Dissertation -...

Dissertation

submitted to the

Combined Faculties for the Natural Sciences and for Mathematics

of the Ruperto-Carola University of Heidelberg, Germany

for the degree of

Doctor of Natural Sciences

presented by

Diplom-Biologist Ruben Strecker

born in Heilbronn, Germany

Oral examination: .....................................................

Toxicity and teratogenesis in zebrafish embryos (Danio rerio)

Referees: Prof. Dr. Thomas Braunbeck

Centre for Organismal Studies, Heidelberg

Prof. Dr. Henner Hollert

Institut für Umweltforschung, RWTH Aachen

Hiermit erkläre ich, dass ich die vorliegende Dissertation selbst verfasst und mich dabei kei-

ner anderen als der von mir ausdrücklich bezeichneten Quellen und Hilfen bedient habe. Des

Weiteren erkläre ich, dass ich an keiner Stelle ein Prüfungsverfahren beantragt oder die Dis-

sertation in dieser oder einer anderen Form bereits anderweitig als Prüfungsarbeit verwendet

oder einer anderen Fakultät als Dissertation vorgelegt habe.

Heidelberg, 24.01.2013 ____________________________

Ruben Strecker

"That which can be asserted without evidence, can be dismissed without evidence."

Christopher Eric Hitchens (1949-2011)

Danksagung

Verschiedene Institutionen und Personen haben zum Gelingen meiner Doktorarbeit beigetra-

gen, denen ich herzlichst danken möchte.

Mein besonderer Dank gilt:

Herrn Prof. Dr. Thomas Braunbeck, für die Möglichkeit meine Doktorarbeit in seiner Ar-

beitsgruppe anzufertigen und für seine Unterstützung über all die Jahre hinweg. Insbesondere

unsere „Dienstreisen“ werde ich nicht vergessen.

Herrn Prof. Dr. Henner Hollert für die unkomplizierte Übernahme des Korreferats.

Christopher „Da Hansel“ Faßbender, meinem Kalauerkumpanen, ohne den das Leben für

mich deutlich langweiliger gewesen wäre (inwiefern das die lieben Kollegen im selben Zim-

mer ebenso genossen haben, sei dahingestellt). Irgendwann einmal werden wir noch unseren

Film drehen …

Patrick „Chuck Gyver/Mac Norris“ Heinrich, das Juniormitglied im Range eines Kalauer-

meisters. Dein Holzlaster sowie auch dein Jägermeistervorrat haben uns wertvolle Dienste

geleistet …

Erik Leist, für die viele Arbeit im Fischkeller und die vielen Fachsimpeleien.

Den Arbeitsgruppen von Prof. Dr. Braunbeck und Prof. Dr. Frings für die entspannte Arbeits-

atmosphäre, den Sekt, das Bier und die vielen Süßigkeiten:

Britta Kais, Stefan Weigt, Daniel Stengel, Katharina „Käthe“ Schneider, Federica „Fede“

Genovese, Kerstin Vocke, Kristin Dauner, Eva Lammer, Weiping Zhang, Svenja Böhler, Se-

mir Jeridi, Philipp Daiber, Florian Schmitt, Franziska „Franzi“ Förster, Kirsten Henn, Sina

Volz, Leon Kreuter, Kristina „Krisi“ Rehberger, Frank Möhrlen, Chrisztina Vincze, Markus

Lutz, Jens Otte, Lisa Baumann, Nadja „Naddl“ Schweitzer, Kerstin Ullrich, Paula Suares-

Rocha, Philipp Kremer, Shoui, Pluto, Cleo, Emma, Natalya „Natascha“ Demir, Melanie

„Mel“ Böttcher, Markus „Maggus“ Braun, Raoul Wolf, Sarah Schnurr, Ann-Marie Oppold,

Annika Batel, Franziska Scheller, Thomas-Benjamin Seiler, Gabriele „Gabi“ Günther, Steffi

Grund, Aennes Abbas, Uta Jürgens, Sebastian „Seba“ Lungu, Susanne Keiter, Susanne „Su-

se“ Knörr, Ulrike „Uli“ Diehl, Malte Posselt, Werner Greulich, Frank Böttger, Frau Marek,

Christina Godel, Gisela Adam (unserem Photoshopgenius) und Herrn Schade.

Meinen Eltern, für den Rückhalt und ihre Unterstützung.

Abbreviations

AAF 2-Acetylaminofluorene

AFB1 Aflatoxin B1

BaP Benzo[a]pyrene

CBZ Carbamazepine

CPA Cyclophosphamide

CSG Chemical Selection Group

CV Coefficient of Variation

CYP Cytochrome Enzymes

DMSO Dimethylsulfoxide

EC Effect Concentration

ECVAM European Centre for the Validation of Alternative Methods

ECHA European Chemical Agency

hpf hours post fertilization

IFO Ifosfamide

IUCLID International Uniform Chemical Information Database

LC Lethal Concentration

LOX Lysyl oxidase

MSDS Material Safety Data Sheet

NC Negative Control

OECD Organisation for Economic Co-operation and Development

PAH Polycyclic Aromatic Hydrocarbon

PC Positive Control

PHE Phenytoin

QSAR Quantitative structure–activity relationship

REACH Regulation, Evaluation, Authorization and Restriction of Chemicals

RT-PCR Real Time-Polymerase Chain Reaction

SC Solvent Control

SOP Standard Operation Procedure

TCDD Tetrachlordibenzodioxine

TEG Tegafur

TI Teratogenic Index

TG Test Guideline

TMO Trimethadione

TT Thio-TEPA (N,N,N-triethylenethiophosphoramide)

UBA Umweltbundesamt (German EPA)

VMG Validation Management Group

WHO World Health Oranisation

ZFET Zebrafish Embryo Toxicity Test

I

Contents

Summary ................................................................................................................................... 1

Zusammenfassung .................................................................................................................... 3

Introduction ............................................................................................................................ 5

Alternatives to animal testing....................................................................................... 5

Zebrafish as a test organism ......................................................................................... 8

General aspects and outline of this thesis .................................................................... 8

Chapter I

1 The Zebrafish (Danio rerio) Embryos Toxicity Test (ZFET) OECD .....................

validation study ........................................................................................................ 12

1.1 Abstract ...................................................................................................................... 12

1.2 Introduction ................................................................................................................ 13

1.3 Materials & Methods.................................................................................................. 14

1.3.1 Test chemicals ............................................................................................................ 14

1.3.2 Zebrafish maintenance and spawning ........................................................................ 16

1.3.3 Zebrafish Embryo Toxicity Test (ZFET) - Principles ................................................ 17

1.3.4 Determination of toxicity - Lethal endpoints ............................................................. 18

1.3.5 Acceptance criteria ..................................................................................................... 19

1.3.6 Chemical analysis....................................................................................................... 20

1.3.7 Statistics ..................................................................................................................... 20

1.4 Results ........................................................................................................................ 21

1.4.1 Phase 1a ...................................................................................................................... 21

1.4.2 Phase 1b ..................................................................................................................... 23

1.4.3 Phase 2 ....................................................................................................................... 28

1.5 Discussion .................................................................................................................. 40

1.6 Conclusions ................................................................................................................ 43

Chapter II

2 Zebrafish (Danio rerio) embryos as a model for testing proteratogens .............. 46

2.1 Abstract ...................................................................................................................... 46

2.2 Introduction ................................................................................................................ 47

2.3 Materials and Methods ............................................................................................... 50

II

2.3.1 Materials ..................................................................................................................... 50

2.3.2 Methods ...................................................................................................................... 50

2.4 Results ........................................................................................................................ 53

2.4.1 Exposure of zebrafish embryos to 2-acetylaminofluorene, benzo[a]pyrene .................

and aflatoxin B1 ......................................................................................................... 55

2.4.2 Exposure of zebrafish embryos to the antiepileptic drugs carbamazepine, ..................

phenytoin and trimethadione ...................................................................................... 56

2.4.3 Exposure of zebrafish embryos to the anticancer drugs cyclophosphamide,

ifosfamide, tegafur and thio-TEPA ............................................................................ 59

2.4.4 LC50, EC50 and teratogenicity index (TI) ................................................................... 62

2.5 Discussion .................................................................................................................. 62

2.6 Conclusions ................................................................................................................ 68

Chapter III

3 Developmental effects of coumarin and the anticoagulant coumarin ....................

derivative warfarin on zebrafish (Danio rerio) embryos ...................................... 72

3.1 Abstract ...................................................................................................................... 72

3.2 Introduction ................................................................................................................ 72


3.3.1 Materials ..................................................................................................................... 74

3.3.2 Methods ...................................................................................................................... 75

3.4 Results ........................................................................................................................ 78

3.4.1 Exposure of zebrafish embryos to coumarin .............................................................. 79

3.4.2 Exposure of zebrafish embryos to warfarin ............................................................... 82

3.5 Discussion .................................................................................................................. 86

Chapter IV

4 Cartilage and bone malformations in the head of zebrafish (Danio rerio) ............

embryos following exposure to disulfiram and acetic acid hydrazide ................ 92

4.1 Abstract ...................................................................................................................... 92

4.2 Introduction ................................................................................................................ 93


4.3.1 Fish maintenance and egg production ........................................................................ 95

4.3.2 Test chemicals ............................................................................................................ 95

III

4.3.3 Zebrafish Embryo Test (ZFET) ................................................................................. 95

4.3.4 Whole-mount Alizarin red and Alcian blue skeletal staining .................................... 96

4.3.5 Cartilage and bone scoring ......................................................................................... 96

