated activity in this area. Moreover, chemical analysis of target compounds provides only limited information on adverse biological effects of complex mixture (Wang et al., 2014b; Floehr et al., 2015a). The concept of effected-directed analysis (EDA), which integrates sample extraction, clean-up, fractionation, bioassays and chemical analysis, has been demonstrated to be a suitable tool for the identification of causative toxicants in various environmental matrices (Brack, 2003; Hecker and Hollert, 2009; Luebcke-von Varel et al., 2012; Simon et al., 2013; Radović et al., 2014; Brack et al., 2015; Di Paolo et al., 2015b). In our previous studies, sampling sites along the TGR were screened according to the triad approach (Chapman, 1996) to achieve a comprehensive perspective on ecotoxicological status of this area (Floehr et al., 2015a; 2015b). Two sites– close to the cities of Chongqing and Kaixian – were identified as regional “hot-spots” with respect to dioxin-like activity and mutagenicity. The present study aimed to characterize and identify individual AhR agonist in the sediments extracts in support of the prioritization and regulation of environmental contaminants present in sediments of the TGR.

4.3 Material and methods

4.3.1 Sample collection and preparation

Sediment samples were collected using a Van-Veen sampler in September 2011. Three samples were collected at the mainstream close to Chongqing – upstream (CNG-U), downstream (CNG-D) and directly at the tributary’s inlet (CNG-T), as well as one sample from Hanfeng Lake (HF-L) in Kaixian. For detailed information see Section 3.3.1. All sediments were freeze-dried, sieved (≤ 0.2 cm), and thoroughly homogenized by using pestle and mortar. Thereafter, the applied EDA strategy followed the flowchart as shown in Fig. 4.1. Detailed information of sediment extraction can be found at Section 3.3.3. Sediments extracts were rotary-evaporated close to dryness and re-dissolved in dichloromethane (DCM) to a final concentration of 20 g sediment equivalents (SEQ) per mL DCM. Subsequently, extracts were purified by gel-permeation chromatography (GPC) (Biobeads SX3, Bio Rad) as described

______146

Chapter 4 Effect-directed analysis of aryl hydrocarbon receptor agonists ______elsewhere (Streck et al., 2008). Corresponding fractions of different runs were pooled, evaporated close to dryness, and re-dissolved in hexane: DCM (9:1; v:v) for fractionation.

Fig. 4.1 Flowchart of the applied EDA strategy. NP-HPLC: Normal Phase-High Performance Liquid Chromatography; F: Fraction; GC/MS: Gas Chromatography-mass spectrometry; LC-HRMS: Liquid Chromatography-High Resolution Mass Spectrometry; QASR: Quantitative Structure-Activity Relationships.

4.3.2 Normal phase fractionation

To reduce the complexity of the extracts, compounds were separated according to their physico- chemical properties, e.g. polarity, hydrophobicity, molecular size, planarity, and the presence of specific functional groups (Brack et al., 2003a). In the present study, the fractionation step was performed on three coupled normal-phase high performance liquid chromatography (NP-HPLC) columns, including cyanopropyl (CN), nitrophenyl (NO) and porous graphitized carbon (PGC), which has been described in detail in Luebcke-von Varel et al. (2008). Polar polycyclic aromatic compounds (PACs) were trapped on and eluted from CN column, while nonpolar compound groups were retained on NO and PGC columns. Extracts were fractioned of 40-60 g sediment equivalents (SEQ) per run. After fractionation, for one sub-fraction a solvent exchange was performed with dimethylsulfoxide (DMSO) and tested for its AhR-mediated activity as described below, the other sub-fraction was stored in amber glass vials with acetone-hexane (1:1; v:v) at -20°C for chemical analysis.

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Chapter 4 Effect-directed analysis of aryl hydrocarbon receptor agonists ______

4.3.3 Ethoxyresorufin-O-deethylase (EROD) induction assay

The AhR-mediated activity was determined by the EROD induction assay using rainbow trout (Oncorhynchus mykiss) liver cells (RTL-W1) according to previously described methods in Section 3.3.5. Each sample was analyzed in three replicates. Bioassay-derived TCDD equivalents (BEQs) were calculated by relating biological activities caused by the samples to those of the standard compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD). Moreover, the measured BEQs in fractions were compared to the TCDD equivalents (TEQs), which were obtained by multiplying the concentration of each compound in each sample by its relative potency (REP) in RTL-W1 cells (Bols et al., 1999; Brack and Schirmer, 2003) and summing up these values. This comparison enables mass-balance estimation of the contribution of the analyzed chemical contaminants to the measured AhR-mediated activity (Van den Berg et al., 2006).

4.3.4 Targeted analysis of PACs by GC-MS

The identification and quantification of PACs were performed with a GC (Agilent 6890) equipped with an auto sampler (Agilent 7683) and coupled with a mass spectrometer (Agilent

5973) with electron ionization source. Aliquots of 1 μl of sample were injected in splitless mode at an injector temperature of 250°C. After sample injection, the analytes were separated on capillary column (HP5MS, 35 m × 0.25 mm, i.d., 0.25 µm, Agilent) using helium as carrier gas at a constant flow rate of 1.3 mL/min. The oven temperature started at 60°C and ramped with 30°C/min until 150°C, then with 6°C/min up to 186°C, followed by 4°C/min to 280°C held for 21.5 min. For priority 16 EPA-PAHs, quantification was performed in selected ion monitoring (SIM) mode using external standards and corrected by means of the injection standards, i.e., 13C benzo[a]pyrene. The concentration of substituted PAHs as well as heterocyclic aromatic compounds were calculated based on methods described elsewhere (Luebcke-von Varel et al., 2011).

4.3.5 Non-target analysis in polar fractions by LC-HRMS

Because of the relatively low effects in samples CNG-D and CNG-T, we focused on characterizing the AhR agonists in the higher effects of fraction F13 – F15 in the samples of Chongqing upstream (CNG-U) and the Kaixian area (HF-L). The liquid chromatography separation was performed with an Agilent 1200 system equipped with a Kinetex Core-Shell C18 ______148

Chapter 4 Effect-directed analysis of aryl hydrocarbon receptor agonists ______column (100 mm × 3.0 mm; 2.6 µm; Phenomenex). A linear gradient elution with water and methanol both containing 0.1 % formic acid at a flow rate of 0.2 mL/min was used. The LC system was connected to an ion trap-Orbitrap hybrid instrument (LTQ Orbitrap XL, Thermo Scientific). Analytes were ionized by electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) in separate runs, both in positive and negative ion mode, respectively. Details on instrument settings are given in the supporting information (Annex II, Table II.1). Full scan spectra were recorded at nominal resolving power of 100,000 (referenced to m/z 400) at a mass range of m/z 100 – 1000. High resolution product ion spectra (HRMS/MS) were acquired data-dependent for the two most intense precursor ions of the full scan at a nominal resolving power of 15,000, using an isolation width of 1.3 m/z, a minimum precursor ion intensity of 50,000 and a dynamic exclusion time of 20 s. Both collision induced dissociation (CID) at 35% and higher-energy collisional dissociation (HCD) at 100% were used for fragmentation.

4.3.6 Processing of HRMS data for compound identification

The MZmine 2.10 software (Pluskal et al., 2010) was used for peak detection and peak lists (consisting of m/z values, retention times, and signal intensities) obtained from full scan chromatograms of the samples, solvent and processing blanks. The processing steps and settings of MZmine were given in Annex II (Table II.2). The peak lists were further processed using a R script to remove background contamination peaks occurring in solvent and processing blanks

(intensity ratio of sample to blank < 10), and those originating from background signals not resembling Lorentzian or Gaussian peak shapes (details are given in Hug et al. (2014)). To further narrow down the peak lists to containing only compounds potentially being AhR agonists, the following filtering steps were applied: (i) all peaks with retention times > 15 minutes were kept, as the active sediment fractions of interest should comprise compounds exerting certain hydrophobicity; (ii) all peaks were kept, which showed an at least five times higher intensity in the active fractions F13 to F15 than in any of the non-active fractions F16 to F18; (iii)all peaks were kept, which had a mass defect below an upper boundary defined by the molecular formula CnH (2n-6-0.5n) (n is an even number), as most AhR receptor agonists should be of polyaromatic nature or contain at least a certain number of double bonds.

This upper boundary corresponds to 6.5 double-bond equivalents (DBEs) for n = 10 and

7.0 for n = 12, etc. ______149

Chapter 4 Effect-directed analysis of aryl hydrocarbon receptor agonists ______

The filtered peak lists of potential AhR agonists of the active fractions were processed using the R “nontarget” package (Schymanski et al., 2014b; Loos, 2015). This allowed searching for bounds of isotope peaks (13C, 15N, 34S, 37Cl, and 81Br) with a rule-based algorithm and peaks of + + + +   relevant adducts (M+H , M+Na , M+K , M+NH4 in positive mode; M-H [M+formate] in negative mode) considering single and double charged ions. Peaks were finally grouped into components, encompassing the monoisotopic peak and its associated isotope or adduct peaks representing an individual chemical compound. The package’s settings are given in the Annex II (Table II.3). Starting from these annotated component lists, a determination of molecular formulas was done based on the raw data file using the QuanBrowser of the Xcalibur software (Thermo Scientific). For obtained molecular formulas, the Chemspider compound database (Royal Society of Chemistry 2015) was searched to retrieve candidate structures. Plausible candidate compounds were identified based on the number of references in Chemspider as an indicator of human use and commercial importance. For these compounds reference standards were obtained if possible and used for confirmation based on retention times and MS/MS spectra. For compounds without reference compound, a tentative identification was done based on an interpretation of MS/MS spectra in comparison to those of known compounds.

4.3.7 QSAR modeling usingVirtualToxLab

The AhR binding affinities of candidate compounds were simulated by VirtualToxLab. The VirtualToxLab is an in silico tool for predicting the toxic potential of chemicals by simulating and quantifying their interactions towards a series of proteins, which are known to trigger biological effects using automated, multi-dimensional QSAR (Vedani et al., 2012; 2015).

4.3.8 Statistical analysis

Statistical analyses were performed using SigmaPlot 12.0 (Systat Software Inc). Non-parametric

Shapiro-Wilk ANOVA on ranks followed by Holm-Sidak’s post hoc test (p ≤ 0.05) were used to determine significant differences in the EROD induction of fractions compared to the process control.

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Chapter 4 Effect-directed analysis of aryl hydrocarbon receptor agonists ______

4.4 Results and discussion

4.4.1 Bioassay-derived induction equivalent quantities

AhR-mediated activity of all parent- (par) and sub-fractions, reconstituted extracts (rec), and arithmetic sum (sum) of all fractions was examined in the EROD assay with RTL-W1 cells (Fig. 4.2). BEQs showed significant fraction-specific differences, but followed the same relative distribution pattern in all four samples. The observation was in accordance with other studies which applied the same automated on-line fractionation method as the present study (Kaisarevic et al., 2009; Luebcke-von Varel et al., 2011; Woelz et al., 2011). The fractions F3 – F5, co-eluting with typical AhR agonists such as polychlorinated naphthalenes (PCNs), coplanar PCBs, polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs), caused no or very low AhR-mediated activity, suggesting low levels of these compounds in TGR sediments. This result was in accordance with previous findings that only low levels of PCBs and PCDD/Fs were detected in water and sediments of the TGR (Wang et al., 2009; Wolf et al., 2013a; Floehr et al., 2015b). Significantly greater EROD inducing potency (p ≤ 0.05) was detected in fractions F7 –F10, characterized by PAHs with four to six aromatic rings, as well as fractions F13 – F15, mostly characterized by intermediately polar to polar PACs. The highest BEQs were detected in HF-L, ranging from 42 to 177 pg BEQ/SEQ g, and 69 to 109 pg BEQ/SEQ g in fractions F7 – F10 and F13 – F15, respectively.

______151

Chapter 4 Effect-directed analysis of aryl hydrocarbon receptor agonists ______

1200

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Fig. 4.2 Aryl hydrocarbon receptor-mediated activities of parent (par), reconstituted (rec) extracts, arithmetic sum of fraction BEQs (sum) and fraction activities (1-18) of four sediment extracts. Asterisks denote significant differences between fractions and process control (Non-parametric Shapiro-Wilk ANOVA on ranks with Holm-Sidak’s post hoc test, p≤0.05).dw: dry weight. N.A.-No data available.

In order to determine eventual losses of activity during sample processing, all fractions were combined to form reconstituted extracts and were tested with the same procedure. Recovery was estimated on the basis of reconstituted fractions of the parent extracts, and was 67 % for HF-L, 57 % for CNG-T , and 93 % for CNG-D . Due to limited sample amount, BEQs of the parent extract CNG-U were not available, and thus no recovery could be determined. Our results indicated good chemical recoveries, in which typical chemically analyzed standard compound concentration recoveries of 60-80 % were reported (Brack et al., 2002). The total response to exposure with these fractions was site-specific. The sample from the mainstream (CNG-U) exhibited higher levels of contaminants than the samples originating from tributaries (CNG-T) and downstream regions (CNG-D), but with similar fractionation patterns. Thus, higher levels of contamination in the upstream of Yangtze river at Chongqing area may be ______152

Chapter 4 Effect-directed analysis of aryl hydrocarbon receptor agonists ______diluted by sediments of the less contaminated tributary, the Jialing River. This results in lower concentrations of contaminants downstream of Chongqing, which is in agreement with previous findings (Wang et al., 2014a; Floehr et al., 2015a). The artificial Hanfeng Lake at Kaixian exhibited the highest AhR-mediated activity, which could be attributed to a lower dilution of discharged contamination compared to the Chongqing area, and a potential accumulation of contamination in the lake (Floehr et al., 2015a).

4.4.2 Target chemical analysis

The PAH fractions (F7-F10), which exhibited significant EROD induction activity (Fig. 4.2), were selected for priority PAH analysis. Moreover, the highest potency fractions HF-L-F9 – F10 were scanned for methylated PAHs as well as heterocyclic aromatic compounds (Annex II, Table II.4). The fractions in the Chongqing area exhibited similar PAHs profiles, which were characterized by four- and five-ringed PAHs. In addition, the concentration of CNG-U showed a higher contamination than the tributaries (CNG-T) and downstream (CNG-D), which is in accordance with the bio-analytical results of the present study. The fractions from the Kaixian area showed a contamination with pyrogenic five- and six- ringed PAHs, together with some methylated PAHs, and their oxygen (O-) and sulfur (S-) heterocyclic aromatic compounds. The total priority EPA-PAHs ranged from 9 – 101 ng/g sediment (Annex II, Table II.4). Among the parent PAHs, fluoranthene was measured at the highest concentrations (56.6 ng/g sediment in CNG-U-F7). Levels of PAHs with EROD induction capacity (Bols et al., 1999), including benzo[k]fluoranthene, dibenzo[a,h]anthracene, benzo[a]pyrene, indeno[1,2,3-c,d]pyrene, benzo[b]fluoranthene, chrysene, and benzo[a]anthracene were detected up to 40.2 ng/g sediment. The concentrations of heterocyclic aromatic compounds were detected up to 5 ng/g sediment. The analytical results revealed that relatively low levels of chemical contamination in TGR, which are one to two orders of magnitude lower in comparison to the European river systems (Brack and Schirmer, 2003; Kaisarevic et al., 2009; Grund, 2011).

4.4.3 Contribution of PACs to the EROD induction potency of fractions

To estimate the fraction of activity explained by priority PAHs, we compared TEQs, calculated using the REP values given by Bols et al. (1999), to BEQs from EROD assay (Fig. 4.3). The results revealed that the contribution of priority PAHs contributed up to 43% of the AhR- mediated activity of responding fractions. Low contributions of chemically derived TEQs were

______153

Chapter 4 Effect-directed analysis of aryl hydrocarbon receptor agonists ______found in F7 and F8 of CNG-U, where priority PAHs such as fluoanthene and pyrene were detected, which have been reported as non-inducers and weak AhR-agonists in previous studies (Bols et al., 1999; Machala et al., 2001). Four to six ringed parent PAHs such as benzo[a]anthracene, chrysene, benzo[b+k]fluoranthene, benzo[a]pyrene were commonly found in F9, and indeno[1,2,3-cd]pyrene, dibenz[a,h]anthracene were found in F10 of CNG-U and HF-L. Especially the four- to six- ringed PAHs, which have been confirmed as AhR agonists (Bols et al., 1999; Machala et al., 2001; Bosveld et al., 2002), accounted for 43% of the BEQs in CNG-U-F9, thus likely being the main contributors. Similar results were observed in CNG-D-F9 (28%) and CNG-T-F9 (40%). In the significant high potency of fractions HF-L-F9 and HF-L-F10, the priority PAHs accounted for only about 9% and 12% of BEQs.

Fig. 4.3 Chemical analyzed priority PAHs-TEQs, BEQs as well as the calculated contribution of PAHs (percentage) to the EROD induction in the selected fractions (based on EROD assay results).

In the faction of HF-L-F10, we hypothesize that me-PAHs and heterocycles detected may account for a part of the observed effects in the fraction. Dinaphtho[1,2-b;1’,2’-d]furan and Dinaphtho[1,2-b;2’,3’-d]furan were identified as potent inducers of EROD acitivity in the RTL- W1 cell line (Brack and Schirmer, 2003) and rat hepatoma H4IIEGud.Luc 1.1 cell line (Vondráček et al., 2004). Based on the REP values from Brack and Schirmer (2003), the TEQ of dinaphthofurans was calculated as 8.7 pg TEQ / g SEQ, corresponding to 7% of the BEQ in HF- L-F10. Methylated benzo[a]pyrenes were detected in the fraction. Due to too many isomers

______154

Chapter 4 Effect-directed analysis of aryl hydrocarbon receptor agonists ______present in the sample, concentrations of each compound were hard to quantify. Methylated PAHs were shown to significantly induce AhR-mediated activity in reporter gene assays (Machala et al., 2008), and have been reported to be responsible for large portions of AhR activity in sediments (Brack and Schirmer, 2003; Kaisarevic et al., 2009). Methylated benzo[a]anthracenes were reported to be significantly more potent AhR inducers than their parent compounds (Marvanová et al., 2008). Brack and Schirmer (2003) identified methylation of chrysene in the 1-position and of benz[a]anthracene in the 9-position to enhance the EROD induction potency by one to two orders of magnitude. Thus, we hypothesize that methylated benzo[a]pyrenes play a role of the AhR-mediated activity in the bioactive fraction. Due to a lack of respective REP values, the AhR binding affinities of heterocyclic PACs were simulated using QSAR (Annex, Table II.4). Most of PACs were predicted to have a binding affinity to the AhR, thus taken as tentative compounds. To confirm the contribution of PACs, the relative potency of heterocycles compounds should be an aim of further research.

4.4.4 Non-target analysis of polar fractions

In addition to the high potency fractions F7 to F10, the polar fractions, mainly eluting with mono- nitro- PAHs (F13) as well as (hydroxyl-) quinones, keto-, dinitro-, hydroxyl- PAHs and N- heterocycles (F14 – F15) showed relatively high AhR-mediated effects. Thus, we also focused on characterizing the AhR agonists in polar fractions (F13 – F15) using LC-HRMS. After peak detection using MZmine 2.10 and the removal of background peaks, typically several hundred peaks remained in the peak lists for ESI+ and APCI+ mode, except for fraction HF-L- F14, in which about 60 and 40 peaks could be detected (Annex II, Table II.5). In APCI- and particularly ESI- mode, a significantly lower amount (< 90) of peaks remained. The numbers of peaks were further reduced by an additional filtering step targeting likely AhR agonists based on retention times, mass defects and absence in the non-active fraction F16 – F18 (Annex II, Table II.5). Nevertheless, in individual fractions more than 100 candidate peaks remained. Thus, priority for identification was given to the most intense peaks in the active fraction HF-L-F13.

4.4.5 Identification and selection of candidate AhR agonists

Based on accurate mass and isotope patterns, a plausible molecular formula could be determined in ESI+ mode of all intense peaks ( > 106 a.u. intensity) in fraction HF-L-F13 (Annex II, Table II.6). No additional compounds could be detected in APCI+ mode. While benzothiazole,

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Chapter 4 Effect-directed analysis of aryl hydrocarbon receptor agonists ______

2-mercaptobenzothiazole and 2-(methylthio)benzothiazole were confirmed by reference standards, MS/MS spectra of several other compounds suggests the occurrence of further benzothiazole derivatives as indicated by fragments characteristic for a benzothiazole or a mercaptobenzothiazole moiety (Annex II, Table II.6, Fig. II1-5). Benzothiazole derivatives are widely used as vulcanization accelerators and antioxidants in rubber (De Wever and Verachtert, 1997; Wik, 2007) and have been detected in water and sediments (Kumata et al., 2000; Kloepfer et al., 2005; Hug et al., 2014). The compound at m/z 300.9923 and RT 27.4 was tentatively assigned as 2,2’-sulfanediylbis-(1,3-benzothiazole), which could be a by-product of mercaptobenzothiazole use. Two isobaric peaks at m/z 239.0669, RT 16.2 and 16.7, respectively, showed almost identical MS/MS spectra with an intensive fragment at m/z 72.0803, which corresponds to a C4H10N group (Annex II, Fig. II.3). This suggested N-t- butyl-2-benzothiazolesulphenamide, which is a vulcanization accelerator as well, and a closely related isomer as further plausible candidates. He et al. identified benzothiazole as AhR-active compounds in tire extracts by a toxicant-identification-evaluation (TIE) approach (He et al., 2011b). The study presented the ability of 2-mercaptobenzothiazole to induce AhR-dependent gene expression, as well as benzothiazole to be a weak AhR agonist in mammalian cell bioassays. In a previous study, both 2-mercaptobenzothiazole and benzothiazole were shown as a relative potent AhR agonist of the human AhR expressed in yeast (Noguerol et al., 2006). Another group of compounds in fraction HF-L-F13 were likely benzylamines, among them the confirmed compound tribenzylamine, and several other compounds showing similar MS/MS + + spectra with characteristic fragments C14H16N and C7H7 (AnnexII, Table II.6, Fig. II.5) were found. The complete absence of the C6H7N+ ion (m/z 93.0573) suggests that no aniline structure was present in these molecules. Among the tentative identified toxicants, 2-mercaptobenzothiazole, benzothiazole 2-(methylthio)benzothiazole and tribenzylamine were predicted to have a binding affinity to the AhR by QSAR (Table 4.1). Thus, we assume benzothiazole and its derivates to be important pollutants in the Kaixian area.