4.3.6 Statistics and data presentation .................................................................................. 97

4.4 Results ...................................................................................................................... 101

4.4.1 Determination of LC50 values and teratogenic index (TI) ....................................... 101

4.4.2 Cartilage and bone malformations following exposure with disulfiram (Fig.4.3) ... 101

4.4.3 Cartilage and bone malformations following exposure with .........................................

acetic acid hydrazide (Fig. 4.4) ............................................................................... 103

4.4.4 Head length of larvae exposed to disulfiram (Fig. 4.5)............................................ 105

4.4.5 Head length of larvae exposed to acetic acid hydrazide (Fig. 4.6) .......................... 105

4.4.6 Notochord and otolith malformations ...................................................................... 106

4.5 Discussion ................................................................................................................ 107

4.5.1 Toxicity, teratology as well as ecological relevance of observations ...................... 107

4.5.2 Osteolathyrism - a potential underlying mechanism for skeletal deformities.......... 109

4.6 Conclusions .............................................................................................................. 111

Chaper V .............................................................................................................................. 113

5 Toxicity and teratogenicity to cartilages and bones of zebrafish embryos ............

(Danio rerio) after exposure to hydrazides and hydrazines .............................. 114

5.1 Abstract .................................................................................................................... 114

5.2 Introduction .............................................................................................................. 115

5.3 Materials and Methods ............................................................................................. 116

5.3.1 Fish maintenance and egg production ...................................................................... 116

5.3.2 Test chemicals .......................................................................................................... 116

5.3.3 Whole-mount alizarin red and alcian blue skeletal staining .................................... 116

5.3.4 Staging of cartilages and bones ................................................................................ 117

5.4 Results ...................................................................................................................... 118

5.4.1 Toxicity, general teratology and notochord malformation ...........................................

after exposure to isoniazid ....................................................................................... 118


after exposure to benzydrazide................................................................................. 119


after exposure to benzylhydrazine ........................................................................... 120

IV


after exposure to phenylhydrazine ........................................................................... 120

5.4.5 Head length of exposed embryos after 6 days ......................................................... 120

5.4.6 Cartilage and bone malformations ........................................................................... 121

5.5 Discussion ................................................................................................................ 123

5.6 Conclusions .............................................................................................................. 124

Chapter VI

6 Oxygen requirements of zebrafish (Danio rerio) embryos ......................................

in embryo toxicity tests with environmental samples ......................................... 126

6.1 Abstract .................................................................................................................... 126

6.2 Introduction .............................................................................................................. 126


6.3.1 Oxygen measurements ............................................................................................. 128

6.3.2 Fish maintenance and egg production ...................................................................... 129

6.3.3 Oxygen consumption of Danio rerio under normoxic conditions ........................... 129

6.3.4 Sediment samples ..................................................................................................... 130

6.3.5 Oxygen measurements of sediment samples............................................................ 131

6.3.6 Temporal and spatial oxygen deprivation profiles of sediments ............................. 131

6.3.7 Sediment contact test with zebrafish (Danio rerio) embryos .................................. 132

6.3.8 Statistics ................................................................................................................... 133

6.4 Results ...................................................................................................................... 134

6.4.1 Oxygen consumption of D. rerio embryos at different developmental stages ........ 134

6.4.2 Survival and performance of D. rerio embryos under low oxygen concentrations . 135

6.4.3 Temporal oxygen deprivation during exposure to selected sediments .................... 137

6.4.4 Spatial oxygen deprivation profile above sediments ............................................... 139

6.4.5 Developmental retardation of zebrafish embryos in consequence of sediment

exposure ................................................................................................................... 140

6.5 Discussion ................................................................................................................ 142

6.5.1 Oxygen requirements of zebrafish (Danio rerio) embryos ...................................... 142

6.5.2 Oxygen consumption of zebrafish embryos in the fish embryo test for chemicals . 143

6.5.3 Compensation of low oxygen supplies to zebrafish embryos .......................................

in the fish embryo test for whole effluents and sediments ....................................... 144

6.5.4 Oxygen deprivation of sediment samples ................................................................ 145

V

6.5.5 Developmental retardation as consequence of oxygen shortage.............................. 146

6.5.6 Agitation as a possibility to improve oxygen availability .............................................

in sediments contact tests ......................................................................................... 146

6.6 Conclusions .............................................................................................................. 147

Chapter VII

7 Sediment-contact fish embryo toxicity assay with Danio rerio to assess ................

particle-bound pollutants in the Tietê River Basin (São Paulo, Brazil) ............ 150

7.1 Abstract .................................................................................................................... 150

7.2 Introduction .............................................................................................................. 151


7.3.1 Sediment sampling and treatment ............................................................................ 153


7.3.3 Sediment contact assay with Danio rerio ................................................................ 155

7.4 Results ...................................................................................................................... 156


7.4.2 Sediment contact assay with Danio rerio ................................................................ 157

7.5 Discussion ................................................................................................................ 162

7.6 Conclusions .............................................................................................................. 167

Final Conclusions ............................................................................................................... 169

Publications ......................................................................................................................... 172

References ............................................................................................................................ 175

VI

Summary

1

Summary

The present thesis gives an overview about the potentials zebrafish embryos can be used for in

the area of ecotoxicology. The first chapter summarizes the outcome of the ZFET (Zebrafish

Embryo Toxicity Test) OECD validation study, an international attempt for the standardiza-

tion and development of an embryo toxicity test as an (animal) alternative test to the acute

(adult) fish test which is a mandatory component of chemical registration worldwide. The

overall reproducibility of the ZFET within all participating laboratories was acceptable and

the comparison of the fish embryo LC50 data calculated in the validation study with available

acute adult fish LC50 data revealed good correlation. Hence, the fish embryo test is a reliable

alternative method which is able to replace conventional acute (adult) fish toxicity testing.

The following chapters deal with more specific questions on toxicity and teratogenicity. Re-

cently, zebrafish embryos have been shown to be a useful model for the detection of direct

acting teratogens. Therefore, chapter II investigated the capability of zebrafish embryos for

bioactivation (via CYP P450) of various proteratogenic substances. Apart from one substance,

which mainly produced lethal effects, all proteratogens were teratogenic in zebrafish embryos.

The test compounds revealed characteristic patterns of fingerprint endpoints.

Chapter III investigated detailed both toxic and teratogenic effects of coumarin and warfarin,

which are intensively metabolized in animals and humans. Both chemicals produced

teratogenic and lethal effects in zebrafish embryos. The comparison of the ratios between the

embryo effect concentrations and human therapeutic plasma concentrations revealed a distinct

teratogenic potential of warfarin, as well as the equivocal status of coumarin.

Since zebrafish embryos are able to (bio)activate proteratogens and also show a promising

correlation to humans, in chapter IV very specific teratogenic endpoints were assessed. A di-

thiocarbamate pesticide (disulfiram) and a hydrazide (acetic acid hydrazide) were selected for

the assessment of cartilage and bone teratogenesis in the head of six day old zebrafish larvae.

Cartilages of the neurocranium proved to be more stable than cartilages of the pharyngeal

skeleton, whereas bones proved more susceptible than cartilages. In chapter V, cartilage and

bone malformations after exposure to additional hydrazides and hydrazines were investigated.

Despite of the different order of acute toxicity, the dose-dependent malformation of cartilages

and decrease of ossification were comparable between all test substances.

Chapter VI deals with oxygen consumption of sediments and embryos. The zebrafish embryo

test is a widely used bioassay for the testing of effluents and sediments, e.g. in Germany it is

now mandatory for effluent testing. In this context, oxygen depletion of sediments and efflu-

ents is very important and may be a confounding factor in the interpretation of apparent tox-

icity. Zebrafish embryos can withstand a broad range of oxygen concentrations, but concen-

trations lower than 0.88 mg/L are 100 % lethal. In the sediment contact test with zebrafish

embryos, native sediments rapidly developed strongly hypoxic oxygen conditions.

Chapter VII then summarizes embryotoxic and teratogenic effects of a sediment contact assay

with specific sediments from locations in the Tietê River Basin (Brazil), providing a compre-

hensive and realistic insight into the bioavailable hazard potential of these sediments. High

embryo toxicity could be found in samples in the vicinity of the megacity São Paulo, but also

Summary

2

downstream. Results confirm that most toxicity is due to the discharges of the metropolitan

area of São Paulo.

Along all findings, the overall results indicate that zebrafish embryos are a useful alternative

method for traditional toxicity and teratogenicity testing. Regarding chemical testing, the

OECD validation study and further work proved that embryos tests are neither better nor

worse than conventional testing with adult fish, the correlation including all data available at

this time being excellent (r² > 0.9). Furthermore, zebrafish embryos proved to be a suitable

model for the determination of teratogenic effects and a potential alternative method for tradi-

tional teratogenicity testing including mammalian species.

Zusammenfassung

3

Zusammenfassung

Die vorliegende Arbeit gibt einen Überblick über die vielfältigen Anwendungsmöglichkeiten

des Zebrabärblings Embryotoxizitäts Tests (ZFET), insbesondere im Rahmen der Testung

sowohl von Umweltproben als auch von Chemikalien. Das erste Kapitel fasst die Ergebnisse

der internationalen ZFET OECD Validierungsstudie zusammen. Hierbei handelt es sich um

den Versuch den ZFET international zu standardisieren und als Alternativmethode zum welt-

weit, im Rahmen der Registrierung von Chemikalien, obligatorischen Akuttest mit adulten

Fischen zu etablieren. Die Reproduzierbarkeit der Testergebnisse sowohl innerhalb als auch

zwischen den teilnehmenden Laboratorien war akzeptabel und die Korrelation zwischen

Fischembryo LC50 und adulten Fisch LC50 Daten sehr hoch. Der ZFET stellt somit eine zu-

verlässige und kostengünstigere Alternative zum klassischen Akuttest mit adulten Fischen

dar.