______156

Chapter 4 Effect-directed analysis of aryl hydrocarbon receptor agonists ______

a

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vulcanization

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251 186 372 278 422 398 276

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167 135 238 181 300 287 197

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a

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Chapter 4 Effect-directed analysis of aryl hydrocarbon receptor agonists ______

4.4.6 Source of contamination and environmental significance

High molecular weight PAHs are the significant AhR-active compounds inducing AhR-mediated activity in the Chongqing section, possibly originating from urban traffic emissions and runoff, coal combustion, air pollution, as well as intensified shipping activities since the impoundment of the reservoir (Floehr et al., 2015b). Further research is required to identify the causative toxicants for unexplained effects in this area. In the Kaixian area, only a minor part of the EROD inducing potency of tested sediment extracts could be explained by the priority PAHs. The bioactive fractions of PAHs were characterized by a broad variety of heterocycles and methylated PAHs. Dinaphthofurans were identified as pollutants to contribute part of the EROD induction potency in the bioactive fraction. The methylated PAHs were hypothesized as contributors to the toxicity in the Kaixian area. PACs originate from incomplete combustion and industrial processes or fossil fuels as well as from natural sources like volcanic eruptions.(Laflamme and Hites, 1978) Methylated benzo[a]pyrenes have been identified as components from cigarette smoke, urban air particulates, gasoline engine, diesel exhaust, and forest fire smoke, as well as in a variety of coal- derived liquids and tars (Grimmer and Böhnke, 1975; McMahon and Tsoukalas, 1978; Jensen and Hites, 1983; Rice et al., 1987). Biomass burning such as wood fuel used for cooking or heating, or the burning of straw residues in the fields has caused serious pollution in the southeast of China (Oregon State University 2011; Pan et al., 2011), and should be considered since the Kaixian area constitutes a rather rural area. Benzothiazole and its derivatives most likely originated from an identified rubber factory nearby the sampling site. Tribenzylamine are well- known extractants in industrial wastewater (Malvankar and Shinde, 1990; Rajesh and Subramanian, 2006; Kalidhasan and Rajesh, 2009). Thus, the compound was hypothesized to be associated with wastewater treatment. Dibenzylamine has been reported to be detected in environment samples, such as surface water (Botalova and Schwarzbauer, 2011) and wastewater (Botalova et al., 2011). It was reported as by-product during the rubber vulcanization process (Helmick and Fiddler, 1994). The present investigation integrated a biological-chemical approach for the characterization of AhR agonists in the sediment of TGR. To the best of our knowledge, this is the first time that AhR agonists were analyzed in TGR in more detail using EDA. Our study strongly supports that focus on prioritized pollutants may result in inadequate assessment of complex environmental mixtures (Brack and Schirmer, 2003; Brack et al., 2005; Kaisarevic et al., 2009). Although ______158

Chapter 4 Effect-directed analysis of aryl hydrocarbon receptor agonists ______relatively low target chemical concentrations were detected in the present study, the vulnerable TGR ecosystem might still be of concern for its absolute pollution mass (Floehr et al., 2013). Long-term monitoring programs including the identified compounds should be employed parallel to the proceeding economic and demographic development due to the rapid industrialization and increased urbanization in TGR. Although EDA has been proven to be useful for toxicant identification in the present study, the approach is still not widely applied in environmental routine monitoring programs (Brack et al., 2016). With respect to a tedious evaporation and solvent exchange steps, as well as large number of fractions for bioassays, the relative laborious work largely limits a wider applicability of EDA (Brack et al., 2016). Thereafter, workflows that can lead to a rapid assessment of the key toxicants are required (Brack et al., 2016). Correspondent studies in high throughput EDA integrating micro-fractionation, effect assessment and chemical identification are currently performed, with the aim to be applied as a routine monitoring program, in order to support the prioritization of environmental contaminants and the regulatory decisions in TGR.

4.5 Acknowledgements

This study has been carried out as part of the Yangtze-Hydro project (No. FKZ 02WT) supported by the Federal Ministry of Education and Research, Germany (BMBF), SOLUTIONS project supported by the European Union Seventh Framework Programme (FP7-ENV-2013-two-stage Collaborative project) under grant agreement No. 603437, and the EDA-EMERGE project supported by the EU Seventh Framework Programme (FP7-PEOPLE-2011-ITN) under the grant agreement No. 290100. We also want to express our gratitude to Dr. Niels Bols and Dr. Lucy Lee from the University of Waterloo, Canada, who kindly provided the CYP1A expressing fibroblast- like permanent cell line RTL-W1 from primary hepatocytes of rainbow trout (Oncorhynchus mykiss). We thank Marion Heinrich and Lena Schinkel for conducting the analysis of polycyclic aromatic compounds by GC-MS. In addition, Hongxia Xiao received a personal grant supported by the scholarship program Chinese Scholarship Council.

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Chapter 5 5 Ecotoxicological evaluation of 3,4,3’,4’-tetrachloroazobenzene (TCAB) in comparison to the parent compound 3,4-dichloroaniline and propanil

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This chapter have been published in a peer-reviewed journals as:

Xiao, H., Kuckelkorn, J., Nüßer, L-K., Floehr, T., Henning, M-P., Roß-Nickoll, M., Schäffer, A., Hollert, H. (2016) The metabolite 3,4,3’,4’-tetrachloroazobenzene (TCAB) exerts a higher ecotoxicity than the parent compounds 3,4-dichloroaniline (3,4-DCA) and propanil. Science of the Total Environment 551: 304-316.

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5.1 Abstract

3,4,3’,4’-tetrachloroazobenzene (TCAB) is not commercially manufactured but formed as an unwanted by-product in the manufacturing of 3,4-dichloroaniline (3,4-DCA) or metabolized from the degradation of chloranilide herbicides, like propanil. While a considerable amount of research has been done concerning the toxicological and ecotoxicological effects of propanil and 3,4-DCA, limited information is available on TCAB. Our study examined the toxicity of TCAB in comparison to its parent compounds propanil and 3,4-DCA, using a battery of bioassays including in vitro with aryl hydrocarbon receptor (AhR) mediated activity by the 7-ethoxyresorufin-O-deethylase (EROD) assay and micro-EROD, endocrine-disrupting activity with chemically activated luciferase gene expression (CALUX) as well as in vivo with fish embryo toxicity (FET) assays with Danio rerio. Moreover, the quantitative structure activity response (QSAR) concepts were applied to simulate the binding affinity of TCAB to certain human receptors. It was shown that TCAB has a strong binding affinity to the AhR in EROD and micro-EROD induction assay, with the toxic equivalency factor (TEF) of 8.7 × 10-4 and 1.2 × 10-5, respectively. TCAB presented to be a weak endocrine disrupting compound with a value of estradiol equivalence factor (EEF) of 6.4 × 10-9 and dihydrotestosterone equivalency -10 factor (DEF) of 1.1 × 10 . No acute lethal effects of TCAB were discovered in FET test after 96 h of exposure. Major sub-lethal effects detected were heart oedema, yolk malformation, as well as absence of blood flow and tail deformation. QSAR modelling suggested an elevated risk to environment, particularly with respect to binding to the AhR. An adverse effect potentially triggering ERβ, mineralocorticoid, glucocorticoid and progesterone receptor activities might be expected. Altogether, the results obtained suggest that TCAB exerts a higher toxicity than both propanil and 3,4-DCA. This should be considered when assessing the impact of these compounds for the environment and also for regulatory decisions. Key words: propanil, 3,4,3’,4’-tetrachloroazobenzene (TCAB), AhR-mediated activity, endocrine disrupting, zebrafish, quantitative structure activity response model (QSAR)

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5.2 Introduction

Propanil (3,4-dichloropropionaniline), which is applied to control barnyard grass in the cultivation of rice, is one of the most extensively used herbicides worldwide. Propanil application after post-emergence of rice is one of the main herbicide treatments for weed control in America and Asia (Labrada, 2003; Grube et al., 2011). In the United States, it maintained the top 20 ranking among the pesticides used for agriculture from 2001 to 2007 (Grube et al., 2011). Until 2006, the estimation for total domestic use of propanil was approximately 3,175 tonnes. It was used to treat around 8,000 km2 of land, which accounts for about 50 – 70% of rice crop in USA (USEPA, 2006). In China, herbicides are used on approximately 75% of the rice acres (Zhang et al., 2007a); among that, propanil is one of the major herbicides that are applied in the southern parts of China (Baki et al., 2000; Zhang, 2003). In contrast to the intensive use of propanil in America and Asia, the herbicide is not approved for the use in the European Union due to a lack of sufficient data for the environmental risk assessment and the toxicity of possible metabolites (EU Regulation 2011; European Commission 2011; EU Commission Regulation 2013). Propanil is not persistent in the environment (with a half-life in soils < 5 days) (Tanaka et al., 1981; WHO, 2004), and enzymatical or hydrolytical processes can form the more persistent metabolite 3,4-dichloroaniline (3,4-DCA) (Deuel et al., 1977; McMillan et al., 1990; Pothuluri et al., 1991). The liberated 3,4-DCA can be further converted to 3,4,3’,4’-tetrachloroazobenzene (TCAB) by microbial peroxidases in the environment (Kearney et al., 1970; Bordeleau and Bartha, 1971; Burge, 1973; Di Muccio et al., 1984). In the areas where propanil had been applied before, environmental contamination by TCAB was often examined. In the study of Chisako and Kearney (1970), TCAB was detected in all soil samples collected from rice paddy fields in Japan. Deuel et al. (1977) monitored trace quantities of TCAB in rice paddy water 24 h after flooding in 1973. However, no more TCAB was found in water samples collected after flooding in 1974 and 1975. Kearney et al. (1970) surveyed propanil treated rice soils from the known point of propanil application and crop rotation in Arkansas, and identified that TCAB existed in soil up to 30 cm depth. TCAB has been taken as a potential environmental toxicant based on its structural and biological behaviour (Hsia et al., 1977; NTP, 2010). TCAB is approximately isosteric to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the well-known aryl hydrocarbon receptor (AhR) agonist (Poland and Knutson, 1982). Thus, there is a high likelihood that TCAB induces ______165

Chapter 5 Ecotoxicological evaluation of 3,4,3’,4’-tetrachloroazobenzene (TCAB) ______

AhR-mediated activity. TCAB has been shown to bind to the AhR with a binding affinity of one- fifth that of TCDD (Poland et al., 1976). Van Birgelen et al. (1999) pointed out that TCAB could account for more dioxin-like activity in the environment than polychlorinated dibenzo-p-dioxins and dibenzofurans together. On the other hand, TCAB has a low aqueous solubility, together with a high logKow (6.69, www.Chemspider.com), a high affinity for adsorption to sediment and organic particles, and thus to sediment-dwelling or feeding organisms are expected. In the study of Allinson and Morita (1995b) the aquatic snail Indohiramakigai (Indoplanorbis exustus) was exposed to detrital TCAB. Significant concentrations of TCAB were detected in some tissues of the snails, indicating the snails accumulated TCAB from the environment. Similar results were also detected in Allinson and Morita (1995a) when exposed detrital TCAB to Japanese Medaka (Oryzia latipes). Humans may potentially be exposed to TCAB through several routes, such as in the manufacturing and application of 3,4-DCA or its herbicidal derivatives, consumption of crops contaminated with dichloroaniline-derived herbicides, which have been degraded by peroxide- producing soil microorganisms (Bordeleau and Bartha, 1971; Taylor, 1979; Hill et al., 1981; Singh and Bingley, 1989; Singh and Bingley, 1991; Bhusari et al., 2014). Occupational chloracne has been reported among employees working in chemical plants manufacturing 3,4-DCA or its derivatives, which was attributed to TCAB exposure (Bunce et al., 1979; Morse et al., 1979; Hill et al., 1981). Information concerning toxicological and ecotoxicological effects of TCAB is still limited. Several toxicity studies for TCAB were conducted on mammals: e.g., carcinogenicity to male rats (Hsia et al., 1980), cytotoxicity to rodent cells (Hsia et al., 1977), histopathology of male and female rats and mice (Van Birgelen et al., 1999; Singh et al., 2010), developmental neurotoxicity to rats (Harry et al., 2014). In addition, mutagenicity with Salmonella typhimurium strains has been investigated as well. TCAB was reported inactive to the S. typhimurium reversion assay (McMillan et al., 1988), but acts as a week mutagen in S. typhimurium strain of TA1538 and TA1532 (Gilbert et al., 1980; NTP, 1991). TCAB can be considered as AhR agonist since it induces the aryl hydrocarbon hydroxylase (AHH) by the same mechanism as TCDD (Poland et al., 1976; Hsia et al., 1977; Van Birgelen et al., 1999). In view of previous reports that AhR agonist induce not only the AhR-mediated activity, but also a broad spectrum of other toxic effects, such as disruption of normal hormone

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Chapter 5 Ecotoxicological evaluation of 3,4,3’,4’-tetrachloroazobenzene (TCAB) ______signalling pathways, reproductive and developmental defects (Bonefeld-Jørgensen et al., 2001; Wassenberg and Di Giulio, 2004; Mandal, 2005). Many studies reported a cross-talk between AhR and estrogen receptor (ER) as well as androgen receptor (AR) signalling pathway (Nilsson et al., 2001; Bradshaw et al., 2002; Safe, 2006). Furthermore, exposure of AhR agonist to the most sensitive fish stage is of ecological concern because of their potential to mortality in further development stage (Walker and Peterson, 1994; Andreasen et al., 2002) and thus cause adverse effects in wild fish populations (Niimi, 1983; Gilbertson, 1992; Whyte et al., 2000; Van der Oost et al., 2003; Grund, 2011). AhR signaling can be activated by TCDD to regulate the induction of cytochrome P450 (CYP) monooxygenase expression in several tissues in embryonic zebrafish, including endocardium and vascular endothelium, as well as ventricular myocardium in the adult zebrafish (Stegeman et al., 1989; Andreasen et al., 2002; Zodrow et al., 2004). As fish are in a particular sensitive stage at early life stages (Di Paolo et al., 2015b), the in vivo Fish Embryo Toxicity (FET) test with eggs of Danio rerio (OECD, 2013) has been proven a suitable tool in ecotoxicological risk assessment (Peddinghaus et al., 2012; Floehr et al., 2015a), and can serve as a replacement to the acute toxicity test with adult fish (Lammer et al., 2009; Embry et al., 2010; Strähle et al., 2012). Environmental chemicals, which induce adverse effects mediated by their binding to the receptor, are assumed to have a quantitative structure-activity relationships to such receptors, like AhR, ER, AR (Safe, 1998). Based on such relationships, the relative equivalent potency (REP) of individual compound relative to a standard (e.g., TCDD, 17ß-estradiol, and dihydrotestosterone) can be experimentally determined. Consequently, when the concentrations of individual compounds in the environment are known, the overall effects on activated receptors by different agonists can be estimated, which is expressed as toxic equivalents (TEQs). This approach has been extensively applied as a probabilistic methodology to assume the associated burden of overall toxicity in environmental matrices, such as soil, sediment etc. (Andersson et al., 2009; Brinkmann et al., 2014) and to evaluate the potential hazard and risk assessment of human exposure to chemicals (Van den Berg et al., 2006; Meyer et al., 2014; Maletz et al., 2015). In silico toxicology prediction based expert systems such as quantitative structure activity responses (QSAR) tools and modeling approaches are widely used to predict the toxicity of environmental chemicals (Cronin and Dearden, 1995; Kruhlak et al., 2007; Christen et al., 2010). This approach can be used as toxicological screening considering mechanistic targets active with

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Chapter 5 Ecotoxicological evaluation of 3,4,3’,4’-tetrachloroazobenzene (TCAB) ______cellular pathways (Christen et al., 2010; Modi et al., 2012; Radović et al., 2014). Numerous commercially available and free web-based programs for toxicity prediction are available and partly reviewed in Muster et al. (2008) and Modi et al. (2012). Previous studies about the toxicological effects of TCAB were more likely exposed to rats and mice. Limited information is available on the effects in mechanism specific cell assays and early vertebrate development. Very little is known about possible counterparts with other receptors instead of AhR. Therefore, to investigate whether TCAB has the potential to receptor-mediated responses, we used in vitro assays with the AhR-mediated 7-ethoxyresofufin-O-dethylase activity (EROD) using permanent fish liver cell line RTL-W1 and H4IIE rat hepatoma cells and the endocrine activity (estrogen and androgen) with U2OS human osteosarcoma cells based on chemically activated luciferase gene expression (ERα-CALUX and AR-CALUX). In addition, the in vivo assay FET was applied to examine the embryotoxic/teratogenic effects of TCAB with embryos of Danio rerio. Moreover, for in silico testing we used the QSAR concepts to simulate the binding affinity of TCAB to human receptors by applying virtualToxLab with a series of 16 proteins (www.biograf.ch) (Vedani et al., 2009). The toxicity of its parent compounds propanil and 3,4-DCA was also evaluated, which facilitates a toxicity comparison with the metabolites. Compounds investigated were suggested by the framework of Yangtze project (Bergmann et al., 2012) as being representative herbicides and metabolites in agriculturally used areas of the Three Gorges Reservoir (TGR) (http://www.yangtze-project.de).

5.3 Material and methods

5.3.1 Chemicals and reagents

TCAB (98%, CAS: 14047-09-7) was purchased from Dr. Ehrenstorfer (Augsburg, Germany). 3,4-DCA (98%, CAS: 95-76-1) and propanil (99.6%, CAS: 709-98-8) both were ordered from Sigma Aldrich Chemie GmbH (Steinheim, Germany). Dimethyl sulfoxide (DMSO) was supplied by Carl Roth GmbH & Co. KG (Karlsruhe, Germany). Stock solutions of test compounds were prepared in DMSO. The maximum concentration of TCAB stock solution was 1 mg/mL according to the low solubility in DMSO. Water bath (37°C, B. Braun Biotech International, Germany) and sonic treatment (Bandelin Sonorex, Germany) were applied in the preparation of the stock solutions.

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5.3.2 EROD assay

The EROD assay indicates the catalytic activity of CYP1A and is measured as a biomarker of responses to dioxin-like compounds (Giesy et al., 2002; Heger et al., 2012; Eichbaum et al., 2014). The neutral red retention assay (NR), which is an acute in vitro assay for the determination of cell cytotoxicity, was used as a pre-test for the EROD assay to avoid masking effects. Both of the assays were carried out with the permanent fish cell line RTL-W1 (rainbow trout liver Waterloo1) (Lee et al., 1993). The NR assay was carried out according to Babich and Borenfreund (1990) modified as published by Keiter et al. (2006). The EROD assay was determined according to previously described methods in Section 3.3.5. The test compounds – TCAB, 3,4-DCA and propanil were diluted with complete L15 medium in seven 1:2 dilution steps with three internal replicates, from 0.4 – 50 µg/L, 39 – 5000 µg/L, 39 – 5000 µg/L.

5.3.3 Micro-EROD assay

In order to compare the CYP1A-inducing potential of selected compounds with different metabolism, micro-EROD assay was performed with the rat hepatoma cell line H4IIE as well. The micro-EROD assay was carried out according to the procedure published by Schiwy et al. (2015a). Cells were cultivated in DMEM medium without phenol red (Life Technology, Carlsbad, USA), which was supplemented with 10% FBS, 2% GlutaMAX solution (Life Technology, Carlsbad, USA ) and 12.5 mL 1 M HEPES buffer (Sigma-Aldrich, Steinheim, Germany) under aseptic conditions. The cells were seeded at a concentration of 2 × 105 cells per mL (104 cells per well) into sterile 96-wells plates (Sarstedt, Nümbrecht, Germany) at least 2 hours prior to exposure. Subsequently, TCAB, 3,4-DCA and propanil were diluted with culture medium in six 1:2 dilution steps with three internal replicates, from 15 – 500 µg/L, 312 – 10,000 µg/L, 312 – 10,000 µg/L, respectively. On each plate, 0.5% DMSO medium and a concentration series of 0-300 pM TCDD were used as negative control and positive control, respectively. After exposure for 72 h, resorufin associated fluorescence was measured in the solution on a multiwell fluorescence reader (Infinite M200, Tecan Austria GmbH, Grödig, Austria) with the excitation/emission wavelength of 530/590 nm. Protein was added with a bicinchoninic acid protein kit solution (BCA, Sigma-Aldrich, Steinheim, Germany), and then measured at a wavelength of 562 nm. Dose-response curves for micro-EROD induction were computed by non-linear regression (Prism

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6.0, GraphPad Software Inc., San Diego, USA) using the log concentration (agonist) vs. response with variable slope as a model equation.

5.3.4 CALUX assay

U2OS human osteosarcoma cells were stably transfected with a reporter plasmid (pEREtata-luc) and an expression plasmid (pSG5-neo-hERα) to detect receptor-mediated estrogen (-like) effects. ERα and AR-CALUX bioassays derived from the U2OS cell line are members of a panel of CALUX reporter cell lines, which are efficient and highly sensitive measurements to detect compounds interfering with the estrogen and androgen receptors (Sonneveld et al., 2005). Once an activated agonist-receptor-complex binds to the responsive element, the cells would produce the enzyme luciferase. Then, the activity of luciferase can be detected by measurement of light emission after applying the substrate luciferin. The CALUX assay was performed according to the standard operating procedure (BDS, 2013), as detailed in Maletz et al. (2013). U2OS cells were cultured in DMEM/F12 medium with phenol red (Gibco, Life Technologies

GmbH, Darmstadt, Germany), which was supplemented with 0.68 g NaHCO3 (Sigma-Aldrich, Steinheim, Germany), 41 mL FBS, 5 mL MEM 100x (GibcoTM, Bleiswijk, Netherlands) and 1 mL penicillin-streptomycin solutions (GibcoTM, Bleiswijk, Netherlands). Cells were seeded at a density of 104 cells per well into sterile 96-well plates 24 hours prior to exposure. Then the compounds to be tested were diluted with medium in ten 1:3 dilution steps with three internal replicates from 0.05 – 1000 µg/L. 17ß-estradiol (E2, Sigma Aldrich, Steinheim, Germany) for ERα-CALUX and dihydrotestosterone (DHT, Sigma Aldrich, Steinheim, Germany) for AR- CALUX were used as positive controls in the dilution series ranging from 0.1 pM to 100 pM and 0.1 pM to 10,000 pM, respectively. Cells were exposed to 0.1% DMSO as negative control. After 24 h of exposure, the medium was removed and the magnitude of luciferase activity was measured after applying 50 µL of lysis mix (PBS with MgCl2 and CaCl2, Sigma-Aldrich, Steinheim, Germany) and 50 µL of illuminate mix (Perkin Elmer Inc., Rodgau, Germany) with a GloMax96 (Promega GmbH, Mannheim, Germany). Luciferase responses were interpolated in the linear range of the standard curve (sigmoidal fit, four parameter logistic functions). Dose- response curves for ERα/AR CALUX were computed by non-linear regression (Prism 6.0, GraphPad Software Inc., San Diego, USA) using the log concentration (agonist) vs. response with variable slope as a model equation.

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5.3.5 Fish Embryo Toxicity (FET) test

To determine the embryotoxic and teratogenic potential of the substances, the FET was carried out according to Section 3.3.5. Zebrafish (Danio rerio) were maintained according to Hollert et al. (2003) and Peddinghaus et al. (2012). TCAB was screened with nine concentrations from concentration 7.7 to 2500 µg/L in FET test. The higher exposure concentrations (2500 – 1250 – 625 µg/L) presented significantly crystal precipitations in exposure medium, thus TCAB was performed in six 1:1.5 dilution steps from 7.7 to 315 µg/L (with ten biological replicates per concentration).

5.3.6 QSAR model according to VirtualToxLab

The VirtualToxLab is an in silico tool for predicting the toxic potential of chemicals by simulating and quantifying their interactions towards a series of proteins, which are known to trigger adverse effects using automated, multi-dimensional QSAR (Vedani et al., 2012; 2015). The model of VirtualToxLab was obtained from free open resource www. Biograf.ch. To estimate the toxic potential of the tested compounds, 16 proteins including 10 nuclear receptors were considered: AhR, AR, ERα, ERβ, glucocorticoid, liver X, mineralocorticoid, peroxisome proliferator-activated receptor γ, progesterone, thyroid α, thyroid β, four members of the cytochrome P450 enzyme family (1A2, 2C9, 2D6, and 3A4), and the potassium ion channel (hERG).

5.3.7 Data analysis

REP is expressed as an equivalent to a reference compound, i.e., as the toxic equivalency factor (TEF) to TCDD for AhR-, the estradiol equivalency factor (EEF) to E2 for ERα-, and the dihydrotestosterone equivalency factor (DEF) to DHT for AR-mediated activity; tests were conducted according to Villeneuve et al. (2000) and Brinkmann et al. (2014). The relative luminescence values of the mean solvent control were subtracted from samples and positive standards, and then converted to a percentage of the mean maximum response of positive standard (positive controlmax), which scales from 0 (solvent control) to 1 (positive controlmax). The resulting values were then plotted using the software Prism 6.0 (GraphPad Software Inc., San Diego, USA) and fitted using four parameter logistic regressions with variable slope. The TEF,

EEF, and DHE were calculated based on the standardized range of EC50, EC20 and EC80, respectively (Equation 5.1). ______171

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퐸퐶푖(푟푒푓푒푟푒푛푐푒 푐표푚푝표푢푛푑) (Equation 5.1) 푅퐸푃푖 = 퐸퐶푖(푆푎푚푝푙푒)

Where REPi is the relative potency determined to the reference compound at a defined level of response i, which TEF to TCDD for AhR-, EEF to E2 for ERα-, and DEF to DHT for AR- mediated activity, ECi (reference compound) equals the concentration of reference compound causing response i, and ECi (sample) is the concentration of samples causing the response i. Data analysis of FET was conducted following the recommendations of Peddinghaus et al. (2012) and recently published by Zhu et al. (2015). The effect rate from triplicates was plotted using the software Prism 6.0 (GraphPad Software Inc., San Diego, USA) in concentration-response curves.

The half maximal effective concentration (EC50) was computed from the curves using log concentration (agonist) vs. response with variable slope, where the top and bottom of the curve was respectively set to 0% (minimum of negative control) and 100% (maximum of positive control) with 95% confidence bands (p < 0.05).

5.4 Results and discussion

5.4.1 AhR-mediated activity

According to the NR assay, TCAB, 3,4-DCA, and propanil presented above 80% vitality compared with negative control. In conclusion, no cytotoxic potential was detected under the maximum exposure concentration of 0.01 mg/mL with the three tested substances (Data not shown). The concentration-response curve for the three substances in the EROD and micro-EROD are given in Fig. 5.1-a, b. Propanil was inactive in both the EROD and micro-EROD assays, i.e. they did not reach 20% induction of the TCDD standard. The TEFs of selected compounds were expressed as fixed-effect level-based on TCDD. For the EROD assay, the TEF50 values of TCAB -4 -4 were 8.7 × 10 with a range of TEF20-80 from (2.9-26) × 10 , i.e., two to three orders of -6 magnitude higher than that of 3,4-DCA (TEF50 2.3 × 10 ) with a range of TEF20-80 (1.7-3.2) × 10-6. These results are in accordance with the findings in the micro-EROD assay, the -5 -5 TEF50 values of TCAB were 1.2 × 10 with a range of TEF20-80 from (0.4-3.3) × 10 , while the -8 -8 TEF50 value of 3,4-DCA were 2.2 × 10 with a range of TEF20-80 from (1.27-3.9) × 10 .

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Fig. 5.1 Dose − response curves and corresponding 95% confidence bands in the (a) EROD assay, (b) micro-EROD assay, (c) ER-CALUX assay, (d) AR-CALUX assay of TCAB, Propanil, 3, 4-DCA (○), and the standard curves (●), i.e.,TCDD in EROD and micro-EROD assay, E2 in ER-CALUX, DHT in AR-CALUX, respectively. Data were measured in n=3 independent assay repetitions. Concentration values on the x-axis refer to nominal medium concentrations of each substance. Dots represent mean values from technical replicates of one respective experiment.

Our results are in accordance with the findings of other studies, where TEF values of TCAB were reported with a range from six to two orders of magnitude less potent than TCDD at other endpoints. Poland et al. (1976) reported that the relative potency for TCAB was two orders of magnitude less potent than TCDD based on the induction of AHH to chicken embryos. TCAB was found about five to six orders of magnitude less potent than TCDD using thymic atrophy as the endpoint (Van Birgelen et al., 1995; NTP, 1998). The TEF of TCAB was in the range of 3 × 10-6 to 10-5 based on dermal CYP450 1A1 induction in the 13-week gavage study in female B6C3F1 mice performed by National Toxicology Program (NTP) (NTP, 1998). The above

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Chapter 5 Ecotoxicological evaluation of 3,4,3’,4’-tetrachloroazobenzene (TCAB) ______discussion indicating a great variety of sensitivities to TCAB among species, which can be explained by the bioavailability of compounds and multiple metabolisms such as its biological half-life, the possible cross-talks of AhR with other signalling pathways, the presence or absence of intracellular/extracellular agonists binding sites, cellular AhR subunit concentrations, AhR functionality etc. (Bank et al., 1992; NTP, 2010; Sorg, 2014). This observation is in agreement with other studies, which found that sensitivity to dioxin-like compounds can largely differ between species (Elonen et al., 1998; Doering et al., 2013). TEF has been applied as a probabilistic methodology to describe the associated burden of dioxin- like activity in environmental matrices (Van den Berg et al., 2006). Fig. 5.2 compares the TEF values of TCAB to other typical dioxin-like compounds. The TEF magnitude of TCAB is comparable to that of some mono-ortho coplanar PCBs and large polycyclic aromatic hydrocarbons (PAHs), e.g., benzo[b]fluoranthene, benzo[a]pyrene, indeno[1,2,3-cd]pyrene. NTP (1998) already stressed the overall toxicity of TCAB induced in the environment. They calculated that, with an estimation amount of 16,000 kg TCAB produced per year in the US together with TCAB six to two orders of magnitude less potent than TCDD, it could release up to 0.016 to 160 kg TEQs to the environment. Based on our finding that the potency of TCAB is four to five orders of magnitude less potent than TCDD, the dioxin-like activity induced by TCAB could be limited to 0.16 to 1.6 kg of TEQs. Nevertheless, for such amounts, it still takes account for 3-30% of 5.2 kg TEQs in total, which was the total annual atmospheric emission, including all the incinerators, traffic, and industry, in the Federal Republic of Germany in the 1980s (Fiedler et al., 1990). Rice is the major crop in China, which makes up to 30% in total of 1.14 million km2 of agriculture land (Zhang, 2003). Zhang (2001) reported that, due to weed competition, 10 million tons of rice are lost annually, which would be sufficient to feed at least 56 million people for 1 year. Herbicides are being rapidly adopted in China to liberate hand weeding labour and to raise crop yields (Zhang, 2003). The herbicide application has increased from 1,067 tons in 1970 to 72,800 tons in 2007 (Lu and Xia, 2008) and the herbicide treated areas have increased steadily from less than 0.01 million km2 from the early 1970s up to more than 0.7 million km2 in 2005 (Zhang, 2001; Gianessi and Williams, 2011). Propanil and propanil mixtures are still largely used as herbicides for weed control in rice paddy in China, particularly in TGR. For the relatively high TEFs of its metabolite TCAB, the potential to adverse human health effects and environmental damage associated with exposure of TCAB should be of public and regulatory concern.

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Chapter 5 Ecotoxicological evaluation of 3,4,3’,4’-tetrachloroazobenzene (TCAB) ______

(1) Chlorinated dibenzo-p-dioxins (2) Chlorinated dibenzofurans (3) Non-ortho-substituted PCBs (4) Mono-ortho-substituted PCBs (5) Polycyclic aromatic hydrocarbon (PAHs) (6) Polycyclic aromatic hydrocarbon (PAHs) (7) TCAB (7) DCA

-8 -7 -6 -5 -4 -3 -2 -1 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 Relative Potency

Fig. 5.2 Distribution of TEFs values for the dioxin-like compounds in comparison to TCAB and DCA as measured in the EROD assay and micro-EROD assay. (1) Chlorinated dibenzo-p-dioxins, include: 2,3,7,8-TCDD, 1,2,3,7,8- PeCDD, 1,2,3,4,7,8-HxCDD, 1,2,3,6,7,8-HxCDD, 1,2,3,7,8,9-HxCDD,1,2,3,4,6,7,8-HpCDD,OCDD (Van den Berg et al., 2006); (2) Chlorinated dibenzofurans include: 2,3,7,8-TCDF, 1,2,3,7,8-PeCDF, 2,3,4,7,8-PeCDF, 1,2,3,4,7,8-HxCDF, 1,2,3,6,7,8-HxCDF, 1,2,3,7,8,9-HxCDF, 2,3,4,6,7,8-HxCDF, 1,2,3,4,6,7,8-HpCDF, 1,2,3,4,7,8,9-HpCDF, OCDF (Van den Berg et al., 2006); (3) Non-ortho-substituted PCBs include: 3,3’,4,4’-tetraCB (PCB77), 3,4,4’,5-tetraCB(PCB81), 3,3’,4,4’,5-pentaCB (PCB126), 3,3’,4,4’,5,5’-hexaCB (PCB169) (Van den Berg et al., 2006); (4) Mono-ortho-substituted PCBs include: 2,3,3’,4,4’-pentaCB (PCB 105), 2,3,4,4’,5-pentaCB (PCB114), 2,3’,4,4’,5-pentaCB (PCB118), 2’,3,4,4’,5-pentaCB (PCB123), 2,3,3’,4,4’,5-hexaCB (PCB156), 2,3,3’4,4’,5-hexaCB (PCB157), 2,3’,4,4’,5,5’-hexaCB (PCB167), 2,3,3’,4,4’,5,5’-heptaCB (PCB189) (Van den Berg et al., 2006); (5) polycyclic aromatic hydrocarbons (PAHs): Benzo[a]anthracene, Chrysene, Benzo[b]fluoranthen, Benzo[k]fluoranthen, Benzo[a]pyrene, Dibenzo[a,h]anthracen, Indeno[1,2,3-cd]pyrene (Bols et al., 1999); (6) polycyclic aromatic hydrocarbons (PAHs): Benzo[a]anthracene, Chrysene, Benzo[b]fluoranthen, Benzo[k]fluoranthen, Benzo[a]pyrene, Dibenzo[a,h]anthracen, Indeno[1,2,3-cd]pyrene (Willett et al., 1997); (7) Present study, TEF50 with range between H4IIE and RTL-W1 cell line.

5.4.2 Estrogen and androgen activity

The concentration-response curve for the tested substances in the ERα-CALUX and AR-CALUX are given in Fig. 5.1-c, d. Both of the parent compounds, propanil and 3,4-DCA did not reach 20% induction of the E2 and DHT standard, i.e. the two parent compounds were inactive in both assays. A similar finding was reported by Salazar et al. (2006) that propanil does neither bind to the ER α nor ß. TCAB did not reach 50% induction of the E2 standard and 20% induction of the DHT standard (Fig. 5.1-c, d), making a slight extrapolation beyond the measured range of response necessary to establish the EEF20-80 and DHT20 ranges as recommended by Villeneuve et -9 al. (2000). Thereafter, the EEF50 values of TCAB were estimated to be 6.4 × 10 with a range of ______175

Chapter 5 Ecotoxicological evaluation of 3,4,3’,4’-tetrachloroazobenzene (TCAB) ______

-9 -10 EEF20-80 from (1.2-33) × 10 . The DEF50 values of TCAB were 1.1 × 10 with a range of -9 DEF201.7 × 10 . The detected EEF and DEF of TCAB were nine to ten orders of magnitude less potent than the corresponding standard compound, which is significantly lower than commonly detected contaminants in environmental media, like bisphenol A, nonylphenol, heterocyclic aromatic hydrocarbons, pesticides and phthalates, as well as testosterone (Table 5.1). Little is known considering the endocrine activity of TCAB. In the frame of NTP, TCAB was exposed to Harlan Sprague-Dawley rats and B6C3F1 mice for 2 years. The incidence of a unique neoplasm, urethral transitional epithelial carcinoma was significantly increased and compromised the survival of male mice. TCAB was also associated with increased incidences of non-neoplastic lesions in the urinary tract and genital system of male mice (NTP, 2010). In view of previous studies that dioxins are typical environmental contaminants that can exert adverse estrogen related effects (Ohtake et al., 2003). TCDD was observed to be a possible endocrine disruptor in rodents by various studies showing the uterine gene expression in mice (Boverhof et al., 2006), a weight loss of uterine in rat (Safe et al., 1991), and an inhibition of mammary and uterine cancer in female rats (Kojima et al., 1994). Three weak AhR agonists from PCB congeners–2,2’,3,4,4’,6-hexaCB, 2,2’,4,4’,5,5-hexaCB, and 2,2’,3,3,3’,5,5-heptaCB – exhibited the binding affinity to both ER and AR with human breast cancer cells and Chinese hamster ovary cells (Bonefeld-Jørgensen et al., 2001). The estrogen and androgen receptor signalling has been shown to be interfaced by AhR signalling pathway (Morrow et al., 2004; Safe, 2006; Sorg, 2014). AhR agonists may induce ER-mediated activity via AhR signalling in the absence of ER agonists through several pathways. For instance, down regulation of the ER, interference of ligand activated ER binding to DNA response elements and or induction of CYP 1A1, 1A2, or 1B1 activities (Spink et al., 1994; Krishnan et al., 1995; Kharat and Saatcioglu, 1996). Jana et al. (1999) previously reported that TCDD inhibited testosterone-induced luciferase activity in LNCaP cells transfected with an androgen-responsive construct containing the mouse mammary tumor virus promoter. Morrow and co-workers (2004) studied the inhibitory AhR-AR crosstalk in LNCaP cells, which was shown that TCDD and the selective AhR modulators 6-methyl-1,3,8-trichlorodi-benzofuran inhibit growth of LNCaP prostate cancer cells and hormone-induced upregulation of AR protein. They concluded that the inhibitory AhR-AR crosstalk in transactivation experiments was promoter-dependent.

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Chapter 5 Ecotoxicological evaluation of 3,4,3’,4’-tetrachloroazobenzene (TCAB) ______

Table 5.1 EEF and DEF of TCAB in comparison with other compounds based on ERα and AR CALUX assay. Compounds EEF Reference Compounds DEF Reference 17ß-estradiol (E2) 1 (Houtman et al., 2009; dihydrotestosterone 1 (Houtman et al., Sonneveld et al., (DHT) 2009; Sonneveld et 2006) al., 2006) 17-ethinylestradiol 1.86 (Houtman et al., 2009; testosterone 0.21 (Houtmann et al. (EE2) Sonneveld et al., 2009) 2006) estrone (E1) 0.02 (Houtman et al., 2009; 0.15 (Sonneveld et al., Sonneveld et al., 2006) 2006) estriol 0.04 (Sonneveld et al., trenbolone 0.94 (Houtman et al., 2006) 2009) nonylphenol 4.6×10-5 (ter Veld et al., 2006) androstenedione 0.06 (Houtman et al., 2009) bisphenol A 2.5×10-5 (ter Veld et al., 2006) androstanediol 0.01 (Houtman et al., 2009; Sonneveld et al., 2006) genistein 5.0×10-5 (van der Woude et al., androstenediol 0.02 (Houtman et al., 2005) 2009) 2×10-4 (Sonneveld et al., androsterone 0.01 (Houtman et al., 2006) 2009) pesticides 1- 0.05 (Thomas et al., 2002) dehydrotestosterone op’-DDT 9.1×10-6 (Legler et al., 2002) 5ß-androstane- 2×10-4 (Thomas et al., 2002) 3ß,11ß-diol-17-one o,p-DDE 2.3×10-6 (Legler et al., 2002) 4-androstendione 0.2 (Thomas et al., 2002) phthalates 5α-androstanedione 0.2 (Thomas et al., 2002) diethylphthalate 3.2×10-8 (Legler et al., 2002) epi-androsterone 0.03 (Thomas et al., 2002) dibutylphthalate 1.8×10-8 (Legler et al., 2002) TCAB 1.1×10-10 present study butylbenzylphthalate 1.4×10-6 (Legler et al., 2002) Heterocyclic Aromatic hydrocarbons 2-methylbenzofuran 2.85×10-7 (Brinkmann et al., 2014) quinoline 1.25×10-7 (Brinkmann et al., 2014) 6-methylquinoline 5.21×10-7 (Brinkmann et al., 2014) 2,3- 1.38×10-5 (Brinkmann et al., dimethylbenzofuran 2014) dibenzofuran 1.82×10-6 (Brinkmann et al., 2014) dibenzothiophene 5.53×10-6 (Brinkmann et al., 2014) acridine 3.18×10-5 (Brinkmann et al., 2014) xanthene 3.94×10-6 (Brinkmann et al., 2014) TCAB 6.4×10-9 present study

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Chapter 5 Ecotoxicological evaluation of 3,4,3’,4’-tetrachloroazobenzene (TCAB) ______

Though the parent compounds-propanil and 3,4-DCA were inactive in both estrogen and androgen activity, TCAB presents the potential as a weak endocrine disrupting compound. Thereafter, when compounds are evaluated for a toxicology endpoint, it should not only conduct on the parent compounds, but also metabolites have to be considered.

5.4.3 Lethal and sub-lethal effects in FET

In the FET test, zebrafish embryos were exposed to TCAB for 24 h, 48 h, 72 h and 96 h to observe lethal and sub-lethal effects. No lethal effects were observed after exposure for 96 h even at the highest exposure concentration of 2500 µg/L, i.e., no valid half lethal concentration (LC50) value of TCAB was obtained. The lowest sub-lethal concentration revealing effects was 7.7 µg/L after 96 h exposure. Early life stage toxicity were below 20% after 24 h and 50% after 48 h, thus, no valid 24 h EC50 and 48 h EC50 value were calculated (Data not shown). Significantly sub- lethal effects were observed when fish eggs were exposed to TCAB after 72 h. The 72 h, 96 h

EC50 values were calculated on the basic of nominal concertation as 113 µg/L, and 33 µg/L, respectively (Fig. 5.3).

Fig. 5.3 Concentration–response curve and corresponding 95% confidence bands in FET for TCAB after 72 h(○), 96 h(□), as well as negative control (NC●) and positive control (PC▪).

In our study, a steep increase in the sub-lethal effects, such as blood flow, heart edema, malformed yolk, and bent spine was observed after 72 h compared to 48 h exposure. This finding ______178

Chapter 5 Ecotoxicological evaluation of 3,4,3’,4’-tetrachloroazobenzene (TCAB) ______is comparable to the studies of Antkiewicz et al. (2005) and Belair et al. (2001), where TCDD exposure reduced the blood flow in zebrafish embryos after 72 h post fertilization. Similar findings were observed in the study of Schrankel et al. (1982), where chick embryo lethality occurred within three to four days after treatment with TCAB, and more embryos died until the twelfth day of incubation. They attributed this effect to an insensitivity of early embryos towards the metabolites or older embryos contained higher levels of CYP enzyme than early embryos. NTP preformed a 2-years study, in which male and female F344/N rats and B6C3F1 mice were exposed to TCAB in corn oil by oral gavage 5 days a week with dose from 0 to 100 mg per kg of body weight and 0 to 30 mg per kg of body weight, respectively. Significant mortality was observed in the 2 year studies rather than in the 3-month studies, while most of the deaths were attributed to the formation of neoplasms (NTP, 2010). Di Paolo et al. (2015a) determined the delayed toxicity in zebrafish larvae exposure to PCB126. No mortality occurred up to 5 days post fertilization (dpf), but various sub-lethal morphological and behavioural effects were observed at 4 and 5 dpf. A particular steep increase in mortality occurred by 8 dpf after larvae were transferred to clean water. They attributed the observation that PCB126 (logKow of 6.98, www.chemspider.com) as hydrophobic chemicals (logKow > 5) are not easily eliminated and have a significant tendency for bioaccumulation (Daley et al., 2009; Di Paolo et al., 2010). When such compound deposited or accumulated to the yolk-sac of embryos, it could accumulate in the lipid-rich yolk. The progression of yolk absorption will further lead to chemical mobilization into the embryo tissues, and the toxic residue concentrations will reached in target organs for showing delayed sub-lethal effects (Di Paolo et al., 2015a). In our study, TCAB with logKow of 6.69 can be expected to have similar biological behaviour with PCB126, therefore, a delayed mortality as well as growth impairment and delayed development might occur after prolonged time exposure. The major malformation in zebrafish embryos detected in our study were heart oedema, heart malformation, yolk malformation, as well as cardiovasular disorders (Fig. 5.4), which are known to be typical effects of AhR-agonists in zebrafish embryos (Grimes et al., 2008; Seok et al., 2008; Di Paolo et al., 2015a). Dioxin-like compounds were reported to induce effects in zebrafish from persistent deregulation of genes controlled by the AhR and its DNA binding partner the aryl hydrocarbon receptor nuclear translocator (ARNT) complex (Teraoka et al., 2002; Carney et al., 2004; Billiard et al., 2006; Otte et al., 2010; McGee et al., 2013). Andreasen et al. (2002) previous studied that TCDD induced CYP1A protein and mRNA expression in the cardiovascular

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Chapter 5 Ecotoxicological evaluation of 3,4,3’,4’-tetrachloroazobenzene (TCAB) ______system and tissues involved in osmoregulation of zebrafish, suggesting that the AhR signalling can be activated by TCDD in the larval tissues involved in TCDD developmental toxicity. Carney et al. 2004 described that AhR ligands could not only bind to AhR, but also induce specific toxicity mediated by mechanisms that extend beyond the regulated transcription of genes belonging to the AhR/ARNT signaling. In order to verify the AhR activation in zebrafish embryos exposed to TCAB, gene expression analysis of AhR pathway by quantitative real-time polymerase chain reaction (qPCR) (Redelstein et al., 2015) or fish embryo EROD (FE-EROD) assay to embryos (Schiwy et al., 2015b) should be of subject in further studies.

Fig. 5.4 Observed sub lethal effects in Danio rerio embryo after 24, 48, 72 and 96 h exposure. (a) no significant effects detected (23 µg/L);(b) heart edema (1’arrow) (104 µg/L) (c) hatched fish with heart edema, malformed yolk (2’ arrow), bent spine (3’ arrow), and no blood circulation (4’ arrow) (156 µg/L) (d) hatched fish with bent spine, malformed yolk, no blood circulation, and heart edema (315 µg/L).