Die folgenden Kapitel behandeln spezifischere Fragestellungen im Hinblick auf Toxizität und

Teratogenität.

Embryonen des Zebrabärblings sind ein ausgezeichneter Modellorganismus zum Nachweis

von direkt wirkenden teratogenen Substanzen, infolge dessen wurde in Kapitel II untersucht

inwieweit die Embryonen selbst dazu in der Lage waren proteratogene Substanzen (erst nach

Aktivierung z.B. über CYP P450 teratogen) zu verstoffwechseln, also zu aktivieren. Neun von

zehn getesteten Proteratogenen zeigten eindeutige teratogene Effekte im Embryotest mit cha-

rakteristischen Mustern an Effekten abhängig von der Substanzklasse.

Kapitel III beschreibt detailliert sowohl sehr starke toxische als auch teratogene Effekte von

Coumarin und Warfarin auf Embryonen des Zebrabärblings. Beide Chemikalien werden so-

wohl von Tieren, als auch vom Menschen stark metabolisiert. Der Vergleich zwischen den

Konzentrationen, die im Test an den Embryonen starke teratogene Effekte auslösten und den-

jenigen, die während einer medizinischen Behandlung im Blutplasma von Menschen gemes-

sen wurden belegt, dass ein erhebliches teratogenes Potential besteht, da es sich um einen

vergleichbaren Konzentrationsbereich handelt

Da Embryonen des Zebrabärblings nicht nur in der Lage sind proteratogene Substanzen zu

aktivieren, sondern auch noch äußerst vielversprechend mit in der Medizin gemessenen Blut-

plasmakonzentrationen korrelieren, werden in den beiden Folgekapiteln sehr spezielle terato-

gene Endpunkte untersucht. Die Kapitel IV und V konzentrieren sich auf die Dosis-Wirkungs

spezifischen teratogenen Effekte ausgewählter Substanzklassen (Dithiocarbamate, Hydrazide

und Hydrazine) auf die Knochen und Knorpelentwicklung im Kopf von 6 Tage alten Em-

bryonen. Die Knorpelelemente des Neurocraniums erwiesen sich als stabiler, verglichen mit

denen der Kiemen- bzw. Unterkieferregion, die bei denselben oder gar geringeren Testkon-

zentrationen deutlich stärkere Effekte aufwiesen. Die Knochenelemente waren jedoch deut-

lich am empfindlichsten und waren häufig nicht mehr erkennbar, während die Knorpel noch

keinerlei Effekte aufwiesen.

Kapitel VI untersucht den Sauerstoffverbrauch sowohl von ausgewählten Sedimenten, als

auch der Embryonen selbst. Im Rahmen der Abwasserprüfung ist der ZFET seit einigen Jah-

ren in Deutschland gesetzlich vorgeschrieben. Insbesondere in diesem Kontext ist der Sauer-

Zusammenfassung

4

stoffbedarf von Sedimenten und Abwässern von zentraler Bedeutung. Bei zu geringen Sauer-

stoffkonzentrationen im zu testenden Medium kann eine normale Entwicklung der Embry-

onen dann nicht mehr gewährleistet werden und eine möglicherweise vorhandene Toxizität

überinterpretiert werden. Die Sauerstoffbedingungen im Sediment-Kontakt-Test mit nativen

Sedimenten konnten mit Hilfe von spezifischen Schüttlern verbessert werden. Diese sorgen

für eine konstante Durchmischung der Wasserphase über dem Sediment und somit für eine

höhere Sauerstoffkonzentration direkt über dem Sediment.

In Kapitel VII wurden dann unter Berücksichtigung der Sauerstoffbedingungen weitere Sedi-

mentproben aus dem Tietê River Becken (Brasilien) auf toxische und teratogene Effekte un-

tersucht. Die Proben aus dem Einzugsgebiet der Stadt São Paulo waren stark toxisch, aber

auch weiter flussabwärts konnten erhebliche Effekte beobachtet werden. Die Ergebnisse bele-

gen, dass der Großteil der Toxizität durch die Abwässer der Stadt selbst verursacht wurde.

Die Ergebnisse aller Kapitel verdeutlichen, dass die Testung von Embryonen des

Zebrabärblings eine äußerst vielversprechende Alternativmethode zu traditionellen ökotoxiko-

logischen Testmethoden darstellt. Insbesondere die ZFET OECD Validierungsstudie hat ver-

deutlicht, dass die Testung von Embryonen weder bessere noch schlechtere Ergebnisse liefert

als der klassische akute Fischtest gemäß OECD TG 203 und somit diesen prinzipiell ersetzen

kann; die Korrelation zwischen beiden Testsystemen ist sehr hoch (r² > 0.9).

Introduction

5

Introduction

Alternatives to animal testing

The systematic generation and evaluation of ecotoxicological data of chemicals is critical for

risk assessment and the safety of man and the environment as well as classification and label-

ing. Due to an enormous amount of chemicals already produced and sold within the European

Union for decades and new developments, as e.g. nanoparticles, a tremendous need for new,

more sensitive test methods and more test animals exists (Basketter et al. 2012). Based on

ethical recommendations and animal welfare restrictions, alternative methods to animal test-

ing are being developed. For the definition of alternative methods to animal testing, the 3 R

principles of Russel and Burch (1959) are of special importance: The three Rs stand for the

replacement of animal tests, the reduction of the number of test animals and the refinement of

whole tests and test strategies, already expanding into European legislation and OECD direc-

tives (Gruber and Hartung, 2004). One major aim of the German federal government has thus

become the development of alternative methods reducing the number of animal tests (animal

welfare report 2005).

In basic research, various successfully tested alternative methods are described, but their ap-

plication has often been restricted to the laboratory where they had been developed (Gruber

and Hartung, 2004). Another problem is the poor standardization and reproducibility of such

methods, even if they are already published (Gruber and Hartung, 2004). The validation of In-

vitro methods, e.g. for the eye corrosion/eye irritation test in adult rabbits (Draize test), was

very challenging (Huggins 2003), because the non-animal alternatives were poorly planned

and evaluated with inappropriate statistical methods (Huggins 2003). Today, the Draize test

has been replaced by the Het-cam-test, which uses chicken embryos for the assessment of eye

corrosion or irritation.

In 2003, the 7th

Amendment (Directive 2003/15/EC) to the Cosmetics Directive (Di-

rective76/768/EEC) was passed by the EU, banning animal testing of cosmetics and their in-

gredients as soon as non animal test methods are available (Basketter et al., 2012). In 2009,

the Directive came into effect and a complete ban for animal tests followed, supported by a

marketing ban in the EU (Basketter et al., 2012). However, for several toxicological end-

Introduction

6

points, the marketing ban has been postponed until 2013 for animal tests performed outside

the EU.

In 2006, the Official Journal of the European Union published REACH (Regulation, Evalua-

tion, Authorization and Restriction of Chemicals; Directive EG 1907/2006). The main reason

for creating such a fundamentally new and extensive regulation was the awareness that for

several thousands of chemicals, circulating and consumed within the EU, no or only very lim-

ited (eco)toxicological data existed (Rovida et al., 2011). In consequence, all substances man-

ufactured or commercialized within the EU at quantities > 1 t/a must be completely investi-

gated regarding the effects they might have on human health or the environment. Depend-

ing on the tonnage produced per year (1, 10, 100 or 1000 t/a), the amount of tests which

have to be carried out, increases. For chemicals, produced > 10 t/a, the registration dossier

must include a Chemical Safety report resulting from an extensive Chemical Safety Assess-

ment (Rovida et al., 2011). Responsible for the whole management of the registration process

is the newly created European Chemical Agency (ECHA) located in Helsinki (Finland). Be-

fore REACH was created, various attempts trying to predict the number of test animals

REACH would require were performed by the European Commission (Rovida et al., 2011).

REACH also implemented several possibilities for the reduction of animal testing:

REACH requires the latest validated test methods, e.g. OECD Test Guidelines (TG),

becoming obligatory if they replace in vivo tests with vertebrates. ECHA (2011) de-

fines alternative tests as follows: “Alternative techniques that can provide the same

level of information as current animal tests, but which use fewer animals, cause less

suffering or avoid the use of animals completely. Such methods, as they become avail-

able, must be considered wherever possible for hazard characterisation and consequent

classification and labeling for intrinsic hazards and chemical safety assessment.”

All existing data on a chemical have to be pulled together and assessed before new

tests are performed. This concept is called “Weight of Evidence”: If more than two in-

dependent studies show the same results, despite the lack of GLP compliance, insuffi-

cient data or full reliability, these data can be accepted.

All registrants have to share their data; they have to complete the whole registration

process together by forming a Substance Information Exchange Forum (SIEF). Hence,

sharing of these data is obligatory, avoiding unneeded additional repeated animal tests.

This procedure applies also for companies which do not want to registry their sub-

Introduction

7

stance but which own the data; the principle of “one substance, one registration” is in-

troduced (Rovida et al., 2011).

The “waiving” option: If, e.g., a complete lack of exposure can be demonstrated, new

tests are neither necessary nor applicable; alternatively, if a more important endpoint

has already been confirmed, new tests can be waived.