Propanil in particular 3,4-DCA (as the positive control for the fish embryo test with the zebrafish) (DIN 38415-6, 2009; OECD, 2013) has been well investigated in other research. The lowest observed effect concentrations (LOECs) of different endpoints exposed to 3,4-DCA were ______180

Chapter 5 Ecotoxicological evaluation of 3,4,3’,4’-tetrachloroazobenzene (TCAB) ______reported in Scheil et al. (2009), while LC50 after 72 h rare minnow (Gobiocypris rarus) were

4.1 mg/L(Zhu et al., 2013). EC50, no observed effect concentration (NOEC), and LOEC of 3,4-DCA after 48 h exposed to zebrafish embryos were estimated to 1,62 mg/L, 0,97 mg/L, and 1,46 mg/L, respectively (Lange et al., 1995). Nagel et al. (1991) estimated LOECs of 0.1 and 0.2 mg/l for survival rates in early life stages of zebrafish after 4 and 2 weeks of exposure to

3,4-DCA, respectively. Schiller et al. (2014) identified the EC50 of propanil exposed to zebrafish embryo after 48 h was 1.8 mg/L. The 96 h LC50 values of propanil was 8.6 mg/L when tested with Fathead minnows (Call et al., 1983) and for 3,4-DCA ranged from 8.4 to 13 mg/L when tested with zebrafish (Braunbeck et al., 2005). No valid 96 h LC50 of TCAB can be calculated in the present study. According to previous discussion, exposure TCAB after 96 h was not likely reached the steady-state in the present study. Therefore, the toxicity potential between TCAB and propanil and 3,4-DCA should be compared of caution. Further researches about the delayed lethal or sub-lethal effects of TCAB are recommended. Propanil and 3,4-DCA are frequently investigated in environmental samples, like in water (Santos et al., 1998; de Almeida Azevedo et al., 2000; Karpouzas et al., 2005; Primel et al., 2007; Sapari and Ismail, 2012), and in soil samples (Santos et al., 1998; Perera et al., 1999). Primel et al. (2007) investigated the concentration of propanil was up to 3600 µg/L, which were sampled one day after the herbicide application. Though the herbicide was degraded rapidly, relatively high concentrations of 3,4-DCA were found, up to 567 µg/L in water and 119 mg/kg in soil samples. The concentration of TCAB in soils appears to be relatively low (up to 0.06 mg/kg) (Kearney et al., 1970; Carey et al., 1980). With the sub-lethal effects recorded in the present study, propanil and the metabolites are indicated to induce rather chronic toxicity to fish under the environmentally relevant concentrations, suggesting hazards and risks in long-term exposure to the environmental pollutant.

5.4.4 Prediction of toxicity potential using VirtualToxLab model

To simulate the binding affinities of TCAB to a set of human receptors, the QSAR model- VirtualToxLab was applied. This model has been examined to predict the toxic potential for over 2500 compounds (Vedani et al., 2012). The estimated “toxic potential” of the analyzed compounds is expressed as total potential (TP). TP is estimated from the individual binding affinities, their standard deviation, and the quality of the underlying model, simulating and quantifying the interactions towards a series of macromolecular targets at the molecular level ______181

Chapter 5 Ecotoxicological evaluation of 3,4,3’,4’-tetrachloroazobenzene (TCAB) ______using automated flexible docking combined with multi-dimensional QSAR (Vedani et al., 2009). High numbers refer to probable interactions with receptors; the higher the number of TP, the higher the risk it might induce. According to Vedani et al. (2012), TP ranges from 0.0 (none) to 1.0 (extreme) and has been split into five classes: TP > 0.8 (Class IV,*****, extreme risk), 0.7 < TP ≤ 0.8(Class III,****, very high risk), 0.6 < TP ≤ 0.7(Class II, ***, high risk),

0.5 < TP≤ 0.6 (Class I, **, elevated risk) while class 0 include 0.4 < TP ≤ 0.5 (*, moderate risk), 0.3 < TP ≤ 0.4 (+,low risk) and TP ≤ 0.3 (-, no risk). In addition to the risk assessment, it also provides specific information at which target protein an elevated binding affinity and which potentially triggered adverse effect might be expected.

Table 5.2 The toxic potential of propanil, 3,4-DCA and TCAB predicated by VirtualToxLab using multi- dimensional QSAR (www. Biograf.ch).

Compound Chemical structure TP TP Estimated main targets Class

NH Cl Propanil H3C 0.247 - - O Cl 3,4-DCA H2N Cl 0.213 - -

Cl TCAB Cl 0.541 ** AhR, AR,ERα, ERß, GR, MR, PR Cl

N N

Cl Cl

** : 0.5 < TP ≤ 0.6 (Class I, **, elevated risk); TP ≤ 0.3 (-, no risk).AhR: aryl hydrocarbon receptor; AR: androgen receptor; ERα: estrogen α receptor; ERβ: estrogen β receptor; GR: glucocorticoid receptor; MR: mineralocorticoid receptor; PR: progesterone receptor

Table 5.2 shows the estimated toxic potential of TCAB and the parent compounds 3,4-DCA and propanil. The overall TP value of TCAB (0.541) suggested an elevated risk, particularly with respect to binding to the AhR, AR and ERα. Apart from the above receptors, TCAB was predicted to bind moderately to the ERß, mineralocorticoid, glucocorticoid and progesterone, which should be further investigated. The parent compounds 3,4-DCA and propanil are suggested for no risk, and no trigger receptor was estimated. It should be noticed that the virtualToxLab is based solely on thermodynamic considerations, i.e. ignoring mechanisms influencing the availability of a compound at the site of action and its adsorption, distribution, metabolism, and excretion properties (Vedani et al., 2012). Regarding ______182

Chapter 5 Ecotoxicological evaluation of 3,4,3’,4’-tetrachloroazobenzene (TCAB) ______the contaminants to environmental impacts, aspects like fate, behavior, disposition, and toxicity with a particular focus on the bioavailability have to be considered. Nevertheless, the toxic potential and predicated trigger receptor by TCAB may be interpreted as a toxic alert and further studies needs to be performed.

5.4.5 Adverse outcome pathways

Adverse outcome pathways (AOPs) are conceptual frameworks that summarize existing knowledge, and link direct molecular-level initiating events and adverse outcomes at level of biological organization relevant to hazard assessment (Ankley et al., 2010; Volz et al., 2011). The AOP framework was developed in part by the National Research Council (NRC) (NRC 2007), and then are included into the work of the OECD (OECD 2013), with the aim to identify alternatives, high-throughput predictive assays and testing strategies for risk assessments, facilitate identification of chemicals responsible for adverse effects or toxicity in a system, provide flexibility approach in testing based on risk assessment, as well as to reduce animal numbers in research and study costs (Ankley et al., 2010; Yozzo et al., 2013; Villeneuve et al., 2014). The environmental risk of AhR agonists has been evaluated by Ankley et al. (2010) and Volz et al. (2011). The research proposed an AOP for TCDD using zebrafish embryo as a model, suggesting a declining trajectory in relevant organism. According to our results and previous literature research, TCAB could result in an AOP similar to that of TCDD, from binding AhR, effects in the embryo, and linkages to AhR effects on higher vertebrates (Fig. 5.5). Here, TCAB could bind to the AhR using QSAR modeling, which resulted in an activation of the AhR in vitro assay. AhR signaling can be activated by TCAB in the cardiovascular system and tissues of zebrafish (Fig. 5.4), which means that TCAB can induce CYP1A protein in the cardiovascular system. This can link to alteration in cardiomycocyte disorders, induced heart malformations and defected cardiovascular system in fish embryo. This would lead to an increase of developmental abnormalities in embryo, and finally decrease the growth in adult fish in ecosystem (Ankley et al., 2010; Volz et al., 2011).

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Chapter 5 Ecotoxicological evaluation of 3,4,3’,4’-tetrachloroazobenzene (TCAB) ______

Fig. 5.5 Adverse outcome pathway for TCAB resulting in fish population declining (cf. Ankley et al. 2010). Dotted line denotes predicative linkages.

5.5 Conclusion

In the present study, we investigated the toxicity of TCAB in mechanism specific cell assays and early vertebrate development, as well as applied an in silico tool to simulate the binding affinity of TCAB to certain receptors. Among the evaluated endpoints, the major effect observed was the AhR-mediated activity, as expected. The TEF values of TCAB were four to five orders of magnitude less potent than TCDD based on fish liver cell line (RTL-W1) and rat hepatoma cell line (H4IIE), which is comparable to those of some mono-ortho coplanar PCBs and large PAHs. With the TEFs established, the associated burdens of dioxin-like activity brought into the environment by propanil and DCA forming compounds should be taken seriously especially since TCAB is a rather persistent compound (corresponding studies in water sediment systems are currently performed and will be reported elsewhere). Apart for that, our study revealed that TCAB possesses both a low estrogen and androgen potential. To our best knowledge, it is the first time to describe the endocrine disrupting activity of TCAB. In FET, the exposure of Danio rerio indicated significant embryotoxicity and teratogenicity of TCAB. However, the steady-state was not likely reached after 96 h of exposure in the present study. Delayed lethal or sub-lethal effects of TCAB can be expected after prolonged exposure time. Together with recorded sub- lethal effects, TCAB exposure might result in adverse outcomes in aquatic systems on the long term. Moreover, the QSAR model provides supplementary information for the overall risk assessment process. An adverse effect potentially triggering ERß, mineralocorticoid, glucocorticoid and progesterone might be expected, which should be the subject for further research. Real-time PCR could be employed in the process. Importantly, the obtained results also

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Chapter 5 Ecotoxicological evaluation of 3,4,3’,4’-tetrachloroazobenzene (TCAB) ______demonstrate that TCAB has a higher toxicity than propanil and DCA. This has to be considered when assessing the environmental impact of DCA forming chemicals. Propanil and propanil mixed with other herbicides are still frequently used for weed control in United States, China, and other countries of Asia (Zhang et al., 2005; Rao et al., 2007; USGS, 2015). Hence, risk assessment of chemicals, such as pesticides, should also include knowledge on the toxicity and fate of their metabolites before they are deliberately brought to our environment (Brack et al., 2015). Moreover, suitable monitoring strategies like a combination of analytical, effect-based screening, and ecological assessment (Altenburger et al., 2015; Wernersson et al., 2015) should be applied to areas with such pesticides applied before to ensure the aquatic and human health.

Conflict of interest

The authors declare no conflict of interest.

5.6 Acknowledgements

The authors acknowledge financial support by the Federal Ministry of Education and Research, Germany (BMBF) with the project framework of “Yangtze-Hydro-Sustainable Management of the Newly Created Ecosystem at the Three Gorges Dam” (No. FKZ 02WT; www.yangtzeproject.de). This study has been carried out as part of the MICROTOX project (“Transformation, Bioaccumulation and Toxicity of Organic Micropollutants in the Yangtze Three Gorges Reservoir”, No. FKZ 02WT1141).We want to express our gratitude to Drs. Niels Bols and Lucy Lee from the University of Waterloo, Canada, who kindly provided the CYP1A expressing fibroblast-like permanent cell line RTL-W1 from primary hepatocytes of rainbow trout (Oncorhynchus mykiss). Our gratitude also goes to BioDetection Systems (Amsterdam, The Netherlands) for supporting the investigations of CALUX assays. In addition, Hongxia Xiao received a personal grant supported by the scholarship program Chinese Scholarship Council.

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Chapter 5 Ecotoxicological evaluation of 3,4,3’,4’-tetrachloroazobenzene (TCAB) ______

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Chapter 6 6 Optimized work-flow for identification of dioxin-like compounds in environmental samples combining High-throughput fractionation and bioassays

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Thias chapter is based on a manuscript to be submitted to Environmental Science &

Technology as:

Xiao, H*& Brinkmann, M*., Thalmann, B., Schiwy, A,, Eichbaum, K,, Gembé, C,, Seiler, T-B., Hollert, H. (2016) Optimized work-flow for identificaiton of dioxin like compounds in environmental samples combining high-throughput fractionaiton and bioassays (in preparation) (* Shared the first coauthorship)

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Chapter 6 High-throughput fractionation and bioassays ______

6.1 Abstract

Effect-directed analysis (EDA) has been demonstrated to be a powerful strategy to identify biologically active compounds in environmental samples. However, in current EDA studies, fractionation and subsequent handling procedures are laborious, consist of multiple steps, and thus bear the risk of contamination and decreased recoveries of the target compounds. The relatively low resolution throughput has been one of the major bottlenecks of EDA. Here, we propose a high-throughput EDA (ht-EDA) work-flow combining reversed phase high- performance liquid chromatography (HPLC) fractionation of samples into 96-well microplates, followed by toxicity assessment in the micro-EROD bioassay with H4IIE cells, and chemical analysis of biologically active fractions. The approach was evaluated using single substances, binary mixtures and extracts of sediment samples collected at the Three Gorges Reservoir, China as well as the rivers Rhine and Elbe, Germany. In addition, we optimized the work-flow by seeding previously adapted suspension-cultured H4IIE cells directly into the microplate used for fractionation, which makes any transfers of fractionated samples unnecessary. The proposed ht-EDA work-flow simplifies the procedure for wider application in ecotoxicology and could become the method of choice for the prioritization of environmental contaminants and support for regulatory decisions.

Keywords: Dioxin-like activity; DLCs; EDA; EROD; High-throughput; suspension culture

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6.2 Introduction

Various kinds of contaminants e.g., endocrine disrupting, genotoxic, or dioxin-like compounds, reach the aquatic environment due to anthropogenic activities such as agriculture, wastewater discharges, industrial manufacturing, and household use (Kolpin et al., 2002; Hilton et al., 2010; Floehr et al., 2013; Xiao et al., 2016), which may pose a risk to aquatic ecosystems and human health. Thus, monitoring the presence of such contaminants in aquatic systems is important to ensure environmental and public safety. In the past decades, numerous studies pointed out that these risks may be significantly underestimated if monitoring strategies only reply on a limited number of priority compounds, resulting regulatory decisions that may potentially be misled (Brack et al., 2002; Hollert et al., 2002; Kaisarevic et al., 2009). To avoid that relevant toxicants are overlooked in this process, effect-based monitoring and toxicant identification are required (Wernersson et al., 2015; Brack et al., 2016). Effect-directed analysis (EDA), which combines fractionation procedures, effect-based testing and chemical analysis, has been demonstrated to be a suitable approach to identify causative toxicants in complex environmental samples (as recently reviewed by Brack et al. (2016)). Although this approach has been successfully used for toxicant identification in various environmental matrices, such as water (Meinert et al., 2010), sediment (Bandow et al., 2009; Luebcke-von Varel et al., 2012), suspended particulate matter (Woelz et al., 2010a; 2010b), soil (Woelz et al., 2011), as well as biota (Simon et al., 2011; 2013), EDA is still not routinely applied in environmental monitoring programs. Several aspects can be the reasons that limit a wider applicability of EDA (Hecker and Hollert, 2009). Fractionation steps that reduce the complexity of environmental mixtures and separate toxic fractions from non- or less- toxic fractions is one of the key procedures in EDA (Weller, 2012). Due to the complexity of environmental samples, lower resolution fractionation is often not sufficient, and thus multistep fractionation procedures are required (Brack and Schirmer, 2003; Lübcke-von Varel et al., 2012). Moreover, the solvents used in fractionation usually interfere with bioassays and chemical analysis. Thus, after each fractionation step, a time-consuming evaporation procedure is necessary. Sometimes, several liters of solvents may need to be evaporated during the process, some of which have negative environmental impacts (e.g. chlorinated solvents). Moreover, multiple evaporation steps can lead to a decrease in recovery of compounds (Booij et al., 2014). Importantly, the iterative fractionation and subsequent handling procedures hinder straightforward biotesting, and thus ______191

Chapter 6 High-throughput fractionation and bioassays ______identification of bioactive fractions or chemicals. In summary, the process is very laborious and can take up to several months; thus, conventional EDA is difficult to apply in routine monitoring programs (Simon et al., 2015; Brack et al., 2016). To overcome these limitations, a number of studies, aiming at high-throughput EDA (ht-EDA) were conducted with special emphasis on microfractionation (Nielen et al., 2006; Pieke et al., 2013; Booij et al., 2014; Ouyang et al., 2015) and nanofractionation (Kool et al., 2011; Jonker et al., 2015). Liquid chromatography (LC) is a straightforward and widespread method-of-choice for simplification of environmental matrices (Booij et al., 2014; Jonker et al., 2015; Ouyang et al., 2015). In combination with post-column fraction collectors, low-volume fractions can be collected into multi-well plates at a high frequency to maintain chromatographic resolution. The coupling of high resolution mass spectrometry with highly sensitive cell-based reporter gene assays for mechanism-specific effects, can significantly enhanced the methods for ht-EDA. One such mechanism-specific effect is the induction of the aryl hydrocarbon receptor (AhR) by environmental pollutants that are collectively referred to as dioxin-like chemicals (DLCs). DLC include polychlorinated dibenzo-p-dioxins and –furans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (dl-PCBs), but also other planar aromatic compounds such as polycyclic aromatic hydrocarbons (PAHs) and heterocyclic PAHs. DLCs are a class of widely distributed and well-studied persistent pollutants of particular interest in context of potential environmental health effects (Hinger et al., 2011; Eichbaum et al., 2014; Sorg, 2014). Nevertheless, numerous studies demonstrated that target chemical analysis only provides little information on the potential adverse biological effects of complex mixtures of DLCs (Wang et al., 2014b; Floehr et al., 2015a), and – due to the plethora of chemicals with dioxin-like potency – the identification of causative toxicants often fails (Wang et al., 2014b; Floehr et al., 2015a). Thus, our study focused on the development of a simplified work-flow for identification of contaminants responsible for the observed AhR-mediated activity of complex mixtures, aiming to provide a rapid method for screening AhR agonists in environmental samples. The work-flow combined (a) reversed phase high-performance liquid chromatography (RP-HPLC) fractionation of samples into 96-well microplates, followed by (b) toxicity assessment in the H4IIE micro- EROD bioassay and (c) chemical analysis of biologically active fractions. In addition, we also optimized and simplified the work-flow by (d) adding H4IIE cells adapted to suspension culture

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Chapter 6 High-throughput fractionation and bioassays ______directly into the microplate used for fractionation, and thus omitting the growth phase of the adherent cells.

6.3 Material and methods

6.3.1 Chemicals and solvents

Acetonitrile (≥ 99.9%, p.a.), hexane (≥ 97%, p.a.), methanol (≥ 99.9%, p.a.) were purchased from Sigma-Aldrich GmbH (Steinheim, Germany), acetone (≥99.8%, p.a.) from Carl Roth GmbH & Co.KG (Karlsruhe, Germany), and ultrapure water (LiChrosolv ® Water for Chromatography) was supplied from Merck KGaA (Darmstadt, Germany). The reference standards used in this study are described in the Supporting Information (Annex III, Table III.1).

6.3.2 Sample collection and preparation

Sediments samples from the Three Gorges Reservoir (TGR), at the Yangtze River (China) were applied in the work-flow to identify the main contributors of dioxin-like activity. Three sampling sites close to the cities of Chongqing (CNG-U) and Kaixian (HAN-D and HAN-C), were chosen, as they were identified as regional “hot-spots” with respect to dioxin-like activity in our previous studies (Section 3.5). For detailed geographic information on the sampling sites, see Chapter 3, Section 3.3.1. Sediments collected at the Yangtze river were freeze-dried, sieved (≤ 2 mm), and thoroughly homogenized. The following sediment preparation, extraction, and cleanup were reported in Section 3.3.3. The final concentration was adjusted to 10 g sediment equivalents (SEQ) per mL acetonitrile. The concentrations of 16 EPA-PAHs in these sediment extracts were determined in Table 3.3 and Table 3.4. Additionally, in order to compare the results of chemical analysis for DLCs with the results of our ht-EDA method, sediment samples collected in Germany were also investigated. Two sampling sites, one at the Rhine River at the harbor Ehrenbreitstein (EBR), and one from the cut- off meander Zollelbe (ZE) in the city of Magdeburg, Elbe River (Eichbaum et al., 2016) were chosen. Sediment preparation and extraction were comparable to the method described above, while the clean-up involved desulfurization with activated copper (24 h), sulfuric acid treatment (24 h), and multilayer silica column clean-up, according to US-EPA method 8290 (US-EPA, 1994) with modifications as described in Eichbaum et al. (2016). The concentrations of 12 dl-

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PCBs and 17 PCDD/Fs in these sediment extracts, as well as the effects of unfractionated extracts in the micro-EROD, were determined in the context of previous studies (Annex III, Table III.2).

6.3.3 Fractionation method

The fractionation method applied was a modification of the method proposed by Suzuki and co- workers (Suzuki et al., 2004). An HPLC system (Agilent 1200 series, Agilent, Waldbronn, Germany) equipped with a reversed-phase LC column (Synergi Hydro-RP 80 Å, 4 µm particle size, 250 × 2 mm, Phenomenex, Aschaffenburg, Germany) was applied to separate standards and samples at 30°C, with the following solvent gradient system at a flow rate of 0.2 mL min-1: 0-4 min, 10 % acetonitrile and 90 % methanol/water (80:20; v./v.); 4.0-7.0 min, a linear gradient of 10 % acetonitrile and 90 % methanol/water (80:20; v./v.) to 100 % acetonitrile;7.0-50.0 min, 100 % acetonitrile. Eluting compounds were detected using a photodiode array detector (DAD; Agilent Technologies). DAD signals in the range from 210 to 360 nm were recorded and used for determination of retention times. A 10 µL sample was injected and then fractionated over a period of 50 min. Fractions were collected at 30 s intervals from 0 to 25 min, and at 60 s intervals from 25.0 to 50.0 min in 96-well V-bottom microplates (Biozym, Hessisch Oldendorf, Germany) using an automatic fraction collector (Agilent Technologies). Subsequently, fractions were evaporated under sterile conditions overnight, and then directly frozen at -20°C for bioassay or chemical analysis.

6.3.4 Micro-EROD assay

For the determination of the CYP1A-inducing potential of individual fractions, the micro-EROD assay with the wild-type rat hepatoma cell line H4IIE was performed. The micro-EROD assay was carried out according to the procedure recently published by Schiwy & Brinkmann et al., with slight modifications (Schiwy et al., 2015a). Cells were maintained in a culture medium consisting of DMEM without phenol red (Life Technologies, Darmstadt, Germany), which was supplemented with 10 % fetal bovine serum (FBS, BioWest, origin: South America), 2 % GlutaMAX solution (Life Technologies, Darmstadt Germany) and 2.5 % HEPES buffer (Sigma-Aldrich, Steinheim, Germany) under aseptic conditions. Prior to the test, micro-well plates containing the previously prepared fractions were taken from the freezer and thawed under sterile conditions. Cells were seeded at a concentration of 2 × 105 cells per mL in 50 µL culture medium (104 cells per well) into sterile 96-well plates

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(Sarstedt, Nümbrecht, Germany) at least 2 hours prior to exposure. Meanwhile, thawed plates containing the fractions were reconstituted with 50 µL culture medium containing 2% DMSO and shaken for 2 hours at 300 rpm. After adherence of the cells, the reconstituted fractions were transferred to the plate containing cells using a multichannel pipette; this resulted in a final concentration of 1 % DMSO in all wells. In order to detect cytotoxicity, each plate performed was compared with 1 % DMSO solvent control as negative control. On each plate, a concentration series of 0 to 1200 g ml-1 TCDD was used as standard. After a 72 h exposure, deethylation of exogenous 7-ethoxyresorufin (ETX, Sigma-Aldrich) to the fluorescent product resorufin was measured fluorometrically after adding ETX working solution, which was supplemented with 8 mM ETX and 10 mM dicoumarol (Sigma-Aldrich) in Dulbecco’s phosphate-buffered saline solution (DPBS, with calcium and magnesium; Sigma- Aldrich). Fluorescence of resorufin was measured using a multi-well spectrofluorometer (Infinite M200, Tecan Austria GmbH, Grödig, Austria) with the excitation and emission wavelengths set to 530 and 590 nm, respectively. Protein concentrations were quantified, using a bicinchoninic acid protein kit (BCA, Uptima, France), at a wavelength of 562 nm. Each sample was analyzed in triplicate. Data from the micro-EROD assay were analyzed following the recommendations of Villeneuve et al. (2014) and Brinkmann et al. (2014). The mean relative fluorescence value of the solvent control wells was subtracted from each well, and then normalized to the mean maximum response to the TCDD standard (TCDDmax); thus, the assay responses scale from 0 (solvent control) to 1 (TCDDmax). A regression analysis for the standard response against concentration was performed in GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, USA) using the ‘log concentration (agonist) vs response curves with variable slope’ regression. The biological TCDD equivalents (BEQs) of each fraction were interpolated from these standard curves including the 95 % confidence bands (p < 0.05) in triplicate and later expressed as mean ± standard deviation.

6.3.5 CDS-µEROD assay

Initially, the adherent H4IIE cell line was adapted to a chemically defined medium (CDM; Thermo Scientific) and shaken suspension culture, providing permanent suspension-grown cells (H4IIE-S) in serum-free medium. Before conducting the assay, an exponentially growing H4IIE- S culture (220 rpm, 20 mm amplitude) was centrifuged (209×g) at room temperature for 5 min and resuspended in fresh room-tempered CDM (5×105 cell mL-1). Using the H4IIE-S cells, the ______195

Chapter 6 High-throughput fractionation and bioassays ______micro-EROD assay (for better discrimination from the regular micro-EROD here termed ‘CDS- µEROD’) was performed by by directly adding 150 µL of the cell suspension to 96-well V- bottom polypropylene plates (Biozym Scientific GmbH, Hessisch Oldendorf, Germany) used for fractionation following evaporation of the solvent under sterile conditions. Plates were incubated for 22 hours at 37°C, 5% CO2 and 95% relative humidity and constantly shaken at 450 rpm with 10 mm amplitude. For the CDS-µEROD, 60 µL of a 6 µM solution of ETX in PBS was directly added to the wells and incubated another 30 minutes (37°C, 5% CO2, 95% relative humidity, shaken at 450 rpm with 10 mm amplitude). The reaction was stopped by addition of 90 µL methanol and the mixture incubated for another 10 minutes at room temperature being measured according to Section 6.3.4.

6.3.6 Statistical analysis

Statistical analyses of micro-EROD data were performed using SigmaPlot 12.0 (Systat Software Inc., San Jose, USA). One-way ANOVA with Holm-Sidak’s post-hoc test, p ≤ 0.05was used to determine significant differences in fractions compared to the process control.

6.4 Results and discussion

6.4.1 Development and validation of the fractionation method

The RP-HPLC fractionation method in the present study was established according to Suzuki and co-workers was established and validated using a total number of 33 single model chemicals with log Kow values ranging from 2.03 to 6.98 (Annex III, Table III.1) (Suzuki et al., 2004). We determined a strong correlation between the retention times and the respective log Kow values of the chemicals with a coefficient of determination R2 of 0.94 (Annex III, Fig. III.1). The following linear equation was obtained from the regression analysis: RT (min) = 3.078 log

Kow - 0.610. The correlation in the present study was found to be stronger compared to that established by Suzuki et al. (2004) (R2 of 0.69), although it should be emphasized that the maximum log Kow of a compound in the present study was 6.98, while Suzuki et al. investigated compounds with log Kow values up to 9.05. Based on data from the model chemicals, fractions of complex environmental samples could be assigned to defined log Kow windows based on their retention time, thereby providing valuable qualitative information about the physicochemical properties of the toxicants present in complex environmental samples.

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6.4.2 Fractionation of single substances

To verify the correlation of separation of single known inducer (Annex III, Table III.1) and their EROD activity , PCB 126 and β-naphthoflavone were separated and analyzed in the micro- EROD assay with H4IIE cells, resulted in sharp peaks with a bioassay response in only few defined fractions (Fig. 6.1). In the first experiment, 10 µL of a 50 ng mL-1 solution of PCB 126 (retention time of 19.4 min) were injected, resulting in two adjacent bioactive fractions (19.0 – 19.5 and 19.5 – 20.0 min). In the second experiment, 10 µL of a 50 µg µL-1 solution of β-naphthoflavone (retention time of 14.5 min) were injected, resulting in three adjacent bioactive fractions (13.5 – 14.0, 14.0 – 14.5, and 14.5 – 15.0 min). It can be concluded that single compounds can be reliably fractionated by the methods of Suzuki et al. (2004) and that the micro-EROD was sufficiently sensitive to detect the trace amounts of known EROD inducers in the resulting fractions. Similar findings using LC as the fractionation method have been previously reported for a number of different analytes, including photosynthesis inhibitors in marine algae (Booij et al., 2014), designer steroids in a yeast-based androgen screening assay (Nielen et al., 2006), as well as carbamate pesticides and PAHs (Ouyang et al., 2015). Other studies have demonstrated that fractionation with subsequent bioassays could also efficiently be performed using gas chromatography (GC) with vapor traps, e.g. for organochlorine pesticides (Pieke et al., 2013), and various other organic contaminants (Meinert and Brack, 2010). Additionally, intended to enhance the recovery of compounds, in a first attempt, DMSO was added into micro-plates before fractionation as a solvent keeper; however, significant responses in blank wells and increased noise were observed in the subsequent biotests (data not shown). Thus, no such solvent keeper was used in the presented experiments to avoid interference with bioassay results. Our study demonstrates that special caution should be taken to avoid interference of such keeper solvents with the bioassay selected (Brack et al., 2016), although keeper solvents have been employed in other studies to enhance the recovery of compounds of interest (Ouyang et al., 2006; Booij et al., 2014).

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- 1 - 1 1 0 0 A 1 0 µ g m L 1 0 0 B 5 0 µ g m L

8 0 8 0

)

% (

6 0 6 0

U A

m 4 0 4 0

2 0 2 0

0 0 0 5 1 0 1 5 2 0 2 5 0 5 1 0 1 5 2 0 2 5

5 1 .5

D C )

1 - 1 - 1

- 4

l 5 0 n g m L 5 0 µ g m L m

1 .0 D

D 3

C

T

g

p 2 (

0 .5

Q E

B 1

0 0 .0 0 5 1 0 1 5 2 0 2 5 0 5 1 0 1 5 2 0 2 5

T im e (m in ) T im e (m in )

Fig. 6.1 HPLC-fractionation of single substances with subsequent micro-EROD bioassay. Normalized UV signals (mAU) relative to the peak maximum of PCB 126 at 10 µg mL-1 (A) and β-naphthoflavone at 50 µg mL-1 (B), in combined with bioanalytical TCDD equivalents (BEQs) determined in HPLC fractions of 50 ng mL-1 PCB 126 (C) and 25 µg mL-1 β-naphthoflavone by means of the micro-EROD assay (D). mAU: milli absorption units.

6.4.3 Fractionation of binary mixtures

In a next step, we evaluated the suitability of the fractionation method to separate individual compounds from binary mixtures (Annex III, Table III.1). Therefore, 10 µL of a binary mixture containing 25 µg mL-1 β-naphthoflavone and 25 ng mL-1 PCB 126 were fractionated according to 2.3 and 2.4. Comparable to the experiments with single compounds, distinct bioactive fractions could be resolved for the two compounds. They corresponded to the same time windows (13.5 – 15.0 min for β-naphthoflavone and 19.0 – 20.0 min for PCB 126) as in the previous measurement (Fig. 6.2). Further experiments were conducted using other binary mixtures (benzo[b]fluoranthene/dibenz[a,h]anthracene, TCDD/benzo[k]fluoranthene, Annex III, Table III.1) with comparable performance. However, a full separation was not achieved (data not shown). Nevertheless, it can be concluded that the fractionation method applied in the present study is applicable to separate mixtures of chemicals into discrete fractions. Full separation may

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Chapter 6 High-throughput fractionation and bioassays ______not be achieved if the physicochemical properties of the chemicals are too similar. However, at this stage of development for the method a full separation was not aimed for. In conclusion, the fractionation into groups of chemicals according to lipophilicity is of greater interest, and the rapid work-flow enables for testing of a greater number of samples.

B N F P C B 1 2 6

1 0 0 A

8 0

)

% (

6 0 - 1 - 1

5 0 µ g m L 1 0 µ g m L

U A

m 4 0

2 0

0 0 5 1 0 1 5 2 0 2 5 3 0

2 .0

B

)

1 -

l 1 .5

m

D - 1

D 2 5 n g m L C

T 1 .0

g - 1

p 2 5 µ g m L

(

Q

E 0 .5 B

0 .0 0 5 1 0 1 5 2 0 2 5 3 0

T im e (m in )

Fig. 6.2 HPLC-fractionation of a binary mixture of PCB 126 and β-naphthoflavone with subsequent micro-EROD bioassay. Normalized UV signals (mAU) relative to the respective peak maximum (A), in combined with bioanalytical TCDD equivalents (BEQs) determined in HPLC fractions of a binary mixture of 25 µg mL-1 β-naphthoflavone and 25 ng mL-1 PCB 126 by means of the micro-EROD assay (B). mAU: milli absorption units.

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6.4.4 Fractionation of environmental samples

After verification of the characteristics of the chromatographic fractionation system and the bioassay, we characterized complex environmental samples. In the sediment extract sample from the Chongqing area, China (CNG-U; Fig. 6.3), bioactive fractions eluted in two main retention time windows ranging (a) from 16.5 to 19 min, and (b) from 20.5 to 22.5 min. These time windows correspond to compounds with a log Kow values ranging from (a) 5.50 to 6.37, and (b)

6.86 to 7.50. High molecular weight PAHs such as chrysene (log Kow: 5.81), benzo[a]pyrene (log

Kow: 5.99), and benzo[k]fluoranthene (log Kow: 6.11) are suspected to be the responsible compounds for the observed effect in (a). This result is in accordance with our previous study which showed these compound to be responsible for other toxic modes of action (Chapter 4, Section 4.4.3). Moreover, for this sampling site a sum concentration for the 16 EPA-PAHs of 1,653 µg kg-1 dw was determined (Chapter 3, Table 3.3). This sum parameter includes the compounds mentioned before an indicated their share in the toxicity. In another study by xxx these compounds showed dioxinö-like acitity. In the sediments extract from Kaixian (HAN-D and HAN-C), the concentrations of Σ16 EPA-PAHs were approx. 3-fold lower compared to CNG-U (c.f. Table 3.3 and Table 3.4). This was reflected by a lower bioactivity in the corresponding fractions (Annex III, Fig. III.2-3). The elevated bioactivity in the retention time window (b) of all three sediment extracts is probably caused by dl-PCBs, PCDD/Fs, and other chlorinated chemicals, respectively (c.f. Section 6.4.1). To further unravel the chemical identitys in these bioactive fractions, additional research about chemical analyses on the bioactive fractions are needed. In the multilayer silica column-treated extract of EBR sediment (Ehrenbreitstein, Rhine, Germany), no elevated bioactivity was observed in any of the investigated fractions (Annex III, Fig. III.4). This result corresponds well with the chemical analyses and micro-EROD results of the unfractionated multilayer silica column-treated extracts investigated in the study by Eichbaum et al. 2016. Furthermore, a recent study by Brinkmann et al. (2015) indicated no significant dioxin-like effects in rainbow trout (Oncorhynchus mykiss) exposed to suspensions of EBR sediment. In this sediment extract only a moderate background contamination was observed (Annex III, Table III.2). In Zollelbe (ZE) sediment (Zollelbe, Elbe, Germany), a significantly elevated bioactivity was observed (a) at 18.0 min and (b) at 25.0 to 26.0 min, the latter of which can be assigned to the elevated concentrations of PCDD/Fs in the sample (Annex III, Table ______200

Chapter 6 High-throughput fractionation and bioassays ______

III.2).The clean-up procedure performed on these samples removes all acid-labile compounds, including many PAHs; only the persistent chlorinated DLCs reside in the sample. Correspondingly, no bioactivity was observed in fractions with retention times below 18.0 min in the two fractionation experiments (Annex III, Fig. III.4-5).

0 .8

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- h ig h e r m o le c u la r w e ig h t P A H s

l 0 .6 E P A -P A H s

m

D * * * * ** D *

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T 0 .4

*

g

p

(

Q

E 0 .2 B

0 .0 0 5 1 0 1 5 2 0 2 5 3 5 4 5 5 5

T im e (m in )

Fig. 6.3 Micro-EROD results of HPLC-fractionated sediment extract CNG-U. The sediment extract was sampled from the Yangtze River, China, and 10 µL of a 1 g SEQ mL-1 extract were fractionated. Asterisks denote significant differences between fractions and control (one-way ANOVA with Holm-Sidak’s post-hoc test, p ≤ 0.05). Shaded areas indicate the typical retention time ranges of the substance classes screened as single substances.

6.4.5 Application potential of suspension bioassays in EDA

The applicability of adherent cell lines in high throughput EDA (ht-EDA) is limited by adherence itself. It requires several washing and pre-incubation steps prior to the addition of samples when working with adherent cell lines in ht-EDA. Furthermore, it is necessary to supplement media with FCS, which has several disadvantages, e.g. masking of sample effects, high batch-to-batch variations and ethical objections (Jochems et al., 2002). Due to the ongoing establishment of new in vitro alternatives to animal testing and the resulting increase in demand for FBS, the price for FBS and conventional media will increase within the next years as the production volume is limited (Brindley et al., 2012). As a solution to this problem we have established a stable H4IIE-derived suspension cell line using serum-free, chemically-defined growth and exposure media. We present a cell line that maintains its organ-derived properties as an analytical cell line in serum-free, chemically defined medium being cultivated in suspension. This is in contrast to other studies where cells lost their ______201

Chapter 6 High-throughput fractionation and bioassays ______analytic properties when cultivated in CDM (Soldatow et al., 2013). This circumstance facilitates the direct application of cell suspensions to fractionated samples after solvent evaporation, without the need for solvent exchange or pipetting steps. Hence, the EDA throughput is massively increased, while still, the H4IIE-S cell line is highly sensitive in detecting TCDD (CDS-µEROD, Fig. 6.4). Concentrations of 0.1, 1, and 10 µg mL-1 of TCDD were injected into the RP-HPLC system and fractionated. A sharp peak in the chromatogram was detected with the UV detector at 1 and 10 µg mL-1 TCDD concentrations (retention time of 20.68 min) (Fig. 6.4a), while no significant signal was found after injection of 0.1 µg mL-1 TCDD. Fractionation time was plotted against the relative fluorescence units (RFU) per fraction to generate reconstructed bioactivity chromatograms as shown in Fig. 6.4b. The bioactivity peaks showed massive tailing at 1 and 10 µg mL-1 TCDD concentrations. Only the fractionation of 0.1 µg mL-1 TCDD presented a sharp bioactivity peak. This observation can be contributed to carryover of TCDD between the fractions at higher concentrations, and the higher sensitivity of the bioassay compared to the DAD. These findings are in correspondence with the study by Pieke et al. (2013). This result is valuable for identification of pollutants at environmentally relevant concentrations of chemicals in environmental samples. Additional benefits of using the CDS-µEROD are reduced or absent batch-to-batch variations. The H4IIE S cell remained stable in growth as well as activity over at least 60 passages (approx. 420 days; data not shown). In addition, suspension cell lines can be grown and exposed in disposables made from various materials other than the widely used and relatively polar polystyrene (PS). Thus, the material used can be efficiently chosen based on the physico- chemical properties of the chemicals of interest, e.g. PS for lipophilic compounds and polypropylene for more hydrophilic compounds. This results in a higher sensitivity and lower limits of detection (LOD) for the bioassays as the assorption behavior of the compounds is accounted for. Moreover, after incubation, the exposed cell suspensions can be separated/split into aliquots to conduct various bioassays in parallel, thus facilitating the investigation of a wider spectrum of endpoints in the workflow. In summary, the presented serum-free suspension culture approach enables higher throughput towards full automation for application in EDA and beyond.

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Fig. 6.4 HPLC-fractionation of single substances with subsequent CDS-µEROD bioassay. Normalized UV signals (mAU) relative to the peak maximum of TCDD at 1 and 10 µg mL-1 subtracted from 0.1 µg mL-1 (A), as well as the relative fluorescence units (RFU) in HPLC fractions of 0.1, 1, and 10 µg mL-1 TCDD determined by means of the CDS-µEROD bioassay (B). mAU: milli absorption units.

6.5 Conclusions and outlook

We conclude, that the use of simple fractionation techniques in combination with bioassay, in particular those which can be performed in suspension culture, can drastically reduce the workload of EDA studies and thus increase the acceptance of EDA in environmental risk assessment and monitoring. The proposed method was successfully applied on sediment extracts from the Yangtze River, China, which gave similar results to a more comprehensive conventional EDA. Furthermore, results of our EDA approach were identical with the conclusions drawn from chemical DLC analysis in multilayer silica column-treated sediment extracts from the rivers ______203

Chapter 6 High-throughput fractionation and bioassays ______

Rhine and Elbe, Germany (Eichbaum et al., 2016). Finally, we proposed an optimized work-flow that reduces the need for tedious evaporation and solvent exchange steps by performing fractionation and biotesting using suspension-cultured cells in only one multi-well plate. This approach further reduces the workload of EDA studies since it makes any transfers and solvent exchanges of fractionated samples unnecessary. We strongly believe that the proposed ht-EDA work-flow simplifies the procedure for wider application in ecotoxicology and could become the method of choice for the prioritization of environmental contaminants and support for regulatory decisions.

6.6 Acknowledgements

This study has been carried out as part of the Yangtze-Hydro project (No. FKZ 02WT) supported by the Federal Ministry of Education and Research, Germany (BMBF), and the SOLUTIONS project supported by the European Union Seventh Framework Program (FP7-ENV-2013-two- stage Collaborative project) under grant agreement No. 603437. The authors acknowledge the German National Academic Foundation (‘Studienstiftung des deutschen Volkes’) for a personal scholarship granted to M.B. In addition, H.X received a personal grant supported by the scholarship program Chinese Scholarship Council.

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Chapter 7 7 General discussion, conclusion and outlook

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7.1 General Discussion

The chapters of the present thesis applied effect-based tools for monitoring the ecotoxicological effects of the Yangtze River, and linked ecological and chemical information for a comprehensive environmental risk assessment. Altogether, this study resulted in a holistic perspective on the ecotoxicological status of the Yangtze River (Chapter 2). By implementing the triad approach to the TGR (in vitro, in vivo, and in situ), the ecotoxicological hazard potential from different sites of the TGR was recorded (Chapter 3). Followed by EDA study, contaminants responsible for the toxic potential observed in Chongqing and Kaixian area were identified (Chapter 4). Furthermore, the ecotoxicological study of propanil, its metabolites 3,4- DCA and TCAB were investigated with a battery of bioassays (in vivo, in vitro) and QASR modeling (in silico) for the support of the regulatory decisions (Chapter 5). Finally, an optimized workflow for the identification of dioxin-like compounds was developed (Chapter 6), with respect to simplifying the procedure for wider EDA application and thus application in environmental monitoring programs.

7.2 Environmental pollution status in the Yangtze River

7.2.1 Overview of ecotoxicological status in the Yangtze River

Based on the methodology of the triad approach, Chapter 2 gave an overview of the environmental pollution status from the upstream of Chongqing until the Yangtze River mouth at the East China Sea, with particular focus on the areas of Chongqing, Wuhan, Nanjing, Shanghai, and the Yangtze Estuary. A large share of measured pollutant levels in water and sediments reported in the literature were below Chinese national standards (Ministry of Environmental Protection - China, 2002) as well as relevant contamination by the ICPR (2009), respectively. Similar results were observed in TGR (Chapter 3 and Chapter 4). The pollution levles of POPs such as PAHs, PCBs, and OCPs showed comparable concentration levels to other major Chinese and European rivers (Section 2.5.1 – 2.5.3). Data about the presence of emerging pollutants like PBDEs, PFCs, PAEs, NP, and BPA in the Yangtze River are still scarce, thus more research is demanded (Section 2.5.5). Specific toxic activities, like mutagenicity, genotoxicity and endocrine activity were widely investigated along the Yangtze River, indicating a potential health risk in the study areas of the river (Section 2.6). Limited research was reported on biomarkers or bioassays

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Chapter 7 General discussion, conclusion and outlook ______displaying the cytochrome P450 activity. With respect to toxicological endpoints in situ, the only response measured were genotoxic effects manifested in the formation of micronuclei in carp and catfish from the Lower Reaches of the Yangtze River (Section 2.7). By considering the concentrations of POPs, mechanism specific-effect assessment, and in situ aquatic organisms reported in literatures, pollution levels of the Upper Reaches including the TGR were found to be lower than in the Middle and Lower sections of the Yangtze River. The sections of Wuhan, Nanjing, and Yangtze Estuary presented a higher pollution level, indicating a potential ecotoxicological impact on the surrounding areas. The study showed that highly industrialized cities presented to be the main contaminated sites, which were likely to be associated with the industrialization, increasing population as well as the pollutants accumulation from upstream.