For substances similar in their chemical entity and properties, additional tests are not

required. The data gained from a single substance can be used for similar ones “read-

across” or new tests can be performed on a selected representative substance.

The application of (one of) these options is relatively complicated, because the scientific ar-

gumentation has to be on firm ground and the (eco)toxicological data already available must

be precise and in no case misleading to protect human and environmental health. For that rea-

son ECHA published the “Guidance on information requirements and chemical safety as-

sessment: endpoint specific requirements” (ECHA, 2008). Hereby, ECHA gives advice how

to perform adaptations for information requirements for the respective endpoints (Rovida et

al., 2011). The REACH regulation set three deadlines for the testing and registering of old

chemicals, now defined as “phase-in-substances”, corresponding to the EINECS (European

Inventory of Existing Commercial Chemical Substances) database:

1. November 30, 2010

All substances imported or manufactured in quantities ≥ 1000 t/a

All CMR class 1 or 2 (Carcinogenic, Mutagenic, or toxic to Reproduction)

substances imported or manufactured in quantities ≥ 1 t/a according to

67/548/EWG

All substances imported or manufactured in quantities above 100 t/a which are

considered as toxic to the environment (R50/53; Directive 67/548)

2. May 30, 2013

All substances imported or manufactured in quantities above 100 t/a

3. May 30, 2018

All substances imported or manufactured in quantities above 1 t/a

Introduction

8

Fig. 1.1: Zebrafish male and female. (Picture: Erik

Leist)

These deadlines are obligatory for phase-in substances, which were on the EU market before

1981. Furthermore, all new substances have to be registered as fast as possible. Between June

1st, 2008, and February 28

th, 2011, 24560 registration dossiers were successfully submitted by

registrants (ECHA, 2011).

Zebrafish as a test organism

Adult zebrafish (Danio rerio) are native

to the rivers of India, Burma and Suma-

tra and their size ranges from 3 to 5 cm.

They belong to the family of the

Cyprinidae within the order of the

Cypriniformes. Males are more slender

and torpedo-shaped with blue stripes on

their longitudinal side and colored or-

ange-golden on their belly and fins. Fe-

males are thicker, especially before spawning. After three months, zebrafish reach sexual ma-

turity, where females can spawn hundreds of eggs a day. Another advantage for the use of

zebrafish is their ability to spawn the whole year, each time with huge amounts of eggs. Addi-

tionally, the chorion is transparent, whereby the whole development can easily be observed

via a microscope until the embryos hatch after 72 hpf (hours post fertilization). Zebrafish are

one of the best-investigated fish species with a fully sequenced genome and an excellent

model for teratogenicity testing (Berry et al. 2007, Brannen et al. 2010, Busch et al. 2011,

Carney et al. 2006, McGraph and Li 2008, Nelson et al. 2010, Selderslaghs et al. 2009, 2012,

Teraoka et al. 2003, Van den Bulk et al. 2011, Weigt et al. 2011, Yang et al. 2009). Further-

more, various official test guidelines exist (e.g. DIN 38415-6, OECD 203, 210, 215, 229, 230,

234), with zebrafish as one of the most important aquatic test species.

General aspects and outline of this thesis

REACH and similar international regulations require a huge number of ecotoxicological test

data for the registration of substances. To avoid and to reduce the total amount of test animals,

alternative test methods have to be developed and validated. Toxicity and teratogenicity stud-

ies using zebrafish embryos have been conducted for decades (Dave, 1984, 1991; Groth et al.,

Introduction

9

1993, 1994; Herrmann, 1993; Laale, 1977; Lange et al., 1995; Nagel, 2002; Van Leeuwen

1990). Therefore, it was about time to conduct an international validation study with zebrafish

embryos, coordinated by the OECD and the ECVAM (European Centre for the Validation of

Alternative Methods) with the final aim to create an OECD test guideline (TG) which can be

used as an alternative to OECD TG 203, where adult fish are used in acute toxicity tests.

Since zebrafish embryos up to 96 h post-fertilization are not considered as test animals, be-

cause they do not feed externally (EU, 2010), the ZFET (Zebrafish Embryo Toxicity Test) is

not regarded an animal test. The council of the European Union published in 2010 a so-called

Interinstitutional File (2008/0211 (COD)) on the directive of the protection of animals used

for scientific purposes. “This Directive shall apply to the following animals: (a) live non-

human vertebrate animals including independently feeding larval forms, foetal forms of

mammals from the last third of their normal development and (b) live cephalopods. Further-

more, this directive should apply to animals used in procedures, which are at an earlier stage

of development than that referred to point (a), if the animal is to be allowed to live beyond

that stage of development …”

The different chapters of this thesis deal with the OECD ZFET validation study and the re-

sults, further applications and adaptations of the ZFET, especially in testing (native) sedi-

ments regarding the oxygen consumption of single embryos and the testing of very specific

compounds in terms of skeletal teratogenicity. In chapter I, the results of the ZFET OECD

validation study of the technical lead laboratory Heidelberg are summarized and discussed in

the context of the overall results of all participating laboratories. Chapter II describes embryo

tests with ten different proteratogenic chemicals, aiming to identify the capacity of zebrafish

embryos for bioactivation, whereas chapter III deals with toxic and teratogenic effects of two

specific substances (coumarin and warfarin) to zebrafish embryos. In chapters IV and V,

teratogenic effects of dithiocarbamates, hydrazides and hydrazines on head cartilages and

bones of 6 d-old zebrafish larvae were assessed. The oxygen consumption of developing

zebrafish embryos, especially regarding the oxygen demands of native sediments are dis-

cussed in chapter VI, aiming to improve the sediment contact assay with zebrafish embryos.

In chapter VII, an example for the application of the improved sediment contact assay is illus-

trated with native sediments from the Tietê River Basin (São Paulo, Brazil).

Introduction

10

Chapter I

11

Chapter I

The Zebrafish (Danio rerio) Embryos Toxicity Test (ZFET) OECD

validation study

Chapter I

12

1 The Zebrafish (Danio rerio) Embryos Toxicity Test (ZFET)

OECD validation study

1.1 Abstract

The ZFET (Zebrafish Embryo Toxicity Test) OECD validation study was divided into two

phases. Within phase I, seven chemicals (triclosan, dibutyl maleate, 2,3,6-trimethylphenol,

3,4-dichloroaniline, 6-methyl-5-heptene-2-one, sodium chloride, ethanol) were tested, each

with three runs, aiming to evaluate the transferability of the results from the technical lead

laboratory Heidelberg to the participating laboratories as well as to assess the intra- and inter-

laboratory reproducibility. The validation management group (VMG) inferred from the results

that both the transferability and the reproducibility were acceptable and that phase II could be

initiated. Within the second phase, thirteen additional substances were investigated

(methylmercury(II) chloride, copper(II) sulphate pentahydrate, 4,6-dinitro-o-cresol, 2,4-

dinitrophenol, merquat 100, luviquat HM 552, tetradecylsulfate sodium salt, malathion,

prochloraz, 1-octanol, carbamazepine, dimethylsulfoxide, triethyleneglycol) to assess more

precisely the intra- and inter-laboratory reproducibility of chemicals from different areas of

use e.g. industrial chemicals, pharmaceuticals, biocides and pesticides. Within the lead labora-

tory Heidelberg, the CVs (coefficients of variation) were all below 30 %, whereas several

other laboratories had problems especially with the solubility of prochloraz, the very high

toxicity of methylmercury(II) chloride and the volatility of 6-methyl-5-heptene-2-one result-

ing in CVs above 30 %. Beside these at least for some laboratories problematic substances,

the overall reproducibility within and between all participating laboratories was generally

acceptable. A comparison of the respective LC50s calculated in the validation study with liter-

ature acute (adult) fish LC50 data reveal that for most of the investigated substances the toxici-

ties were very similar or at least within the same order of magnitude. Nevertheless, some

chemicals are more toxic to zebrafish embryos than to adult fish, e.g. merquat 100 or

tetradecyl sulfate sodium salt, and some are less toxic to embryos, e.g. malathion, prochloraz

and carbamazepine. As expected, the chorion acts as a barrier for both polymers, merquat 100

and luviquat HM 552. The molecular weight of both chemicals is too high to enter via the

pores within the chorion, toxicity can only occur after hatch (about 72 h). Generally, all other

chemicals tested in both phases were slightly more toxic after 96 h than at 48 h. Additional

statistical analyses revealed that 10 embryos per concentration are not enough and 20 embryos

are more suitable to ensure statistically robust results.

Chapter I

13

1.2 Introduction

Within European legislation, the acute fish toxicity test (OECD TG 203) is a mandatory com-

ponent of chemical testing. Already in 1986 the European Commission published the former

Council Directive 86/609/EEC on the protection of laboratory animals and subsequently in

2010 Directive 2010/63/EU which in particular regards chemical legislation (REACH, EC

2007). The ZFET is one of the most promising alternatives to the classical acute (adult) fish

toxicity test. Hence, to achieve international acceptance and to assess the reproducibility and

comparability between different laboratories, the ZFET has to be validated.