7.2.2 Ecotoxicological potential in the TGR

An integrative evaluation of the ecological potential in TGR was shown in Table 3.9. Regarding the chemical pollution of sediments in TGR, only 16 EPA-PAHs could be identified out of 54 organic compounds selected on basis of the European Water Framework Directive (EWFD Directive 2000/60/EC, 2000), ranging from 165-1,653 ng/g in 2011 (Table 3.3) and 127-590 ng/g in 2013 (Table 3.4). Representing the “worst-case scenarios”, the toxic effects detected in in vitro bioassay ranged from lower to medium levels in comparison to other river systems (Section. 3.4.2), which is in accordance with the findings in Chapter 2. By applying in vivo bioassays with Danio rerio eggs, it was shown that sediments induced embryotoxic/teratogenic effects, particularly on the cardiovascular system of fish embryos. However, the bioavailability of particle-bound pollutants presented to be rather low (Section 3.4.3). The in situ investigations suggested rather poor conditions of chosen monitoring fish species Pelteobagrus vachellii, with histopathological alterations in liver and excretory kidney (Section 3.4.4). By accounting all endpoints in combination with the chemical data, it is concluded that adverse effects might be induced to the fish at long term, which will have an influence on the overall fitness of fish and other aquatic organisms. Additionally, significant genotoxic impacts could be observed on erythrocytes of Pelteobagrus vachellii from Chongqing, as well as from Hanfeng Lake determined with the micronucleus assay in two sampling campaigns. Together with PAH burden recorded as well as a number of toxic effects (Table 3.9), Chongqing and Kaixian were taken as regional “hot-spots” with respect to higher potential risk areas in TGR. ______208

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7.2.3 Can the pollution problem be solved by dilution

A decrease of chemical pollution (Section 3.4.1) and dioxin-like activites (Section 3.4.2 and Section 4.4.1) were observed from the upper to the lower part of the reservoir. Similar observations were reported for water samples by Wang et al. (2009). Thereby, it was hypothesized that higher levels of contamination in the upstream of Yangtze River at some sites may be diluted by sediments or water from the less contaminated tributary, bringing about lower concentrations of contaminants downstream from corresponding sections. To ensure the public and environmetnal health, it is reasonable to ask whether there is actually a pollution problem due to the high mass transport of water and sediment of the Yangtze River despite all contamination sources. Is the pollution problem solved by simple dilution? Is the Yangtze River capable to dilute pollutant levels to a non-toxic degree, without considerable consequences for wildlife and humans? Regarding the above questions, the pollutants load between the Yangtze River and the Rhine was compared (Section 2.7). It was shown that although concentrations of organic pollutants were reported to be lower in the Yangtze River than those in the Rhine River, one should keep in mind that water discharge of the Yangtze River is approximately 14 tims greater than that of the Rine River (Huisman et al., 2000). Low environmental relevant pollutants can still result in comparably larger amounts of organic pollutants that end up in the Yangtze River’s receiving water body. Furthermore, the mass balance of PAH load in TGR was estimated. Considering annually 151 – 172 Mt of sediment remain in the TGR (2003 – 2008) (Hu et al., 2009; Yang et al., 2007) and the average PAHΣ16 burden in sediment along the reservoir detected in our study, a deposition about 216 – 636 kg PAH/day (0.2 – 0.6 mgPAH/m2day) in TGR were expected (Section 3.4.1), indicating an ecotoxicological risk in TGR. In conclusion, these findings suggested that, the Yangtze River’s immense amount of water and sediments can reduce the ecotoxicological risk from pollution all along the river, but does not eliminate it. Instead of acute toxic effects, sublethal toxic effects to fish and other aquatic organisms are more likely to be induced in the long term of the river (cf. Section 3.4.4). By that, long-period monitoring programs are highly demaded for the protection of public health and aquatic system in the river.

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7.3 Identification of the causative pollutants and possible source of contamination in TGR

Among the hazard pollutants, PAHs appeared to be the dominant pollutants for the Yangtze river (Chapter 2), and also for TGR (Chapter 3, Chapter 4 and Chapter 6). According to PAH sources analysis (Section 3.4.1), the PAH pollution was associated with petrogenic sources relate to crude oil, coal, and their products, mainly released by spillage in rather remote areas like Yunyang, Wushan, while the urban city Chongqing originated from pyrogenic sources relate to incomplete combustion of biomass, like wood burning, and fossil fuels. The contamination at the Hanfeng Lake exhibited a mixture of petrogenic and combustion sources. By the further implemented EDA approach, high molecular weight PAHs presented to be significant AhR-active compounds at Chongqing section, which accounted for 28 - 43% of the significant bioactive fractions in Chongqing section (Section 4.3.2). The possible pollution source was assumed to be originated from urban traffic emissions and runoff, coal combustion, as well as intensified shipping activities. In Kaixian area, the relatively high potency fractions were characterized by PAHs and methylated derivatives thereof and heterocyclic polycyclic aromatic compounds (PACs) such as dinaphthofurans. PACs normally originate from incomplete combustion and industrial processes or fossil fuels. Biomass burning such as wood fuel used for cooking or heating, or the burning of straw residues in the fields has caused serious pollution in the southeast of China (Oregon State University 2011; Pan et al., 2011) and should be considered since the Kaixian area constitutes a rather rural area. Moreover, benzothiazole and its derivatives were identified as suspected toxicants by non-target analysis, which most likely originated from an identified rubber factory nearby the sampling site. Long-term monitoring programs including the identified compounds are recommended to be included in the long-term monitoring programs in TGR.

7.4 Is EDA a suitable monitoring strategy?

In previous view of research, monitoring strategies may have failed for monitoring environmental concentrations of priority pollutants alone without considering combined effects of these pollutants and their possible consequences to the environment. This can lead to the monitoring of irrelevant compounds (Kaisarevic et al., 2009; Simon et al., 2015; Brack et al., 2016). EDA has shown to be particularly useful for identifying potential toxic unknown substances, that cannot be ______210

Chapter 7 General discussion, conclusion and outlook ______established on the basis of conventional chemical monitoring of singular compounds (Simon, 2013). The present thesis applied the EDA approach in order to characterize and identify the AhR agonists in the sediments of Chongqing and Kaixian area (Chapter 4 and Chapter 6). Besides priority compounds, the major contributors to AhR mediated activity in the Kaixian area were identified in success (Chapter 4). In addition, with a combination of simple fractionation techniques with the rapid bioassay, in particular with suspension culture, the workload of conventional EDA was drastically reduced (Chapter 6). The proposed method was also successfully applied on sediment extracts in TGR. Thus, the proposed ht-EDA work-flow simplifies the procedure for wider application in ecotoxicology and could become the method of choice when rapid estimations and source-tracking are needed. In summary, with regard to identifying the causative agents from chemical mixtures that are responsible for major effects to human health and the environment, incorporating the EDA approach into monitoring programs would be a prudent and promising approach.

7.5 Recommend environmental monitoring strategies

Environmental deterioration and pollution can pose a serious threat to human health. World Bank (2007) declared in its report, that in China the lack of monitoring data makes it difficult to assess adverse effects to the public induced by pollution. Effect-based tools applied in the thesis present to be useful tools for the assessment of the impact of chemicals as well as the evaluation of ecotoxicological effects in the aquatic environment. Using the triad approach (Chapman, 1990; Chapman and Hollert, 2006), a holistic ecotoxicological assessment of the Yangtze River along with the TGR area was achieved. Moreover, integrating several lines of evidences, the approach helped to identify “hot-spot sites” with respect to ecological relevant endpoints of the study areas. Thus, the approach can be deployed to evaluate the relevance of pollutants for organisms and to gain a comprehensive view on the pollution status in aquatic environments. Besides, EDA can be applied as follow up research, in order to identify the causative agents in the identified “hot-spot” areas paired with its ecological relevant endpoints (Hecker and Hollert, 2009), thus is suggested to be included in the routine monitoring program in order to support the prioritization of environmental contaminants (Section 7.4).

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Furthermore, the thesis showed that the knowledge on the toxicity and fate of metabolites should also be included before chemicals like pesticides and herbicides are deliberately brought into the environment (Chapter 5). The integrative approach AOP is able to link mechanism responses on the cellular levels with whole organisms (Section 5.4.5), which would be desirable for a comprehensive assessment of the ecotoxicological risk of individual chemicals. Overall, due to the rapid industrialization and increased urbanization in the Yangtze River as well as TGR area, long-term monitoring programs are recommended to preserve and improve the aquatic system. Several approaches here are recommended: (1) The Yangtze should be seen in context of the River Continuum Concept (Vannote et al., 1980), an approach to describe and evaluate a river from its source to its estuary, considering it as an open and holistic ecosystem. It integrates biotic parameters, physical and hydrological factors, energy and nutrient input as well as output. This has also been applied for European river systems in the European Union Water Framework Directive (EWFD Directive 2000/60/EC).

(2) Chemical/ecotoxicological and hydraulic approaches should be combined, e.g., to estimate the risk of remobilization of pollutants during flood events (Woelz et al., 2009; Brinkmann et al., 2010). This appears to be even more important compared to the Rhine River due to higher mass transport of water and sediments in the Yangtze River.

(3) In situ investigations of mechanism-specific toxicity, like genotoxicity in blood cells from fish caught in the river, can serve as additional lines of evidence in weight of evidence studies, in order to prove the relevance of lab-based assays for the situation in the field (Chapman and Hollert, 2006; Boettcher et al., 2010). The chosen monitoring fish species Pelteobagrus vachellii in the study, was proven to be well suited for biomonitoring. It is particularly suitable due to its wide distribution along the TGR and other sections of the Yangtze River. Thus, the biomarkers in combination with the chemical analysis are recommended as furture monitoring approach.

(4) To avoid overlooking the relevant toxicants, EDA is a suitable monitoring strategy for toxicant identification, which should be performed as a routine monitoring strategy for the prioritization of environmental contaminants and support for regulatory decisions.

(5) Additionally, toxicity reduction evaluation should be applied to areas with elevated toxicity levels. Advanced methods of wastewater treatment and integrated strategies to minimize the impact of point and non-point sources should be further developed – e.g., by transfer of

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Chapter 7 General discussion, conclusion and outlook ______knowledge from national research programs and applications (Huckele and Track, 2013; Triebskorn et al., 2013) to bilateral and international joint projects in China (e.g. (Bergmann et al., 2012) and Clean Water Initiative, China).

7.6 Conclusion and Outlook

The present study demonstrated an overview of the pollution status in the Yangtze River, investigated the ecotoxicological potential in the TGR in conjunction with the EDA approach for the identification of AhR agonists in the sediments of Chongaing and Kaixian areas. Our study addressed that monitoring priority pollutants alone is inadequate. By using micro-fractionation in combination with high-sensitive bioassays, EDA procedure can be accelerated for the identification of toxicants in environmental samples. Thus, the presented optimized work-flow is suggested to be included in environmental monitoring program. Morever, effect-based tools are recommended to be suitable methods for monitoring the ecotoxicological effects in the aquatic environment of the Yangtze River as well as other similar river systems globally. The optimized workflow of ht-EDA that reduces the need for tedious evaporation and solvent exchange steps by performing micro-fractionation and biotesting, in particular using suspension-cultured cells, can give a rapid assessment of AhR agonists in environmental risk assessment and monitoring. Further, to address the causitive agents of a comprehensive biological effect, more toxicological endpoints such as in vivo bioassays should be included in the metholdology of ht-EDA. Here, the Fish Embryo Toxicity test using Danio rerio would have the advantage that it takes the bioavailability of contaminants in environmental samples into account. Thus, integrating a wider spectrum of endpoints in the workflow should be included in further research.

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______246

Annex I – Solution by dilution? ______

Annex I – Solution by dilution?

I.1 Drainage basin definitions of the geographical regions

Jinsha River: The Yangtze River from Yushu (Qinghai Province) to Yibin in Sichuan province is called the Jinsha River, which has a total length of 3,496 km. Tuotuo River: The Yangtze River arises from glaciers south of the Geladandong Mountain in Qinghai province and flows west for 343km where it meets with another origin river Danqu and forms the Tongtian River. Yangtze Estuary: The Yangtze Estuary occupies 700 km of the river’s 6,300 km length (Fig. I.1). Chongming Island ramifies the estuary into the South Branch and the North Branch. The South Branch constitutes the main stream of the estuary and receives above 95% of the total estuarine runoff, whereas the North Branch accounts for only about 5%. The main stream of the southern branch is divided into the south channel and north channel by Changxing Island. There are more than 30 branches in the Yangtze estuary, such as Baimao River, Yanglingtang River, Liuhe River, Gujing River, Huangpu River, Xinjianghai River and so on. The longest branch is Huangpu River. The sedimentation rate of the Yangtze estuary is 6.3-6.6 cm yr-1 (Wang et al. 2012b). Yangtze River Delta: including Shanghai, southern Jiangsu province and northern Zhejiang province of China. The Yangtze River drains into the East China Sea. It covers an area of 99,600 km2 and is home to over 105 million people as of 2010, of which an estimated 80 million is urban. Water Period: In general, the water of the River is at the lowest level in the dry season (December to April), at the highest level in rainy season (from July to September, potential flood events), and at the middle level in normal seasons (May to June and October to November) (Sun et al. 2002).

______247 Annex I – Solution by dilution? ______

Fig. I.1 Map of the Yangtze Delta region

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Table I.1 Concentrations of PCDDs/DFs in water and sediments from the Yangtze River and other water bodies in the world. Number of papers: Total papers: 7; PCDDs/DFs in the Yangtze River: 3 (Water: 1; Sediment: 2).

Sampling Location Concentration WHO1998-TEQ Reference Date Water (pg/L) Yangtze River Drainage

Basin Three Gorges Reservoir 2004-2005 2-96 (Ø 12) 0.001-0.3 (Ø 0.1)a (Chen et al. 2008) Sampling Location Concentration WHO1998-TEQ Reference Date Sediment (pg/g) Yangtze River Drainage

Basin Yangtze Estuary 2003-2004 25-374 (Ø 170) 0.4-1 (Ø 0.8)b (Wen et al. 2008) Yangtze Estuary 2004 n.a 0.3-1 c (Sun et al. 2005) Other Water Bodies 2,004-4,314 Pearl River 2000 1.50-6.23 (Ø 3.25)d (Zhang et al. 2009) (Ø 2,794) Yellow Estuary 2004 n.a 0.11-1e* (Hui et al. 2009a) Mondego Estuary 2009 110 0.88 f (Nunes et al. 2011) Mai Po Wetland n.a. 4,826-7,885 10.8-16.4g (Müller et al. 2002) a- 2,3,78-TCDD; 1,2,37,8-PeDD; 1,2,3,4,7,8-HxCDD; 1,2,3,6,7,8-HxCDD; 1,23,7,8,9-HxCDD; 1,2,3,4,6,7,8-HpCDD; OCDD, 2,3,7,8-TCDF; 1,2,3,7,8-PeCDF; 1,2,3,4,7,8-HxCDF; 1,2,3,6,7,8- HxCDF; 1,2,3,7,8,9-HxCDF; 2,3,4,6,7,8-HxCDF; 1,2,3,4,7,8-HpCDF; 1,2,3,4,7,8,9-HpCDF; OCDF b- 2,3,7,8-TCDD; 1,2,3,7,8-PeCDD; 1,2,3,4,7,8-HeCDD; 1,2,3,6,7,8-HeCDD; 1,2,3,7,8,9-HeCDD; 1,2,3,4,6,7,8-HpCDD; OCDD, 2,3,7,8-TCDF;1,2,3,7,8-PeCDF; 2,3,4,7,8-eCDF; 1,2,3,4,7,8- HeCDF; 1,2,3,6,7,8-HeCDF; 2,3,4,6,7,8-HeCDF; 1,2,3,7,8,9-HeCDF; 1,2,3,4,6,7,8-HpCDF; 1,2,3,4,7,8,9-HpCDF c- 2,3,7,8-TCDF; 1,2,3,7,8-PeCDF; 2,3,4,7,8-PeCDF; 1,2,3,4,7,8-HxCDF; 1,2,3,6,7,8-HxCDF; 2,3,4,6,7,8-HxCDF; 1,2,3,7,8,9-HxCDF; 1,2,3,4,6,7,8-HpCDF; 1,2,3,4,7,8,9-HpCDF; OCDF; 2,3,7,8,-TCDD;1,2,3,7,8-PeCDD;1,2,3,4,7,8-HxCDD; 1,2,3,6,7,8-HxCDD;1,2,3,7,8,9-HxCDD; 1,2,3,4,6,7,8-HpCDD; OCDD d- 2,3,7,8-TCDD; 1,2,3,7,8-PeCDD; 1,2,3,4,7,8-HxCDD; 1,2,3,6,7,8-HxCDD; 1,2,3,7,8,9-HxCDD; 1,2,3,4,6,7,8-HpCDD; 2,3,7,8-TCDF; 1,2,3,7,8-PeCDF; 2,3,4,7,8-PeCDF; 1,2,3,4,7,8-HxCDF; 2,3,4,6,7,8-HxCDF; 1,2,3,7,8,9-HxCDF; 1,2,3,7,8,9-HxCDF; 1,2,3,4,6,7,8-HpCDF; 1,2,3,7,8,9- HpCDF e- 2,3,7,8-TCDD; 1,2,3,7,8-PeCDD; 1,2,3,4,7,8-HxCDD; 1,2,3,6,7,8-HxCDD; 1,2,3,7,8,9-HxCDD; 1,2,3,4,6,7,8-HpCDD; OCDD; 2,3,7,8-TCDF; 2,3,7,8-PeCDF; 2,3,4,7,8-PeCDF; 1,2,3,4,7,8-

______249 Annex I – Solution by dilution? ______

HxCDF; 1,2,3,6,7,8-HxCDF; 2,3,4,6,7,8-HxCDF;1,2,3,7,8,9-hxCDF; 1,2,3,4,6,7,8-HpCDF; 1,2,3,4,7,8,9-HpCDF; OCDF f- 2,3,7,8-TCDD; 1,2,3,7,8-PeCDD; 1,2,3,4,7,8-HxCDD; 1,2,3,6,7,8-HxCDD; 1,2,3,7,8,9-HxCDD; 1,2,3,4,6,7,8-HpCDD; OCDD; 2,3,7,8-TCDF; 1,2,3,7,8-TCDF; 1,2,3,7,8-peCDF; 2,3,4,7,8- PeCDF; 1,2,3,4,7,8-HxCDF; 1,2,3,6,7,8-HxCDF; 1,2,3,7,8,9-HxCDF; 2,3,4,6,7,8-HxCDF; 1,2,3,4,6,7,8-HpCDF, OCDF g- 2,3,7,8-TCDD; 1,2,3,7,8-PeCDD; 1,2,3,4,7,8-HxCDD; 1,2,3,6,7,8-HxCDD; 1,2,3,7,8,9-HxCDD; 1,2,3,4,6,7,8-HpCDD; OCDD; 2,3,7,8-TCDF; 1,2,3,7,8-PeCDF h- 2,3,4,7,8-TCDF; 1,2,3,7,8-PeCDF; 2,3,4,7,8-PeCDF; 1,2,3,4,7,8-HxCDF; 1,2,3,6,7,8-HxCDF; 1,2,3,7,8,9-HxCDF; 2,3,4,7,8-HxCDF; 1,2,3,4,6,7,8-HpCDF; 1,2,3,4,7,8,9-HpCDF * -WHO – 200; n.a. - not available; Ø-mean value

______250 Annex I – Solution by dilution? ______

Table I.2 Concentrations of PBDEs in sediments from the Yangtze River and East China Sea. Number of papers: Total papers: 4; PBDEs in the Yangtze River: 3 (Water: 0; Sediment: 3).

Location Sampling Na Concentration (ng/g) Reference Date ƩPBDEs BDE209 TGR 2010 26b n.d.-146 n.d.-502 (Zhao et al. 2013) Yangtze Delta n.a. 12c 0.1-0.55 0.2-95 (Chen et al. 2006) Yangtze Delta n.a. 14d 2-4 (Ø 3) n.a. (Shen et al. 2006) East China Sea 2006 7e n.d.-8 0.3-45 (Li et al. 2012b) a-Number of PBDE congers except BDE-209 b- BDE 3, 7, 15, 17, 28, 49, 71, 47, 66, 77, 100, 119, 99, 85, 126, 154, 153, 138, 156, 184, 183, 191, 197, 196, 207, 206 c-BDE 7, 11, 15, 17, 28, 47, 66, 100, 99, 164, 153, 183 d-BDE 3, 15, 28, 47, 60, 100, 99, 85, 154, 153, 138, 183, 197, 207 e- BED 28, 47, 99, 100, 153, 154, 183 . n.a. - not available; Ø-mean value

______251 Annex I – Solution by dilution? ______

Table I.3 Concentrations of PFCs, PFOS, PFOA in water and sediments from the Yangtze River and other water bodies in China. Numbers of papers: Total papers: 10, PFCs in the Yangtze River: 7 (Water: 5; Sediment: 3)

Location Sampling Concentration Reference Date ƩPFCs PFOS PFOA Water (ng/L) Yangtze River Drainage Basin Three Gorges Reservoir 2003 n.a. 0.1-38 (Ø 3) 0.2-298 (Ø 5) (Jin et al. 2006) Wuhan section (MS) 2003 n.a. 2.3-25.5 3.5-5 (Jin et al. 2006) Three Gorges Reservoir 2003 n.a. 0.2-38 0.2-298 (Jin et al. 2009) and Wuhan section (MS) Chongqing, Yichang, 2005 n.a. 0.01-14 22-260 (So et al. 2007) Nanjing, Shanghai section (MS) Yangtze Estuary (SB) 2008 n.a. 36-144 n.a. (Pan &You 2010) Yangtze Delta 2008 42.4-170 1-13.5 31-119 (65) (Lu et al. 2011) Tai Lake 2009 18-448 4-394 (Ø 26.5) 11-37 (Ø 22) (Yang et al. 2011) (Ø 528)a Dianchi Lake 2010 n.a. 2-15.12 (Ø 8) 3-35 (Ø 10) (Zhang et al. 2012b) Other Water Bodies Pearl River n.a. n.a. 1-99 0.8-13 (So et al. 2007) East to South China Sea 2010 0.133-3.3b n.a. n.a. (Cai et al. 2012a) Liao River 2009 1.4-131 n.d.-6.6 (Ø 0.33) n.d.-28 (Ø 11) (Yang et al. 2011) (Ø 43.6)a Location Sampling Concentration Reference Date ƩPFCs PFOS PFOA Sediment (ng/g, dry weight) Yangtze River Drainage Basin Tai Lake 2009 0.2-1.3 (Ø 0.06-0.3 (Ø 0.02-0.5 (Ø (Yang et al. 2011) 0.7)a 0.15) 0.2) Huangpu River 2007 62.5-276c 1.6-9 5-203 (Li et al. 2010) Huangpu River 2009 0.25-1d n.d-0.5 0.2-0.6 (Bao et al. 2010) Yangtze Estuary (SB) 2008 n.a. 73-537 n.a. (Pan &You 2010) Dianchi Lake 2010 0.2-2.5e 0.1-0.8 (Ø 0.25) n.d.-0.7 (Ø (Zhang et al. 0.1) 2012b) Other Water Bodies Pearl River 2009 0.1-4d n.d.-3 n.a. (Bao et al. 2010) Liao River 2009 0.3-1 (Ø 0.5)a 0.04-0.5 (Ø 0.02-0.18 (Ø (Yang et al. 2011)

______252 Annex I – Solution by dilution? ______

0.15) 0.1) Abbreviations: Pefluoropetanoate (PFPA), perfluorohexanoate (PFHxA), perfluoroheptanoate (PFHpA), PFOA, perfluorononanoate (PFNA), perfluorotridecanoate (PFTriDA), PFBS, PFOS, N-ethyl perfluoroocatane sulfonamide (EtFOSA) PFBS, PFHxS, PFOS, PFDS, PFHpA, PFOA, PFNA, PFDA, PFUdA, PFDoA PFPA, PFHxA, PFHpA, PFNA, PFTriDA, PFBS, PFOS, EtFOSA PFPA, PFHxA, PFHpA, PFOA, PFNA, PFTriDA, PFBS, PFOS, EtFOSA PFBS, PFOS, PFOA, PFDA, PFDoA n.a. – not available; n.d. – not detected; MS - Yangtze River main stream; SB – South Branch; Ø-mean value

______253 Annex I – Solution by dilution? ______

Table I.4 Summary of other organic pollutants in water and sediment of the Yangtze River Drainage Basin. Numbers of papers: Total papers: 10; PAE: 4; NP: 3; BPA: 2. Sampling Location Sampling Date Concentration Reference Ʃ5 PAEs DBP DEHP PAEs (Water: µg/L) Chongqing section (MS) 2008 n.a. n.d.-42 n.d.-12 (Luo et al. 2009 ) Wuhan section (MS) 2005/07 0.034-0.456 0.014-0.126 0.01-0.28 (Wang et al.2008) Wuhan section (MS) 2005/12 35.75-91.22 n.d.-35 4-54 (Wang et al.2008) Wuhan section (TB) 2005/07 0.114-1.259 n.a. n.a. (Wang et al.2008) Wuhan section (TB) 2005/12 0.25-132.12 n.a. n.a. (Wang et al.2008) Jiangsu section (MS) 2004-2005 0.178-1.474 a 0.105-0.286 <0.010-0.836 (He et al. 2011) Yangtze Delta 2010 0.061-28.550 (Øn.d. -7 n.d-28 (Zhang et al. 2012a) 4.536)a PAEs (Sediment: µg/g) Wuhan section (MS) 2005/07 157.1-450 11.7-246 76-221 (Wang et al.2008) Wuhan section (MS) 2005/12 76.3-275.9 25.4-84.3 50.8-192.6 (Wang et al.2008) Wuhan section (TB) 2005/12 6.6-478.9 n.d.-154.8 0.4-323.5 (Wang et al.2008) Sampling Location Sampling Date Concentration Reference NP (Water: ng/L) Chongqing section (MS) 2000/4 20-1,1200 (Shao et al. 2002) Chongqing section (MS) 2000/7 1,550-6,900 (Shao et al. 2002) Chongqing section (MS) 2000/04,07,12 1,700-7,300 (Shao et al. 2005) Yangtze Estuary and2008 13-186 (Ping 2011) adjacent area NP (Sediment: ng/g) Yangtze Estuary 2003 2-36 (Bian et al. 2010) BPA (Water: ng/L Huangpu River n.a. 184-782 (Wang et al. 2012a) BPA (Sediment: ng/g) Yangtze Estuary and2003 1-13 (Ø 3.3) (Bian et al. 2010) adjacent East China Sea Abbreviations: di-methyl phthalate (DMP), di-n-butyl phthalate (DBP), di (2-ethylhexyl) phthalate (DEHP), di-n- octyl-phthalate (DnOP), di-ethyl phthalate (DEP), di-isononyl phthalate (DiNP), , benzyl butyl phthalate (BBP) a-Ʃ5PAEs+BBP n.a. - not available; MS – Yangtze River main stream; TB – tributaries, Ø-mean value

______254 Annex I – Solution by dilution? ______

Reference (Shu et al. 2002) (Qiu et al. 2003) (Yuan et al. 2005) (Lu et al. 2004) (Dong2010) et al. (Shen et al. 2003a) (Li et al. 2006 ) (Wu 2005) (Zhu2003) et al. (Dong2010) et al. (Lu et al. 2010a) (Shi et al. 2011)

er

Compartment Source water Source water, Chlorinated drinking water Chlorinated drinking water Chlorinated drinking water Source water, tap water Source water, tap water Source water Source water Source water, tap water Source wat Source water

Number ofNumber papers: Tota papers: 20; Mutagenicity: 8; Endocrine activity: 4; Other

River.