A draft Test Guideline (TG) for the “Fish Embryo Toxicity Test (FET)” was submitted to the

OECD Test Guideline Programme together with a Background Paper Braunbeck et al. (2005)

by the German Federal Environmental Agency (UBA) in 2005. In the following process the

ad hoc Expert Group for the Fish Embryo Toxicity Test was established while the documents,

already submitted, were reviewed for a first evaluation of the FET. Lammer et al. (2009)

evaluated all existing literature data for the FET and demonstrated that the FET correlates

very well with acute adult fish data. At this time point, a lot of FET data existed already, but

sufficient data demonstrating the reproducibility of the test were lacking. In 2008, ECVAM

(European Centre for the Validation of Alternative Methods) was asked by OECD to manage

and coordinate the Zebrafish Embryo Test Performance Study. The VMG (Validation Man-

agement Group), created in the same year, decided that the study should be divided into two

parts. The test should first show the transferability of the ZFET Standard Operation Procedure

(SOP) from the “Technical Lead Laboratory” Heidelberg (Phase 1a) to the other participating

laboratories as well as the intra- and interlaboratory reproducibility (Phase 1b and 2). The

very first chemical tested in the study (Phase 1a, conducted between April and October 2009),

was 3,4-dichloroaniline (DCA), which was later used as positive control in every test. Within

the first step of Phase 1a, DCA was tested in a single run, and in the second step the same

concentrations of DCA were tested again, but in three independent runs. The results of both

steps were used to determine the final concentration of DCA (4 mg/L) as was then used as

positive control in the further tests. In Phase 1b, conducted between November 2009 and Oc-

tober 2010, six chemicals (triclosan, dibutyl maleate, 2,3,6-trimethylphenol, 6-methyl-5-

heptene-2-one, sodium chloride, ethanol) were tested. All four laboratories, which joined for

Phase 2 and did not participate in Phase 1, had to test DCA as a training exercise (Phase 2a)

before beginning with the actual test chemicals (Phase 2b – 13 chemicals). The training exer-

Chapter I

14

cise for the new laboratories (Phase 2a – three independent runs with DCA) was conducted

between October 2010 and February 2011. Phase 2b was then conducted between February

2011 and November 2011.

Before starting the validation process, the technical lead laboratory provided a draft SOP,

commented and reviewed by the VMG. Furthermore, all substances tested in the validation

study were analyzed in range-finding tests performed by the lead laboratory and Procter and

Gamble.

1.3 Materials & Methods

1.3.1 Test chemicals

All test chemicals were chosen based on the recommendations of the ad hoc Expert Group for

the FET. The final selection of the 20 chemicals to test was one of the most critical steps;

therefore the VMG decided to reactivate the Chemical Selection Group (CSG), which had

been already established at the first meeting of the OECD FET Expert Consultation Meeting

at Berlin (9 - 11.10.2007). Partly, the test substances were sponsored by the participating la-

boratories, or they were purchased by ECVAM. The selection of the test chemicals was also

based on the toxicity level (fish very toxic (“+++“): < 1 mg/L, fish toxic (“++”): 1-10 mg/L,

fish moderately toxic (“+”): 10-100 mg/L, fish non-toxic (“-“): > 100 mg/L), the modes of

action (as far as data are available) and their use (industrial, pharmaceutical, pesticides and

biocides).

1.3.1.1 Test chemicals Phase 1

All chemicals were purchased from Sigma-Aldrich (Tab. 1.1). DCA was aliquoted and dis-

tributed by the lead laboratory, whereas the six remaining substances were aliquoted and dis-

tributed by ECVAM.

Chapter I

15

- = non-toxic (LC50 > 100 mg/L); + = moderately toxic (LC50 = 10 - 100 mg/L); ++ = toxic

(LC50= 1 - 10 mg/L; +++ = very toxic (LC50 < 1 mg/L); NA = Not available; a = experimental

database match; b = estimated using EPISUITE 4.0 (2008) except when measured values

were available (cited within EPISUITE) (table modified according to OECD ZFET Phase 2

Final Report).

Chemical Fish Toxicity CAS Num. Cat. Num. Lot Num. Log Kow

Methylmercury (II) chloride +++ 115-09-3 33368 szba172x 0.41a

Copper (II) sulphate pentahydrate +++ 7758-99-8 209198 mkbd0338 NA

4,6-Dinitro-o-cresol +++ 534-52-1 45464 sze6159x 2.13a

2,4-Dinitrophenol +++ 51-28-5 34334 sze9167x 1.67a

Merquat 100 ++ 26062-79-3 409022 mkbf20418v -2.49b

Luviquat HM 552 ++ 95144-24-4 59059 1322472 1.38b

Tetradecylsulfate sodium salt ++ 1191-50-0 293938 0600LC 2.67b

Malathion ++ 121-75-5 PS86 447-115b 2.36a

Prochloraz ++ 67747-09-5 45631 sze6220x 4.1a

1-Octanol + 111-87-5 293245 stbb5181 3.00a

Carbamazepine + 298-46-4 C4024 119k1317v 2.45a

Dimethylsulfoxide - 67-68-5 10282 215 -1.35a

Triethyleneglycol - 112-27-6 T59455 stbb7542 -1.75b

Tab. 1.2: Test substances used for Phase 2. DMSO was purchased at Grüssing GmbH

(Filsum) the 12 remaining chemicals were purchased at Sigma-Aldrich (Deisenhofen).

1.3.1.2 Test chemicals Phase 2

- = non-toxic (LC50 > 100 mg/L); + = moderately toxic (LC50 from 10 to 100 mg/L); ++ =

toxic (LC50 from 1 to 10 mg/L; +++ = very toxic (LC50 < 1 mg/L); Log Kow and solubility

were estimated using EPISUITE 4.0 (2008) except when measured values were available (cit-

ed within EPISUITE) (table modified according to OECD 157 ZFET Phase 1 Final Report).

Tab. 1.1: Test substances used for Phase 1.

Chemical Fish Toxicity CAS Num. Cat. Num. Lot Num. Log Kow Solubility (mg/L)

Triclosan +++ 3380-34-5 72779 1412854 4.76 4.621

Dibutyl maleate ++ 105-76-0 D47102 07715ch 4.16 8.709

2,3,6-Trimethylphenol ++ 2416-94-6 92693 1290095 3.15 1580

3,4-Dichloroaniline ++ 95-76-1 35827 6080x 2.69 337.9

6-Methyl-5-heptene-2-one + 110-93-8 67320 S52972-429 2.06 4364.1

Sodium chloride - 7647-14-5 S7653 106K0081 -0.46 359000

Ethanol - 64-17-5 34923 sze91380 -0.31 1 x 106

Chapter I

16

Fig. 1.1: Male and female zebrafish during

spawning. Photo: Erik Leist

DMSO was purchased from Grüssing GmbH (Filsum), all other chemicals were purchased

from Sigma-Aldrich (Tab. 1.2).

1.3.2 Zebrafish maintenance and spawning

Adult zebrafish (Westaquarium strain) were kept in 150 L aquaria under constant flow

through conditions at 26 ± 1 °C and 14:10 h light/dark cycle. The fishes were free of diseases

or other macroscopically discernible malformations. The pH varied between 7.9 and 8.3, the

oxygen content was > 95 % and the ammonia (n.d), nitrite (~ 0.05 mg/L) and nitrate (~

5 mg/L) levels were far below the acceptance criteria. The total hardness varied from 230 to

250 mg/L and the conductivity from 600 to 700 µS.

The fishes selected for spawning, were

transferred a few weeks before usage

into smaller aquaria (approx. 15 L) with

7 males and 7 females inside. The

aquatic ecology and toxicology group at

the University of Heidelberg (Lead la-

boratory) maintains 12 spawning groups

permanently, whereas 4 groups can be

prepared simultaneously for spawning.

If the fertilization rate of the embryos or

the egg quality was decreasing over

3 weeks, the fishes were ill or the animals of a specific group were too old, the whole group

was replaced. The fishes were fed with artemia (Great Salt Lake Artemia Cysts, Sanders

Brine Shrimp Company, Ogden, USA) and additionally TetraMinTM

dry flasks twice per day.

On the afternoon the day before embryos were needed, four complete spawning groups were

transferred into four specific spawning aquaria. The spawning aquaria (~15 L volume) con-

tained plastic inserts (11 L, 33 cm x 18 cm x 19 cm) in which the fishes were transferred. The

bottom of these inserts consisted of a stainless steel grid with a mesh size of 1.25 mm and a

tray beneath. As spawning stimulant a green plastic dummy (artificial plant) was used

(Fig. 1.1). Immediately after onset of light in the next morning the fishes began with mating

and spawning. Depending on the strain a laboratory is working with, spawning is completed

within 30 min up to 1 h and the egg trays can be removed out of the aquarium.

Chapter I

17

1.3.3 Zebrafish Embryo Toxicity Test (ZFET) - Principles

All chemicals were tested in 5 concentrations with a spacing factor of 2 or lower and three

independent runs. The test concentrations were prepared with dilution water according to

OECD TG 203 Annex 2 (1992, final concentrations: 294.0 mg/L CaCl2•2 H2O; 123.3 mg/L

MgSO4•7 H2O; 64.7 mg/L NaHCO3; 5.7 mg/L KCl). The resulting degree of total hardness

was between 230 and 250 mg/L, the pH varied 7.7 ± 0.1 and the temperature was maintained

at 26 ± 0.1 °C. The highest test concentration should result in 100 % mortality, whereas the

lowest concentration should cause no observable effects. Pure dilution water was used as ex-

ternal and internal negative control and DCA at a concentration of 4 mg/L as positive control.

Every substance was tested with 20 embryos per concentrations and controls (except the ex-

ternal negative control with 24 embryos) and 2 ml test volume per well. The test procedure is

semi-static, all test concentrations are renewed each day with freshly prepared solutions.