7 cells)

-

) in )the Yangtze

micronucleus (Vivia fabatest root tip

in vivo in

and

assay (Human peripheral blood

estradiol induction, gonad atrophy

-

in vitro hyroid hormone reporter assay (Green gene

Assay method Ames assay (S. typhimurium) SCGE/Comet assay (HepG2 cells) SCGE/Comet assay (HepG2 cells) SCGE/Comet assay (HepG2 cells), Micronucleus test Ames assay (S. typhimurium) Ames, Ara, SOS/umu test Comet lymphocytes), cells) Ames (S. typhimurium) proliferationCell (MCFtest YES assay Adult male goldfish (C. auratus), serum vitellogenin, 17ß T monkeykidney fibroblast)

Lake

Summary bioassayof studies (

5

.

I

Table

endpoints: 7 Sample source Mutagenicity Chongqing section (MS), Jialing River Chongqing section (MS), Jialing River Wuhan section (MS), Dong Lake, Han River Wuhan section (MS), Dong Wuhan section (MS), Han River Shanghai area Nanjing section (MS) Yangtze Estuary Endocrine activity Chongqing section (MS), Jialing River Wuhan section (MS), Han River Nanjing section (MS) Jiangsu section (MS)

______255 Annex I – Solution by dilution? ______

Reference (Cui et al. 2009) (Li et al. 2006) (Zhao2011) et al. (Zhao2009) et al. (Wang et al. 2010) (Wang et al. 2011) (Wu et al. 2010)

water

Compartment Source water Drinking Tap water Source water Source water Source water Sediment

Dawley

-

transferase

-

ATPase

S

-

-

deethylase (EROD),

-

O

Sediment contact assay, zebra

-

ufin

deethylase(EROD) activity, CYP1A1

-

) embryos

Electrophoreticmobility shift assay

ethoxyresor

-

cytotoxity assays with sertoli, leydig and

Danio rerio

Assay method Ethoxyresorufin O mRNA expression, aryl hydrocarbonreceptor binding capacity, activationof xenobiotic response element, H4IIE cells; (EMSA). (Sub)chronic toxicity, rats, multiple endpoints Male reproductive system test, Mus musculus Male reproductive system test, Mus musculus Acetylcholinesterase (AChE), glutathione (GST), 7 glutathioneperoxidase (GPx)and Na+/K+ activities, goldfish (Carassius auratus) In vitro spermatogenic cells from maleadult Sprague rats Fish embryo toxicity test, fish (

Yangtze RiverYangtze main stream

–

not available; MS

-

Sample source Other Endpoint Chongqing section (MS), Jialing River Jialing River Jialing River Nanjing section (MS) Nanjing section (MS) Tai Lake section (MS) in Jiangsu Province Yangtze Estuary n.a.

______256 Annex I – Solution by dilution? ______

Reference (Greenpeace 2010) (Shao et al. 2005) (Xian et al. 2008) (Su et al. 2010) (Su et al. 2012) (Lu et al. 2011) (Wen et al. 2008) (Hu et al. 2009)

PBDEs

-

studies: Total publications: 13

Chemical/Endpoint NP, OP, PFOS NP, NPOEs PBDEs/HBCDs MeOPBDEs, PBDEs PFOS, PFOA PCDDs/DFs PCBs, PAHs, HCHs, DDTs

in situ

Numbers ofNumbers publications in

atus, Ictalurus punctatus, Pseudorasbora parva,

thus

fed fed chicken and ducks

-

studies in studies the Yangtze River.

Species Cyprinus carpio carpio, soldatovi Silurus meridionalis Coreius guichenoti, Coreius heterodon, Leptobotia elongate, Rhinogobio typu, Rhinogobioventralis Hypophthalmichthys molitrix, Ctenopharyngodon idella, Aristichthys nobilis, Carassius auratus, Cyprinus carpio, Coreius heterodon,Parabramis pekinensis, Siniperca chuatsi, Channa argus Coilia nasus, Coilia mystus, Coilia nasus taihuensis, Coilia brachygna Cyprinus carpio, Carassius auratus, Pelteobagrus fulvidraco, Hemicculter leuciclus, Coilia macrognathos Bleeker, Silurus spp., Sinonovacula constrzcta, Drepane punctata, Ilisha elongate, Suggrundus meerdervoortii, Pseudosciaena polyactis Carassius aur home Limnoperna lacutris, Cobicula fluminea Cyprinus carpio, Hypophthalmichthys molitrix, Ctenopharyngodon idellus, Bithynia fuchsiana, Bellamya aeruginosa

in situ

Date 2010/03 2000/04,2000/07,2000/ 12 2006 07.04.2007 n.a. 2007, 2008 2003/12, 2004/11 2004/09, 2004/12, 2005/03

Summary reportedof research about

Estuary,Tai

6

.

I

Table Sampling sites Chongqing, Wuhan and Ma’anshan and Nanjing (MS) Chongqing Section (MS), Jialing River Nantong/Shanghai (MS) Nanjing (MS) to Yangtze Lake and LakeHongze Lower Yangtze Reaches (MS),Yellow Sea Shenyang, Yangtze Delta Yangtze Estuary Jiangning to Haimen (MS)

______257 Annex I – Solution by dilution? ______

2002)

Reference (Yang et al. 2006) (Liu et al. 2004) (Ma et al. 2008) (Chen et al. 2002) (Li et al. 2010) (Chen et al.

Volatile

Chemical/Endpoint DDTsHCHs, DDTs,HCHs, PCBs HCH, DDT Petroleum hydrocarbons, phenols Micronuclei formation Micronuclei formation

,

Pelteobagrus vachellii

Sinonovacula constricta, tributaries

Scapharcasubcrenata

–

Ctenopharyngodon idellus,

Species Corbicula fluminea, Sesarma dehaani Corbicula fluminea, Sinonovacula constricta, Bullacta exarata, Potamocorbula ustulata, Sesarma dehaani, Exopalaemon carinicauda, Mugil cephalus Oyster, Mussel, Mactra veneriformis Reeva, Meretrix meretrix Linnaeus, Coilia ectenes, Eriocheir Anguilla sinensis, japonica, Leiocassis longirostris, Silurus asotus, Siniperca ,chuatsi Cyprinus carpio, Megalobrama amblycephala,Coreius heterodon auratusCarassius Cyprinus carpio, Leiocassis longirostris

1999 1998

- -

Yangtze RiverYangtze main stream; TB

Date 2002/07 2002/07 2006/05, 2007/04 1998 2004/04, 2004/12, 2005/03 1997

–

Nanjing,

not available; MS

-

Sampling sites Yangtze Estuary, coastal areas Yangtze Estuary, coastal areas Yangtze Estuary, coastal areas Anqing, Zhenjiang(MS), Yangtze Estuary Jiangsu province (MS) Anqing, Nanjing, Zhenjiang(MS), Yangtze Estuary n.a.

______258 Annex II – Effect-directed of aryl hydrocarbon receptor agonists ______

Annex II – Effect-directed of aryl hydrocarbon receptor agonists

II.1 Material and methods

II.2 LC-HRMS

With focus on EROD activity, polar fractions F13 – F18 of CNG-U and HF-L were analysed. Separation analysis of bio-active fractions was performed on an Agilent LC system series 1200 HPLC, as published by Hug et al. This system was coupled to an LTQ-Orbitrap hybrid instrument (Thermo Fisher Scientific, Bremen, Germany), equipped with an APCI or ESI source and controlled by Xcalibur software (Thermo Scientific). Volumes of 10 µl were injected for each sample. The lowest detected concentrations of compounds in positive and negative APCI and ESI modes were determined by injecting 5 µl of standard solutions of different concentrations ranging from 1 to 2 µg/mL. Details on instrument settings and HRMS data analysis are given in Table II1-3

Table II.1 Settings of the LTQ Orbitrap XL instrument Equiment Mass spectrometer specification Mode of analysis Autosampler: HTS PAL, CTC Analysis, Ionization: positive ion mode Zwingen, Switzerland Spray voltage: 400 V Column Oven: at 30.C, Jones, Omilab, Full scan mode, Sheath gas: 40 arbitrary units Mettmenstetten, Switzerland m/z 120-600, mass Auxillary gas: 10 arbitrary units Pump: Rheos 2000, Flux Instruments, Basel, resolution 60,000 Ion transfer capillary temperature: Switzerland <5 ppm mass 300.C Mass spectrometer: LTQ Orbitrap, Thermo accuracy Capillary valtage: 20 V Scientific, Waltham, MA Tube lens: 60 V Ionization: Electrospray

______259 Annex II – Effect-directed of aryl hydrocarbon receptor agonists ______

Table II.2 Processing steps and settings used for MZmine 2.10 MZmine step Settings Mass detection Noise cutoff 1000 FTMS Shoulder peak detection Mass resolution 120,000; Peak model function: Lorentzian extended Chromatogram building Min. time span 0.2 min, min. height 10,000 a.u, mass tolerance 0.001 m/z or 8 ppm Smoothing Filter width of 7 Peak deconvolution Local minimum search; chromatographic threshold 89%; search minimum in retention time range 0.4 min; minimum relative height 5%, minimum absolute height 100,000 a.u.; minimum ratio of peak top/edge 1.68; peak duration range 0.2-5 min Peak list alignment Join aligner, m/z tolerance 0.001 or 8 ppm; weight for m/z 80, retention time tolerance 0.4 min; weight for RT 20 Filter for duplicates m/z tolerance 0.001 or 8 ppm; retention time tolerance 0.3 min

Table II.3 Settings used for the R “nontarget” package Step Settings Pattern search (rule based) Cut off intensity = 5000; Isotopes: 13C, 15N, 34S, 37Cl, 81Br; RT tolerance = 0.08 min; m/z tolerance 2.5 ppm, intensity tolerance = 0.2; small m/z tolerance 0.5 ppm; rules 1-11 true (see details in package documentation) Adduct search Adducts: ESI+/APCI+ =M+H, M+Na, M+K, M+NH4, M+2H, M+H+Na, M+2 Na, M+H+NH4 ESI-/APCI- = M-H, M+FA-H, M-2H RT tolerance = 0.08 min; m/z tolerance 3 ppm

______260 Annex II – Effect-directed of aryl hydrocarbon receptor agonists ______

II.2 Results and discussion

II.2.1 Target chemical analysis

Table II.4 Polycyclic aromatic hydrocarbons (PAHs), and hecterocyclic PAH concentrations together with AhR mediated activity in selected fractions of the TGR sediment samples Fr. PAHs Concentration ng/g SEQ AhR Mediated CNG - CNG - CNG - HF - Activity U D T L 7 Fluoanthene 56.6 n.a n.a n.a (-) a Pyrene 44.4 n.a n.a n.a (-) a ∑PAHs 101 8 Benzo[a]anthracene 40.2 n.a 9 n.a (+)a Chrysene n.d n.a n.d n.a (+)a ∑PAHs 40.2 9 9 Benzo[a]anthracene n.d 7.4 6.8 14 (+)a Chrysene 29.2 11.8 7.6 18.8 (+)a Benzo[b]fluoranthene 32.6 13.6 14.8 37.6 (+)a Benzo[k]fluoranthene 16.2 8 6.8 9.4 (+)a Benzo[a]pyrene 21.6 10 9.4 11.4 (+)a ∑PAHs 99.6 50.8 45.4 91.2 10 Indeno[1,2,3-c,d]pyrene 20.4 n.a n.a 21.8 (+)a Dibenz[a,h]anthracene 5.2 n.a n.a 7.6 (+)a Benzo[g,h,i]perylene n.d n.a n.a n.d Benzo[b]fluoranthene n.a n.a n.a 9.4 (+)a Benzo[b]naphtho[2 ,1 -d]furan n.a n.a n.a 0.1 (+)b Benzo[b]naphtho[1,2-d]furan n.a n.a n.a 0.3 (+)b Benzo[b]naphtho[2,3 -d]furan n.a n.a n.a 0.5 (+)b 1-Methylpyren n.a n.a n.a 0.5 (+)b Benzo[b]naphtho[2,3-d]thiophen n.a n.a n.a 2.8 (+)b 1-Phenyldibenzothiophen n.a n.a n.a 0.4 / Benzo[b]naphtho[1,2-d]thiophene n.a n.a n.a 2.2 (-)b 2-[1-Naphthalenyl]benzothiophen n.a n.a n.a 3.8 (+)b 4-Methylbenz[a]anthracen n.a n.a n.a 1.2 (+)b 2-Phenyldibenzothiophene n.a n.a n.a 3.0 / 5-Methylbenz[a]anthracen n.a n.a n.a 0.9 (+)b 3-Phenyldibenzothiophene n.a n.a n.a 3.1 (-)b 2,2’-Binaphthyl n.a n.a n.a 4.0 (+)b 2-[2-Naphthyl]-1-thiaindene n.a n.a n.a 3.4 (+)b Dinaphtho[1,2-b;1’,2’-d]furan n.a n.a n.a 4.2 (+)c

______261 Annex II – Effect-directed of aryl hydrocarbon receptor agonists ______

Dinaphtho[1 ,2 -b; 2',3 '-d]furan n.a n.a n.a 3.6 (+)c 1,3,6-trimethylcgrysene n.a n.a n.a n.d / 3-Methylcholanthren n.a n.a n.a × (+)b 9-Methylbenzo[a]pyrene n.a n.a n.a × (+)b 8-Methylbenzo[a]pyrene n.a n.a n.a × (+)b Dinaphtho[2,1-b;1’2’-d]thiophene n.a n.a n.a 5.0 (+)b ∑PAHs 38.8 ×: compound detected but not quantified; n.d: below detection limit; n.a: not analyzed. -: Not AhR agonist reported in literature or predicted to have no binding affinity to the AhR by QSAR modeling; +: AhR agonist reported in literature or predicted to have binding affinity to the AhR by QSAR modeling; /: inconclusive. a: Bols et al. b: QSAR modeling, c: Brack and Schirmer 2003

______262 Annex II – Effect-directed of aryl hydrocarbon receptor agonists ______

II.2.3 Non-target analysis of polar fractions

Table II.5 Number of peaks detected in the active fractions and candidate filtering for potential AhR agonists based on retention time, mass defects, and absence in non-active fractions Sample Fr. Detected peaks (after removal of Remaining peaks > 105 a.u. intensity after blank peaks) > 105 a.u. intensity applying filter criteria of AhR agonists ESI+ ESI- APCI+ APCI- ESI+ ESI- APCI+ APCI- CNG-U 13 163 23 70 26 62 8 37 23

14 252 12 163 27 131 9 99 27

15 452 13 339 29 108 11 114 29

HF-L 13 483 12 417 87 292 10 287 86 14 62 0 43 11 21 0 5 8 15 510 4 504 32 137 3 87 17

______263 Annex II – Effect-directed of aryl hydrocarbon receptor agonists ______

.

3 3 4 4 4

- - - - -

2 2 4 1 4 1 2 1 4 4 4 4 4 4 1

2 2 3 3 3

ID level

ing ing to (Schymanski et al

benzothiazole)

-

iazole

(1,3

-

isomer isomer

Identification

ribenzylamine

benzoth

ID level is given accord

t

ercaptobenzothiazole

ethylthio)benzothiazole

benzothiazolesulphenamideor benzothiazolesulphenamideor

- -

m

m

-

2 2

The

(

- -

dibenzylamine or similar dibenzylamine or similar

2

-

ulfanediylbis

2

s

-

utyl utyl

’

unknown benzothiazolederivative unknown benzothiazolederivative unknown benzothiazolederivative

b b

- -

t t

LF13.

2,2

- -

-

n n

0 6

11 18 27 36

389 389 138 133 799 257 257 920 615 496 105 122

1246 1110

Chemspider

# of structures in

C7H5NS+H+

C14H15N+H+ C14H15N+H+ C21H21N+H+

C7H5NS2+H+ C8H7NS2+H+

C15H15ON+H+ C21H25ON+H+

C22H22N2S+H+ C14H8N2S3+H+ C17H18N2S+H+ C15H16N2S+H+ C17H15NS5+H+ C20H27NS2+H+

C22H22N2O+H+ C23H29O2N+H+

C10H9NOS2+H+ C22H23NOS+H+

molecualr formula

C11H14N2S2+H+ C11H14N2S2+H+

a.u. intensity) detected in the active fraction HF

6

58E+07

3.04E+07 2.10E+07 1. 1.23E+07 1.06E+07 9.25E+06 9.04E+06 3.86E+06 3.56E+06 3.56E+06 3.16E+06 2.98E+06 2.33E+06 2.27E+06 1.87E+06 1.63E+06 1.56E+06 1.40E+06 1.21E+06 1.08E+06

Signal intensity (a.u.).

16.0 15.2 23.8 21.5 26.3 20.9 27.4 20.3 18.7 25.6 16.7 16.2 18.3 20.2 23.2 22.7 21.4 24.4 31.8 34.0

RT RT (min)

List List of most intense peaks (>10

6

.

II

): 1= confirmed, 2=probable structure, 3=tentative candidate, 4=unequivocal molecular formula, 5=accurate mass.

m/z

288.1745 347.1575 167.9933 300.9923 136.0214 283.1262 331.1804 239.0669 239.0669 308.2007 352.2269 257.1106 224.0196 350.1570 182.0091 393.9880 346.1658

198.1274 198.1274 226.1224

Table 2014a

______264 Annex II – Effect-directed of aryl hydrocarbon receptor agonists ______

Fig. II.1 Extracted ion chromatograms of benzothiazole and related compound in fraction HF-L-F13. (A) ESI+ run showing the protonated molecules and (B) APCI+ run showing the benzothiazole and mercaptobenzothiazole in-source fragments of the compounds detected in ESI+ mode. NL: signal intensity at 100% relative abundance.

______265 Annex II – Effect-directed of aryl hydrocarbon receptor agonists ______

Fig. II.2 MS/MS spectra of benzothiazole and related compounds in fraction HF-L-F13 acquired in APCI+ mode using HCD fragmentation (collision energy 100 a.u.). For two compounds the MS/MS spectra of the in- source fragments resembling the 2-mercapto-benzotzhiazole moiety are shown. NL: signal intensity at 100% relative abundance.

______266 Annex II – Effect-directed of aryl hydrocarbon receptor agonists ______

Fig. II.3 Extracted ion chromatograms and MS/MS spectra of three probable benzothiazole derivatives (m/z 239.0669 at RT 16.2 min; m/z 239.0662 at RT 16.7 min; m/z 346.1658 at RT 34.0 min) in fraction HF-L-F13 acquired in ESI+ mode using HCD fragmentation (collision energy 100 a.u.). NL: signal intensity at 100% relative abundance.

______267 Annex II – Effect-directed of aryl hydrocarbon receptor agonists ______

Fig. II.4 Extracted ion chromatograms and MS/MS spectra of 2-(methylthio)benzothiazole derivatives (m/z 182.0091 at RT 24.3 min) and a related compound (m/z 224.0196 at RT 22.7 min) in fraction HF-L-F13 acquired in ESI+ mode using HCD and Cid fragmentation. NL: signal intensity at 100% relative abundance.

______268 Annex II – Effect-directed of aryl hydrocarbon receptor agonists ______

Fig. II.5 MS/MS spectra of tribenzylamine and related compounds in fraction HF-L-F13 acquired in ESI+ mode using CID fragmentation (collision energy 35 a.u.). NL: signal intensity at 100% relative abundance.

______269 Annex II – Effect-directed of aryl hydrocarbon receptor agonists ______

______270 Annex III – High-throughput fractionation and bioassays ______Annex III – High-throughput fractionation and bioassays

III.1 Material and methods

4 8

water water

. 55 83 29 77 47 59 .

min)

-

72 98 96 75 29 75 96 61 46 ......

......

5 4 6 6 8 8 6 9 8 11 13

13 10 12 12 13 13

RT RT (

)

Octanol

-

(

ow

3 4

03 14 57 67 12 22 29 . . 63 92 94 98 05 18 23 29

......

K

3 3

2 2 2 2 3 3 3 3 3 3 3 4 4 4 4

log

)

1

-

2 2

16 15 19 14 . 16 21 17 22 19 21 . 21 19 22 22 26

......

(g mol

134 152

129 117 143 118 132 167 128 179 146 154 154 168 166 182 184

MW

and retention time (RT).

ow

K

log

uhe, Germany(~95%)

EPA EPI Suite. Experimentaldata were preferredover predicted values.

- Karlsruhe, Germany(>98%)

Supplier (purity)

Aldrich, Steinheim, Germany(~98%)

Aldrich, Steinheim, Germany(>99%) Aldrich, Steinheim, Germany(≥96%) Aldrich, Steinheim, Germany(≥97%) Aldrich, Steinheim, Germany(>99%)

-

- - - -

Merck, Darmstadt, Germany (>98%)S

andards, Wesel, Germany (analytical standard)

abcr chemicals, Karlsr

abcr chemicals, abcr chemicals, Karlsruhe, Germany(>99%) abcr chemicals, Karlsruhe, Germany(>98%) abcr chemicals, Karlsruhe, Germany(>98%) abcr chemicals, Karlsruhe, Germany(>98%)

Sigma

Sigma Sigma Sigma Sigma

LGC LGC Standards, Wesel, Germany(analytical standard)

LGC LGC Standards, Wesel, Germany (analytical standard) LGC Standards, Wesel, Germany (analytical standard) LGC St LGC Standards, Wesel, Germany (analytical standard)

- -

9 6 6 8 9 0

5 3 8 8 3 9 4 7 1

------

------

) were obtainedusing US

25 00

- -

72 89 94 96 64 65

ow

22 62 15 74 20 32 52 73 83

2 1

------

------

K

91 91 95 86 91 83 92 86 92

CAS No.CAS 120 271 4265 260 3782 208 132 132

log

-

2,3

List List of single compounds used for establishing the relationship between

Indole

Comp.

Acridine

Fluorene

Biphenyl

Xanthene

Quinoline

Carbazole

1

.

Benzofuran

Naphthalene

Dibenzofuran

Acenaphthene

Methylquinoline

- Benzothiophene Acenaphthylene

Methylbenzofuran

III

-

Dibenzothiophene

6

2

Dimethylbenzofuran

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17

No.

Table partitioning coefficients (

______271 Annex III – High-throughput fractionation and bioassays ______

14,5

14,16 13,84 15,48 14,96 18,71 16,16 17,39 16,17 18,15 17,48 20,59 18,32 19,79 20,68 19,42

RT RT (min)

)

-

(

ow

K

6,7 6,8

4,45 4,46 4,68 4,88 5,16 5,73 5,76 5,78 5,81 5,99 6,11 6,75 6,76 6,98

log

)

1

-

272,3

178,23 178,23 202,25 202,26 284,78 228,29 252,31 228,29 252,32 252,32 276,33 278,35 276,33 321,97 326,43

MW (g mol

Germany(analytical standard)

Supplier (purity)

Wesel, Wesel, Germany (analytical standard)

ds, Wesel, Germany (analytical standard)

Aldrich, Steinheim, Germany(≥98%)

-

Aldrich, Steinheim, Germany(analytical standard) Aldrich, Steinheim, Germany(analytical standard)

Sigma

- -

LGC LGC Standards, Wesel,

LGC LGC Standards, Wesel, Germany (analytical standard) LGC Standards, Wesel, Germany (analytical standard) LGC Standards, Wesel, Germany (analytical standard) LGC Standards, Wesel, Germany (analytical standard) LGC Standards, Wesel, Germany (analytical standard) LGC Standards, LGC Standards, Wesel, Germany (analytical standard) LGC Standards, Wesel, Germany (analytical standard) LGC Standards, Wesel, Germany (analytical standard) LGC Standards, Wesel, Germany (analytical standard) LGC Standar

Sigma Sigma

Dr, Ehrenstorfer, Augsburg, Germany(analytical standard)

8

2 6

-

7 0 0 1 2 9 9 2 5

- -

8 3 8 3

------

- - - -

28

87 01

-

12 00 44 74 99 01 08 24 39

- -

01 55 32 70

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- - - -

85 56 50 53

CAS No.CAS 120 129 206 118 205 218 207 191 193

6051 1746

57465

]pyrene

cd

-

]perylene

TCDD

]pyrene

]anthracene

-

a

]antracene

a

]fluoranthene

]fluoranthene

ghi

a,h

Comp.

Pyrene

k

b

1,2,3

PCB 126

Chrysene

Anthracene

Fluoranthene

Phenanthrene

Naphthoflavone

2,3,7,8

-

Benzo[

β

Benzo[

Hexachlorobenzene Benzo[

Benzo[

Benzo[

Dibenz[

Indeno[

18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

No.

______272 Annex III – High-throughput fractionation and bioassays ______

III.2 Results and discussion

III.2.1 Development and validation of the fractionation method

2 5 )

n 2

i R = 0 .9 4

m 2 0

(

e

m 1 5

i

t

n

o 1 0

i

t

n e

t 5

e R 0 2 3 4 5 6 7

lo g K o w

Fig. III.1 Linear regression of chromatographic retention time versus the n-octanol/water partitioning coefficient (log Kow) for a total number of 33 chemicals. The straight line is the regression line; the shaded area indicates the 95% prediction band.

______273 Annex III – High-throughput fractionation and bioassays ______

III.2.2 Fractionation of environmental samples

Fig. III.2 Micro-EROD results of HPLC-fractionated sediment extract HAN-D (Yangtze River, China). 10 µL of a 1 g SEQ mL-1 extract were fractionated. Asterisks denote significant differences between fractions and control (one- way ANOVA with Holm-Sidak’s post-hoc test, p ≤ 0,05). Shaded areas indicate the typical retention time ranges of the substance classes screened as single substances.

Fig. III.3 Micro-EROD results of HPLC-fractionated sediment extract HAN-C (Yangtze River, China). 10 µL of a 1 g SEQ mL-1 extract were fractionated. Asterisks denote significant differences between fractions and control (one- way ANOVA with Holm-Sidak’s post-hoc test, p ≤ 0.05). Shaded areas indicate the typical retention time ranges of the substance classes screened as single substances.

______274 Annex III – High-throughput fractionation and bioassays ______

Table III.2 Characteristics of and concentrations of 17 PCDD/Fs and 12 dl-PCBs in sediments from Ehrenbreitstein (EBR) and Zollelbe (ZE). Modified from Eichbaum at al, 2016.

Ehrenbreitstein harbor Zollelbe

Acronym EBR ZE

River system Unit km Rhine main stream 591.4 Elbe cut-off meander 0.1

Longitude 7.60792 11.65087

Latitude 50.35400 52.13256

Sampling date 12,04,2012 10,04,2012

Grab (Max, sampling depth) Van-Veen grab (15 cm) Van-Veen grab (15 cm)

TOC (g kg-1) 49.6 64.3

Loss on ignition (%) 10.6 ± 0.4 14.1 ± 0.3

Concentration of 12 dl-PCB 4.38 ng g-1 dw 9.72 ng g-1 dw

Concentration of 17 PCDD/F 1.06 ng g-1 dw 3.70 ng g-1 dw

-1 Micro-ERODEC25TEQ of PCB fraction (pg g dw) 16.7 ± 7.1 76.4 ± 18.8

-1 Micro-ERODEC25TEQ of PCDD/F fraction (pg g dw) 63.7 ± 8.8 159.0 ± 32.1

______275 Annex III – High-throughput fractionation and bioassays ______

0 .8

C h lo rin a te d c o m p o u n d s a n d

) 1

- h ig h e r m o le c u la r w e ig h t P A H s

l 0 .6 E P A -P A H s

m

D D

C H e te ro -P A H s

T 0 .4

g

p

(

Q

E 0 .2 B

0 .0 0 5 1 0 1 5 2 0 2 5 3 5 4 5 5 5

T im e (m in )

Fig. III.4 Micro-EROD results of HPLC-fractionated multilayer silica column-treated sediment extract (Ehrenbreitstein, Rhine, Germany). 10 µL of a 1 g SEQ mL-1 extract were fractionated. No significant differences between fractions and control were found (one-way ANOVA with Holm-Sidak’s post-hoc test, p > 0.05). Shaded areas indicate typical retention time ranges of the indicated substance classes.

0 .8

C h lo rin a te d c o m p o u n d s a n d

) 1

- h ig h e r m o le c u la r w e ig h t P A H s

l 0 .6 E P A -P A H s

m

D * D

C H e te ro -P A H s

T 0 .4

g *

p *

(

Q

E 0 .2 B

0 .0 0 5 1 0 1 5 2 0 2 5 3 5 4 5 5 5

T im e (m in )

Fig. III.5 Micro-EROD results of HPLC-fractionated multilayer silica column-treated sediment extract (Zollelbe, Elbe, Germany). 10 µL of a 1 g SEQ mL-1 extract were fractionated. Asterisks denote significant differences between fractions and control (one-way ANOVA with Holm-Sidak’s post-hoc test, p ≤ 0.05). Shaded areas indicate typical retention time ranges of the indicated substance classes.

______276 Acknowledgments

Acknowledgments

First of all, I would like to express my profound gratitude to Prof. Dr. Henner Hollert, for giving me this opportunity to carry out my PhD studies at the Institute for Environmental Research, RWTH Aachen University. I would like to express my gratitude to you, for your patience and trust, especially for your splendid hospitality to take care of our Chinese students. Thank you for the tireless support in science and living in Germany throughout my whole study.

I am grateful to Prof. Dr. Andreas Schäffer for having supervised my dissertation. I specially thank you for the scientific support you gave me, for your input for my manuscripts, and because you did not spare any effort to help me to apply for the Young researcher scholarship at the University.

Special thanks deserves Prof. Dr.Xiaowei Zhang from Nanjing University. Thank you for introducing me to Prof. Hollert, thus I was able to start this great journey. I am also appreciating your unfalilingly support during my whole study.

The present study received financial support from China Scholarship Council (CSC), which is gratefully acknowledged. The Federal Ministry of Education and Research (BMBF) was greatly appreciated, for financing the joint environmental research program “Yangtze Project” (Project number: FKZ 02WT1141).

I am grateful to Prof. Dr. Xingzhong Yuan, and Dr. Bo Li from Chongqing University, Prof. Dr. Lingling Wu from Tongji University, and Prof. Dr. Junli Hou from the East China Sea Fisheries Research Institute in China, for their patience and friendly assistance during the sampling campaign.

My stays in Leipzig in 2012 and 2014 were the sources of much enlightening moments. The stay in UFZ was a great treasure for me during my EDA study. I am also appreciating PD Dr. Werner Brack for his support of my stay without reservation, for good advices regarding the experiments, and the constructive comments which greatly improved our manuscript. I would particularly like to thank Dr. Martin Krauss for his professional input about chemical analysis in my dissertation, his extraordinary kindness for the corporation, and tireless support for the manuscript. Special thanks also go to Dr. Arnold Bahlmann, who introduced me to the procedures of fractionation, and helped me performing the experiments.

______277 Acknowledgments

Thanks also go to Dr. Martina Roß-Nickoll, Dr. Björn Scholz-Starke, and the entire team of the BMBF Yangtze Microtox-Project. Thanks for all your constructive input. It was my great pleasure working together with you.

My sincere thanks are also addressed to Dr. Tilman Floehr, for his great assistance by introducing me to the lab work, for his continuous support on the project and for his patience when dealingwith my many questions. I am very glad for being able to work together with you on the same project.

My dear friends Dr. Kathrin Eichbaum and Leonie Katharina Nüßer. M.Sc., I am so grateful for your friendship and helpful assistance throughout all the years. You are the most kind, warm- hearted, and lovely girls I have met. Your enthusiasm casted out the cold in winter, and also warmed me in the institute.

Dr. Markus Brinkmann, I have always learned a lot by talking with you. Thanks for providing so many wonderful ideas in research, and fruitful input for the manuscript. It was a great pleasure to work together with you. Dr. Beat Thalmann and Dipl.-Biol. Andreas Schiway, thank you for your tireless in answering my questions and your kind help for the lab work. It is my honour to be one member of the “Ewomis” team. I do believe, we are doing the right thing.

I am grateful to Carolina Di Paolo, M.Sc., my friend and lunch partner. There were so many interesting topics we discussed together during lunch break. We cheered for each other when we were down and were happy for each other if any improvements for our research could be achieved. Finally, we made it!

I also want to thank Jochen Kuckelkorn. M.Sc, for your kind support for the ER-Calux test. I am gratefull to Henriette Meyer-Alert. M.Sc for the nice talking, laughing and working togehther in the same office.

Thanks go to my colleagues for their assistance for my lab work: Sebastian Heger, Carolin Gembe, and Josef Koch. Many thanks are also addressed to Simone Hotz. Thank you for your support of my work in the lab. I sincerely thank Dr. Thomas-Benjamin Seiler, Edith Schröder and Hercht Hendrik, for saving my computer more than once.

______278

Acknowledgments

Special thanks are addressed to Dennis Goebele, Lubczyk, Leonie and in particular Telse Bauer. Thank you very much for your patience with all the administrative work you assisted me along my study. Thanks go to my bachlor student Oliver Thelen and my master student Yan Yan. It has been a great honour supervising you.

I would like to thank my Chinese friends, Jianhua Yang, Haihong Guo, Zhen Li, Jinyu Li, Yumei Wang, Liyan Zhu, Miaomiao Du, Peng Zhang, Jing Zhao, Feng Cheng, who make my life colorful in Aachen.

Most of all, I would like to express my gratitude to my partents, my young sister and brother. Because of your support, I had the courage to start the journey.

Lastly, it is my greatest fortune to meet you, Sven Schumann. Thanks for your suggestions, and language correction of the thesis. Thanks for your love, understanding, patiences, company, and care taking. You never let me head down. You lead me to be a better one, and you are the best I can image in my life. Without your support and trust, I would not have been able to finish my PhD thesis.

______279

Acknowledgments

______280

Hongxia Xiao • Worringer Weg 1 • D-52064 Aachen, Germany Phone +49 (0) 241/ 80-26686 • E-mail: [email protected] Curriculum vitae

Name Hongxia Xiao Date of Birth 12th of September 1988 Place of Birth Henan, China Nationality People’s Republic of China

EDUCATION Since 04/2012 Ph.D student in Ecotoxicology and Toxicology Institute for Environmental Research (Biology V), RWTH Aachen University, Germany Thesis: Effect-directed Analysis and mechanism-specific bioassays to assess the toxicity of sediments of the Yangtze River (China) 09/2009-03/2012 M.Sc. in Environment Science and Engineering School of Resource and Environmental Engineering, East China University of Science and Technology, China Thesis: Study on Anaerobic and Post Treatment of Wastewater from Cane Molasses Distillery 09/2005-07/2009 B.Eng in Environmental Engineering School of Chemistry and Environment Engineering, Shanghai Institute of Technology,China Thesis: Study on Synthesis and Application of the Compound Dust Suppression RESEARCH EXPERIENCE 11/2012-12/2012 Research visit at the Helmholtz Centre for Environmental Research, 10/2014-11/204 PD Dr. Werner Brack group, Leipzig, Germany 04/2009-02/2012 Internship at the Technology Development Department, Rhodia (China) Co.Ltd, Shanghai, China ADDITIONAL EXPERIMENCE 10/2009-12/2010 Subeditor of The Graduate Student of ECUST 05/2010-06/2010 Volunteer of Shanghai Expo 10/2007-04/2009 Volunteer member of the Shanghai Red Cross Association

______281

______282

Scientific Contributions Scientific Contributions

*Publiscations contributing to this thesis are highlighted with asterisks Research articles in international peer-reviewed journals: *Xiao, H., Kuckelkorn, J., Nüßer, L-K., Floehr, T., Henning, M-P., Roß-Nickoll, M., Schäffer, A., Hollert, H. (2016): The metabolite 3,4,3’,4’-tetrachloroazobenzene (TCAB) exerts a higher ecotoxicity than the parent compounds 3,4-dichloroaniline (3,4-DCA) and propanil. Science of the Total Environment 551: 304-316. *Floehr, T., Scholz-Starke, B., Xiao, H., Koch, J., Wu, L., Hou, J., Wolf, A., Bergmann, A., Bluhm, K., Yuan, X., Roß-Nickoll, M., Schäffer, A., Hollert, H. (2015): Yangtze Three Gorges Reservoir, China: A holistic assessment of organic pollution, mutagenic effects of sediments and genotoxic impacts on fish. Journal of Environmental Sciences 38: 63-82. *Floehr, T., Scholz-Starke, B., Xiao, H., Hercht, H., Wu, L., Hou, J., Schmidt-Posthaus, H., Segner H., Kammann, U., Yuan, X., Roß-Nickoll, M., Schäffer, A., Hollert H. (2015): Linking Ah receptor mediated effects of sediments and impacts on fish to key pollutants in the Yangtze Three Gorges Reservoir, China – A comprehensive perspective. Science of the Total Environment 538: 191-211 Zhu, L., Santiago-Schübel, B., Xiao, H., Tiele, B., Zhu, Z., Qiu, Y.,Hollert, H,. Küppers, (2015) A laboratoray workflow for simulation and ecotoxicological evaluation of advanced oxidation processes Electrochemistry-mass spectrometry linked to toxicity testing. Chemosphere131:34-40 Liu, L., Chen, L., Floehr, T., Xiao, H., Bluhm, K., Hollert, H., Wu, L. (2015) Assessment of the mutagenicity of sediments from Yangtze River Estuary using Salmonella typhimurium/microsome assay. PLoS ONE 10, (11), e0143522. Liu, L., Chen, L., Shao, Y., Zhang, L., Floehr, T., Xiao, H., Yan, Y., Eichbaum, K., Hollert, H., Wu, L. (2014): Evaluation of the Ecotoxicity of Sediments from Yangtze River Estuary and Contribution of Priority PAHs to Ah Receptor-Mediated Activies. Plos one 9 (8): e104748 *Floehr.T., & Xiao, H. (shared first authroship), Scholz-Starke, B., Wu, L.,Hou, J., Yin, D.,Zhang, X., Ji, R., Yuan, X., Ottermanns, R., Roß-Nickoll, M., Schäffer, A., Hollert, H (2013). Solution by dilution? – A review on the pollution status of the Yangtze River. Environmental Science and Pollution Research 20 (10): 6934-6971. Zhu. L., Santiago-Schübel. B., Xiao. H., Hollert. H., Kueppers. S., (2016) Electrochemical oxidation of fluoroquinolone antibiotics: Mechanism, antibacterial activity and ecotoxicity. Water Research 102: 52 – 62.

______283

Scientific Contributions Research articles accepted or submitted to international peer- reviewed journals: Xie, Y., Floehr, T., Hollert, H., Xiao, H., Xia, P., Yang, J., Burton, A., Yu, H., Zhang, X. (2016) In situ microbiomes distinguish aryl hydrocarbon receptor (AhR) mediated activity in the contaminated sediments from Three Gorges Reservoir. Submitted to Multidisciplinary Journal of Microbial Ecology. Shi, P., Zhou, S., Xiao, H., Qiu, J., Sebastian, H., Zhou, Q., Pan, Y., Jia, S., Chen, X., Hollert, H., Li, A. (2016) A holistic assessment on the toxicological profiles of main drinking water sources in Jiangsu Province, China. Submitted to Chemosphere. *Xiao, H., Krauss, Martin., Floehr T., Yan, Y., Bahlmann, A., Eichbaum, K,. Brinkmann, M.,Zhang, X,. Yuan, Y,. Brack, W,. Hollert, H. (2016) Effect-driected of aryl hydrocarbon receptor agonists in sediments from the Three Gorges Reservoir, China. Submitted to Environmental Science & Technology. Platform Presentations:

Xiao, H. (2015) Effect-directed Analysis (EDA) to assess the toxicity of sediments of the Yangtze River (China) and High-Throughput EDA Study. Institute for Environmental Research Anaual Meeting, Aachen, Germany Floehr, T., & Hollert, H., Scholz-Starke, B., Xiao, H., Koch, J., Wu, L., Hou, J., Wolf, A., Yuan, Y., Roß-Nickoll, M., Schäffer, A. (2014) A glimpse in the black box- Ecotoxicological impacts on the Yangtze Three Gorges Reservoir, China. . SETAC Europe 24rd Anaual Meeting, Basel, Switzerland Floehr, T., Xiao, H., Yuan, X., Wu, L., Hou, J., Scholz-Starke B., Roß-Nickoll, M., Schäffer, A., & Hollert, H. (2013): Ecotoxicological effects in the newly created ecosystem of the Yangtze Three Gorges Reservoir, China. Invited speaker, SURF Seminar Dr. Müller, 28th October 2013, EAWAG Kastanienbaum/Lucerne, Switzerland. Poster presentations:

Xiao, H.,& Brinkmann, M., Thalmann, B., Eichbaum, K., Gembé, C., Seiler, T-B., Hollert, H. (2015) Micro-fractionation in 96-well plates combined with the micro-EROD assay to identify the dioxin-like activity in environmental samples. EDA-Emerge Conference. Leipzig, Germany Zhu, L., Santiago-Schuebel, B., Xiao, H., Hollert, H., Kueppers, S. (2015) Risk assessment of fluoroquinolone antibiotics in the environment by electrochemical simulation method.

______284

Scientific Contributions SETAC Europe 25rd Annual Meeting, Barcelona, Spain Floehr, T. & Hollert, H., Scholz-Starke, B., Xiao, H., Koch, J., Wu, L., Hou, J., Wolf, A., Yuan, Y., Roß-Nickoll, M., Schäffer, A. (2014) A glimpse in the black box- Ecotoxicological impacts on the Yangtze Three Gorges Reservoir, China. . SETAC Europe 24rd Annual Meeting, Basel, Switzerland Yan, Y., & Xiao, H.(shared first authroship), Floehr, T., Krauss, M., Brack, W., Hollert, H. (2014): Effected analyisis of dioxin-like activities in the sediments of Yantze River. SETAC German Language Branch, Gießen und Homberg (Ohm), Germany Xiao, H., Di Paolo, C., Heger, S., Thalmann, B., Hollert, H. (2014): Miniaturization of the micronucleus assay to 96-well plate setup presents advantages for application in Effect Directed Analysis. SETAC Europe 24rd Anaual Meeting, Basel, Switzerland Xiao, H., Floehr, T., Scholz-Starke, B., Roß-Nickoll, M., Brack, W., Schäffer, A., Hollert, H. (2013) Effect-directed analysis and mechanism-specific bioassays to assess the toxicity of sediments in Yangtze River (China) .Young Environmental Scientists (YES) the 3rd Anaual Meeting,Krakow, Poland. Xiao, H., Floehr, T., Scholz-Starke, B., Roß-Nickoll, M., Brack, W., Schäffer, A., Hollert, H. (2013): Effected-directed analysis of mutagenic compounds and dioxin-like activity inducers in Yangtze River (China) sediments. SETAC Europe 23rd Anaual Meeting, Glasgow, United Kingdom

______285