Fig. 1.2 illustrates the ZFET test procedure. Within one hour after spawning and fertilization

of the eggs, at least twice the number of eggs needed, have to be transferred into glass vessels

with an appropriate volume of the respective test concentrations and controls. Within the next

two hours, the fertilization success has to be recorded and the embryos must be transferred

into the 24-well plates which are already pre-exposed with the respective test concentrations

and controls to avoid possible binding effects of the chemicals to the plastic surface.

The glass vessels containing the embryos were examined under an inverted microscope or a

dissection microscope with a minimum magnification of 25x to identify the fertilized embryos

and to assess the fertilization rate. Fertilized embryos can be distinguished without a doubt at

the 4 cell stage onwards from non fertilized ones. All embryos with observable abnormalities

or irregular cell divisions were discarded.

For each test concentration including the positive control and, if needed, the solvent control

20 embryos were needed. The 4 remaining wells on each 24-well plate (row 6) were filled

with dilution water and each with one embryo - the internal negative controls. For the external

negative control a complete 24-well plate (24 embryos) was used.

Chapter I

18

1.3.4 Determination of toxicity - Lethal endpoints

To match the best possible correlation between acute Fish Toxicity testing (OECD TG 203)

and the ZFET, four lethal endpoints were recorded. These lethal endpoints are the same as in

DIN 38415-6 (2001) for sewage water treatment investigation in Germany. An embryo is de-

fined to be dead if one of the following endpoints is observed: (A) Coagulation of the embryo,

(B) non-detachment of the tail, (C) non-formation of somites and (D) non-detection of heart-

beat. The endpoints A-C are recorded each day (24, 48, 72 and 96 h), endpoint (D) is recorded

after 48 h onwards because at 24 h a normal developed embryo has no heartbeat. The first

very faint heartbeats can be recorded after approx. 26-30 hpf.

Fig. 1.2: Test procedure of the ZFET from left to right modified after Lammer et al. (2008).

Selection and transfer of the fertilized embryos into glass vessels within 1 hour post fertiliza-

tion (hpf) with subsequent control of fertilization and transfer into the pre-exposed 24-well

plates; n = number of eggs needed for the respective concentration.

100 ml Crystallization dish with respective concentrations and

controls

10 Fertilized eggs

per concentration

Control of fertili-zation success

Spawning unit

20 Eggs

per conc.

Waste

Spawning unit100 ml Crystallization dish with respective concentrations and

controls

10 Fertilized eggs

per concentration


Spawning unit

20 Eggs

per conc.

Waste

2n Eggs

per conc.

Crystallization dishes with

respective concentrations

and controls100 ml Crystallization dish with respective concentrations and

controls

10 Fertilized eggs

per concentration


Spawning unit

20 Eggs

per conc.

Waste


controls

10 Fertilized eggs

per concentration


Spawning unit

20 Eggs

per conc.

Waste

n Fertilized eggs

per concentration

Control of fertili-

zation success

0 hpf ≤ 1 hpf ≤ 3 hpf


controls

10 Fertilized eggs

per concentration


Spawning unit

20 Eggs

per conc.

Waste


controls

10 Fertilized eggs

per concentration


Spawning unit

20 Eggs

per conc.

Waste

24 h Pre-

exposure

Chapter I

19

1.3.5 Acceptance criteria

For a valid test, several acceptance criteria must be fulfilled. If one of these is not met, the

respective test has to be repeated.

The parent fertility rate should be ≥ 70 %.

The dissolved oxygen within the test plates should be ≥ 80 % air saturation.

The water temperature should be maintained at 26 ± 1 °C in all test plates all over the

total duration of the test.

F E D

C B A

Fig. 1.3: Coagulated embryos (A) are white; however observed under the microscope they

appear dark. (B) shows an embryo with no somites, whereas embryo (C) has already formed

weak somites (arrow bottom), but the tail is not detached from the yolk (arrow left). Embryo

(D) illustrates an embryo with edema and blood accumulation on the yolk sac (arrows), indi-

cating heartbeat reduction - an embryo without heartbeat cannot be shown in a photo. For

recording an absence of heartbeat, the embryo has to be observed for at least 1 min with a

minimum magnification of 80x. Embryos (E) and (F) show normal developed embryos after

24 and 48 hpf.

Chapter I

20

Overall survival rate in the external negative control and, if needed, in the solvent con-

trol should be ≥ 90 % until the end of exposure.

Lethality within the positive control (4 mg/L DCA) should be ≥ 30 % at the end of the

test (96 h).

All stock solutions must be renewed each day.

If more than one dead embryo is observed in a specific internal negative control, the plate

might be rejected. Within the validation study all runs of our laboratory met the acceptance

criteria.

1.3.6 Chemical analysis

In Phase 1, Procter and Gamble (USA) performed the chemical analysis for all 6 chemicals

(OECD 157 ZFET Phase 1 Report, ), whereas in Phase 2 they analyzed tetradecylsulfate sodi-

um salt, 1-octanol and copper(III)sulfate pentahydrate. Two further chemicals, carbamazepine

and prochloraz, were analyzed by Ipo-Pszczyna (Poland) (OECD 179 ZFET Phase 2 Report).

The reports of Phase 1 and 2 (including annexes) can be found at the OECD homepage:

http://www.oecd.org/chemicalsafety/testingofchemicals/seriesontestingandassessmentpublicat

ionsbynumber.htm

1.3.7 Statistics

The statistical analysis for the validation study of all data in Phase 1 was performed by Andre

Kleesang (ECVAM) and in Phase 2 by Gregory Carr (Procter & Gamble). Additional infor-

mation about the statistical methods chosen can be found in the final reports and annexes of

the respective phases at the OECD homepage (see chapter 1.3.6).

For this thesis, the data of the lead laboratory Heidelberg were reprocessed and rearranged to

give a more comprehensive overview of the outcome of the validation study. The results of

the other participating laboratories are not presented in the results part, but are discussed later

in context with our findings. For the processing of our data (quantal, dose-response), ToxRat

using a two-parametric logistic regression model was chosen for the calculation of LC50 val-

ues according to the proposals of OECD Series on Testing and Assessment No.54. A compre-

hensive graph of all three runs per chemical was then created using GraphPad Prizm 4 either

http://www.oecd.org/chemicalsafety/testingofchemicals/seriesontestingandassessmentpublicationsbynumber.htm

http://www.oecd.org/chemicalsafety/testingofchemicals/seriesontestingandassessmentpublicationsbynumber.htm

Chapter I

21

with a two- or three-parametric regression model (a detailed explanation of the advantages

and disadvantages of the two models can be found in the ZFET OECD Validation reports for

both phases (e.g. OECD 157 ZFET Phase 1). The reproducibility of the independent runs was

assessed via the coefficient of variation (CV) - the standard deviation compared to the mean

in percent.

1.4 Results

1.4.1 Phase 1a

In Phase 1a - Step 1,3,4-dichloroaniline, later used as positive control in every test, was tested

in one run in each lab, followed by Phase 1a - Step 2 with three independent runs.

1.4.1.1 Results of 3,4-Dichloroaniline (3,4-DCA)

Pretests with 3,4-DCA

In order to determine the appropriate test concentrations for 3,4-DCA in the validation study,

four pretests were conducted (data not shown). As the final test concentrations, 0.5, 1. 2, 3.7,

4 and 8 mg/L were chosen (spacing factor 2.0). The concentration of 3.7 mg/L was added,

because this concentration is used as positive control in the routine fish egg test for waste wa-

ter assessment in Germany according to DIN 38415-6 (DIN 2001).

Chapter I

22

First step - One run with 3,4-DCA

The LC50 calculated for one single run (Fig. 1.4) with 3,4-DCA was 2.75 mg/L (2.52 mg/L)

after 48 h (96 h). Reproducibility was assessed in the second step with three independent runs

with 3,4-DCA.

Second step - Three independent runs with 3,4-DCA

The mean LC50 of all three independent runs (Fig. 1.5) with 3,4-DCA resulted in

3.08 ± 0.76 mg/L (mean ± standard deviation) after 48 h (A) and 2.62 ± 0.47 mg/L after 96 h.

The reproducibility of the runs was acceptable with a CV of 24.6 % (17.9 %) after 48 h

(96 h).

A B

Fig. 1.5: Combined graphs of three independent runs with 3,4-DCA after 48 h (A) and 96 h

of exposure (B) including negative controls (NC) on the right x-axis.

Fig. 1.4: Graphs of one run with 3,4-DCA after 48 h (A) and 96 h of exposure (B).

B A

Chapter I

23

B A

Fig. 1.6: Combined graphs of three independent runs with triclosan after 48 h (A) and 96 h

of exposure (B) including negative (NC), solvent (SC) and positive controls (PC) on the

right x-axis.

1.4.2 Phase 1b

1.4.2.1 Results for triclosan

Pretests with triclosan

The pretests with triclosan were performed in the lab of Procter and Gamble (Miami Valley

Innovation Center, Cincinnati, USA). As final test concentrations, 0.075, 0.15, 0.3, 0.6 and

1.2 mg/L were chosen with ethanol as solvent with 0.1 % in every test concentration including

a solvent control.

Three independent runs with triclosan

The mean LC50 of all three independent runs with triclosan (Fig. 1.6) resulted in

0.40 ± 0.03 mg/L after 48 h (A) and 0.27 ± 0.02 mg/L after 96 h. The reproducibility of the

runs was acceptable with a CV of 8.61 % (6.28 %) after 48 h (96 h).

1.4.2.2 Results for dibutyl maleate

Pretests with dibutyl maleate

For dibutyl maleate, one pretest was performed. As test concentrations 0.25, 0.5, 1, 2 and 4

mg/L were chosen (spacing factor 2.0). At 0.25 and 0.5 mg/L no significant mortality oc-

Chapter I

24

curred, at 1 mg/L 20 % and both at 2 and 4 mg/L 100 % of the embryos were dead after 96 h.

Therefore, these test concentrations were finally used in the validation study.

Three independent runs with dibutyl maleate

The mean LC50 of all three independent runs with dibutyl maleate (Fig. 1.7) resulted in



1.4.2.3 Results for 2,3,6-trimethylphenol

Pretests with 2,3,6-trimethylphenol

Two pretests were performed with 2,3,6-trimethylphenol. For the first one 0.08, 0.4, 2, 10 and

50 mg/L (spacing factor 5.0, no significant mortality at 0.08, 0.4 and 2 mg/L, 15 % at

10 mg/L and 100 % at 50 mg/L after 96 h) were chosen as test concentrations, whereas for

the second pretest the testing range was narrowed by choosing 8, 12, 18, 27, 40.5 and

50 mg/L (spacing factor 1.5, no significant mortality at 8 and 12 mg/L, 50 % at 18, 90 % at 27

and 100 % at 40.5 and 50 mg/L after 96 h). The concentrations of the second pretest were

finally used in the validation study, only waiving the 50 mg/L.

Fig. 1.7: Combined graphs of three independent runs with dibutyl maleate after 48 h (A) and

96 h of exposure (B) including negative (NC) and positive (PC) controls on the right x-axis.

B A

Chapter I

25

Three independent runs with 2,3,6-trimethylphenol

The mean LC50 of all three independent runs with 2,3,6-trimethylphenol (Fig. 1.8) resulted in



1.4.2.4 Results for 6-methyl-5-hepten-2-one

Pretests with 6-methyl-5-hepten-2-one

For 6-methyl-5-hepten-2-one, one pretest was performed. As test concentrations, 10, 20, 40,

80 and 160 mg/L were chosen (spacing factor 2.0, after 96 h no significant mortality at 10 -

40 mg/L, 25 % at 80 mg/L and 95 % at 160 mg/L). Hence, for the validation study the range

was narrowed by choosing 25, 42.5, 72.25, 122.825 and 208.03 mg/L (spacing factor 1.7) as

test concentrations.

Three independent runs with 6-Methyl-5-hepten-2-one

The mean LC50 of all three independent runs with 6-methyl-5-hepten-2-one (Fig. 1.9) resulted

in 138.14 ± 1.72 mg/L after 48 h (A) and 136.93 ± 3.13 mg/L after 96 h. The reproducibility

of the runs was acceptable with a CV of 1.25 % (2.28 %) after 48 h (96 h).

A B

Fig. 1.8: Combined graphs of three independent runs with 2,3,6-trimethylphenol after 48 h

(A) and 96 h of exposure (B) including negative (NC) and positive (PC) controls on the right

x-axis.

Chapter I

26

Fig. 1.9: Combined graphs of three independent runs with 6-methyl-5-hepten-2-one after

48 h (A) and 96 h of exposure (B) including negative (NC) and positive (PC) controls on the

right x-axis.

A B

1.4.2.5 Results for sodium chloride

Pretests with sodium chloride

Two pretests were performed with sodium chloride. For the first pretest 2, 4, 8, 16 and 32 g/L

were chosen as test concentrations (spacing factor 2.0, after 96 h no mortality at 2 and 4 g/L,

70 % at 8 and 100 % both at 16 and 32 g/L). The test concentrations for the second pretest

were selected as 0.5, 1, 2, 4, 8 and 16 g/L (spacing factor 2.0, after 96 h no significant mor-

tality between 0.5 and 4 g/L, 85 % at 8 and 100 % at 16 g/L). The test concentrations of the

second pretest were also used in the validation study, waiving the lowest concentration

(0.5 g/L).

Three independent runs with sodium chloride

The mean LC50 of all three independent runs with sodium chloride (Fig. 1.10) resulted in

6.53 ± 0.41 g/L after 48 h (A) and 6.39 ± 0.57 g/L after 96 h. The reproducibility of the runs

was acceptable with a CV of 6.25 % (8.96 %) after 48 h (96 h).

Chapter I

27

A B

Fig. 1.11: Combined graphs of three independent runs with ethanol after 48 h (A) and 96 h

of exposure (B) including negative (NC) and positive (PC) controls on the right x-axis.

A B

Fig. 1.10: Combined graphs of three independent runs with sodium chloride after 48 h (A)

and 96 h of exposure (B) including negative (NC) and positive (PC) controls on the right x-

axis.

1.4.2.6 Results for ethanol

Pretests with ethanol

For sodium chloride, one pretest was performed. As test concentrations 3.5, 5.3, 8, 12, 18 and

27 g/L were chosen (spacing factor 1.5, after 96 h no significant mortality at 3.5 - 8 g/L, 55 %

at 12 and 100 % both at 18 and 27 g/L). These test concentrations were also used in the vali-

dation study, waiving the lowest concentration (3.5 g/L).

Three independent runs with ethanol

Chapter I

28

A B

Fig. 1.12: Combined graphs of three independent runs with methyl(II)mercury chloride after

48 h (A) and 96 h of exposure (B) including negative (NC) and positive (PC) controls on the

right x-axis.

The mean LC50 of all three independent runs with ethanol (Fig. 1.11) resulted in

12.90 ± 0.76 g/L after 48 h (A) and 11.33 ± 0.58 g/L after 96 h. The reproducibility of the


1.4.3 Phase 2

1.4.3.1 Results for methyl(II)mercury chloride

Pretests with methyl(II)mercury chloride

Two pretests were performed with methyl(II)mercury chloride. For the first one 10, 40, 70,

100, 130, 160 and 190 µg/L were chosen as test concentrations (after 96 h no significant mor-

tality at 10 µg/L, 20 % at 40 and 100 % at 70 to 190 µg/L). For the second pretest the testing

range was narrowed by choosing 1, 2, 4, 8, 16, 32 and 64 µg/L (spacing factor 2.0, after 96 h

no significant mortality between 1 and 32 µg/L, 100 % at 64 µg/L). Hence, for the validation

study, 6.25, 12.5, 25, 50 and 100 µg/L were selected as test concentrations (spacing factor

2.0).

Three independent runs with methyl(II)mercury chloride

The mean LC50 of all three independent runs with methyl(II)mercury chloride (Fig. 1.12) re-

sulted in 69.72 ± 0.00 µg/L after 48 h (A) and 46.34 ± 2.84 µg/L after 96 h. The reproducibil-

ity of the runs was acceptable with a CV of 0 % (6.12 %) after 48 h (96 h).

Chapter I

29

A B

Fig. 1.13: Combined graphs of three independent runs with copper(II)sulfate pentahydrate

after 48 h (A) and 96 h of exposure (B) including negative (NC) and positive (PC) controls

on the right x-axis.

1.4.3.2 Results for copper(II)sulfate pentahydrate

Pretests with copper(II)sulfate pentahydrate

The pretests with copper(II)sulfate pentahydrate were performed within the lab of Procter and

Gamble (Miami Valley Innovation Center, Cincinnati, USA). As the final test concentrations

in the validation study 0.15, 0.3, 0.6, 1.2 and 2.4 mg/L were chosen.

Three with independent runs with copper(II)sulfate pentahydrate

The mean LC50 of all three independent runs with copper(II)sulfate pentahydrate (Fig. 1.13)

resulted in 0.78 ± 0.10 mg/L after 48 h (A) and 0.78 ± 0.10 mg/L after 96 h. The reproducibil-

ity of the runs was acceptable with a CV of 13.27 % (13.27 %) after 48 h (96 h).

1.4.3.3 Results for dinitro-o-cresol

Pretests with dinitro-o-cresol

One pretest was performed with dinitro-o-cresol. As test concentrations 0.1, 0.18, 0.32, 0.58

and 1.05 mg/L were chosen (spacing factor 1.8, after 96 h no significant mortality between

0.1 and 0.32 mg/L, 40 % at 0.58 and 100 % 1.05 mg/L). For the validation study this concen-

tration range was slightly shifted by choosing 0.18, 0.32, 0.58, 1.05 and 1.89 mg/L as test

concentrations.

Chapter I

30

A B

Fig. 1.14: Combined graphs of three independent runs with dinitro-o-cresol after 48 h (A)


axis.

Three independent runs with dinitro-o-cresol

The mean LC50 of all three independent runs with dinitro-o-cresol (Fig. 1.14) resulted in



1.4.3.4 Results for 2,4-dinitrophenol

Pretests with 2,4-dinitrophenol

Four pretests were performed with 2,4-dinitrophenol. For the first one 0.25, 0.5, 1, 2 and 4

mg/L were chosen as test concentrations, whereas 4 mg/L were not high enough to result in

100 % mortality. Therefore, three additional runs were performed with varying exposure con-

ditions (light dark rhythm vs. complete darkness, data not shown) with higher test concentra-

tions (1, 2, 4, 8, 16 mg/L). 2,4-Dinitrophenol concentrations of 8 mg/L and higher resulted in

100 % mortality after 96 h. Hence, for the validation study 0.625, 1.25, 2.5, 5 and 10 mg/L

were chosen as test concentrations.

Chapter I

31

A B

Fig. 1.15: Combined graphs of three independent runs with 2,4-dinitrophenol after 48 h (A)


axis.

Three independent runs with 2,4-dinitrophenol

The mean LC50 of all three independent runs with 2,4-dinitrophenol (Fig. 1.15) resulted in



1.4.3.5 Results for merquat 100

Pretests with merquat 100

Two pretests were performed with merquat 100. For the first one 2, 4, 8, 16 and 32 mg/L were

chosen as test concentrations. 100 % Mortality was observed after 96 h at the two lowest con-

centrations, whereas the mortality of the higher test concentrations was significantly lower

(8 mg/L: 55 %, 16 mg/L: 25 %, 32 mg/L: 60 %). Merquat 100 is a polymer, which is too big

to enter the chorion via the pores, therefore lethal effects can only occur after hatch. The low-

er mortality at higher concentrations can be explained by a malformed chorion (Fig. 1.16) and

a reduced hatching rate at the higher test concentrations. The concentration range was lowered

for the second pretest in choosing 0.05, 0.1, 0.2, 0.4, 0.8 and 1.6 mg/L. The three lowest test

concentrations showed no significant mortality whereas at 0.4 mg/L 85 %, at 0.8 90 % and at

1.6 mg/L 100 % of the test embryos were dead after 96 h. Hence, for the validation study

these concentrations were used, only the lowest concentration (0.05 mg/L) was waived.

Chapter I

32

B A

Fig. 1.17: Combined graphs of three independent runs with merquat 100 after 48 h (A) and


Fig. 1.16: Zebrafish embryos after exposure to merquat 100. (A) 24 h old embryo exposed to

8 mg/L with a malformed chorion. The inner chorion layer(s) are detached from the outer

layer(s) (arrows). (B) shows the outside of the chorion with merquat 100 accumulated at the

chorion (32 mg/L).

B A

Three independent runs with merquat 100

The mean LC50 of all three independent runs with merquat 100 (Fig. 1.17) resulted in

0.52 ± 0.09 mg/L after 96 h. The reproducibility of the runs was acceptable with a CV of

17.18 % after 96 h. After 48 h an LC50 could not be calculated, because the molecule is too

big to enter the chorion. Lethal effects only occur after hatch.

Chapter I

33

B A

Fig. 1.18: Combined graphs of three independent runs with Luviquat HM 552 after 48 h (A)


axis.

1.4.3.6 Results for luviquat HM 552

Pretests with luviquat HM 552

Two pretests were performed with luviquat HM 552 - the second polymer next to merquat

100 which was tested in the validation study. For the first one 1, 2, 4, 8 and 16 mg/L were

chosen as test concentrations. Within all test concentrations the mortality was ≥ 90 %, there-

fore a second pretest with lower concentrations was performed (0.05, 0.1, 0.2, 0.4, 0.8 and

1.6 mg/L). Embryos exposed to 0.05 - 0.4 mg/L showed no significant mortality, whereas at

0.8 mg/L 90 % and at 1.6 mg/L 95 % of all embryos were dead after 96 h. For the validation

study 0.125, 0.25, 0.5, 1 and 2 mg/L were selected as test concentrations.

Three independent runs with luviquat HM 552

The mean LC50 of all three independent runs with luviquat HM 552 (Fig. 1.18) resulted in

0.83 ± 0.06 mg/L after 96 h. The reproducibility of the runs was acceptable with a CV of

7.72 % 96 h. According to merquat 100, after 48 h an LC50 could not be calculated, because

the molecule is too big to enter the chorion. Lethal effects only occur after hatch.

Chapter I

34

A B

Fig. 1.19: Combined graphs of three independent runs with tetradecyl sulfate sodium salt

after 48 h (A) and 96 h of exposure (B) including negative (NC) and positive (PC) controls

on the right x-axis.

1.4.3.7 Results for tetradecyl sulfate sodium salt

Pretests with tetradecyl sulfate sodium salt

The pretests with tetradecyl sulfate sodium salt were performed within the lab of Procter and

Gamble (Miami Valley Innovation Center, Cincinnati, USA). As the final test concentrations

in the validation study 0.156, 0.3125, 0.625, 1.25 and 2.5 mg/L were chosen.

Three independent runs with tetradecyl sulfate sodium salt

The mean LC50 of all three independent runs with tetradecyl sulfate sodium salt (Fig. 1.19)

resulted in 0.33 ± 0.01 mg/L both after 48 and 96 h. The reproducibility of the runs was ac-

ceptable with a CV of 4.44 %.

1.4.3.8 Results for malathion

Pretests with malathion

One pretest was performed with malathion. As test concentrations 0.5, 1, 2, 4, 8 and 16 mg/L

were chosen (spacing factor 2.0, after 96 h no significant mortality between 0.5 and 2 mg/L,

95 % at 4 and 100 % both at 8 and 16 mg/L). For the validation study the same concentrations

were used but the 16 mg/L was passed.

Chapter I

35

A B

Fig. 1.20: Combined graphs of three independent runs with malathion after 48 h (A) and


Three independent runs with malathion

The mean LC50 of all three independent runs with malathion (Fig. 1.20) resulted in

4.75 ± 0.55 mg/L after 48 h and 3.62 ± 0.27 mg/L after 96 h. The reproducibility of the runs

was acceptable with a CV of 11.58 after 48 h and 7.33 % after 96 h.

1.4.3.9 Results for prochloraz

Pretests with prochloraz

One pretest was performed with prochloraz. As test concentrations 0.5, 1, 2, 4 and 8 mg/L

were chosen (spacing factor 2.0, after 96 h no significant mortality between 0.5 and 2 mg/L,

25 % at 4 and 100 % at 8 mg/L). For the validation study the same concentrations were used.

Three independent runs with prochloraz

The mean LC50 of all three independent runs with prochloraz (Fig. 1.21) resulted in

4.65 ± 0.55 mg/L after 48 h and 4.62 ± 0.52 mg/L after 96 h. The reproducibility of the runs

was acceptable with a CV of 11.80 % after 48 h and 11.32 % after 96 h.

Chapter I

36

A B

Fig. 1.21: Combined graphs of three independent runs with prochloraz after 48 h (A) and


1.4.3.10 Results for 1-octanol

Pretests with 1-octanol

Two pretests were performed with 1-octanol. For the first pretest 2, 4, 8, 16, 32 and 64 mg/L

were chosen as test concentrations (spacing factor 2.0, after 96 h no significant mortality oc-

curred at 2 to 8 mg/L, at 16 mg/L 20 % were dead, whereas at 32 and 64 mg/L 100 % mortali-

ty was achieved). A shortened second pretest with only three concentrations (10, 20 and

40 mg/L) was performed, with no mortality at 10 mg/L, 70 % at 20 mg/L and 100 % at

40 mg/L after 96 h. The test concentration in the validation study were selected as 2.5, 5, 10,

20 and 40 mg/L.

Three independent runs with 1-octanol

The mean LC50 of all three independent runs with 1-octanol (Fig. 1.22) resulted in

19.01 ± 0.80 mg/L both after 48 h and 96 h. The reproducibility of the runs was acceptable

with a CV of 4.21 %.

Chapter I

37

1.4.3.11 Results for carbamazepine

Pretests with carbamazepine

Three pretests were performed with carbamazepine. For the first pretest 10, 20, 40, 80 and

160 mg/L were chosen as test concentrations (spacing factor 2.0, after 96 h no significant

mortality occurred at 10 to 80 mg/L, at 160 mg/L 50 % showed lethal effects). Due to lack of

full mortality in the highest test concentration, the aim of the second pretest was the determi-

nation of the maximum solubility of carbamazepine without solvent and the respective mor-

tality. Therefore, 170, 200, 230 and 260 mg/L were tested (after 96 h 60 % mortality at

170 mg/L, 100 % at 200 to 260 mg/). Especially at the two highest concentrations, slight pre-

cipitation of carbamazepine was observed, hence, it was decided that in third pretest

210 mg/L will probably be sufficient and the test concentration 54.7, 76.5, 107.1, 150 and

210 mg/L were chosen (spacing factor 1.4). After 96 h, at 210 mg/L all embryos were dead,

whereas at 150 mg/L 30 %, at 107.1 mg/L 15 % and at the remaining concentrations no sig-

nificant mortality could be observed.

Three independent runs with carbamazepine

The mean LC50 of all three independent runs with carbamazepine (Fig. 1.23) resulted in

177.97 ± 3.22 mg/L after 48 h and 162.74 ± 7.65 mg/L after 96 h. The reproducibility of the


A B

Fig. 1.22: Combined graphs of three independent runs with 1-octanol after 48 h (A) and


Chapter I

38

A B

Fig. 1.23: Combined graphs of three independent runs with carbamazepine after 48 h (A)


axis.

1.4.3.12 Results for triethylene glycol

Pretests with triethylene glycol

One pretest was performed with triethylene glycol. As test concentrations 29.63, 44.44, 66.67,

100 and 150 g/L were chosen (spacing factor 1.5, after 96 h no significant mortality at 29.63

and 44.44 g/L was observed, whereas concentration of 66.67 g/L and higher resulted in 100 %

mortality). For the validation study the concentration range was slightly narrowed to 20, 30,

45, 67.5 and 101.25 g/L.

Three independent runs with triethylene glycol

The mean LC50 of all three independent runs with triethylene glycol (Fig. 1.24) resulted in

69.08 ± 2.82 g/L after 48 h and 52.83 ± 3.65 g/L after 96 h. The reproducibility of the runs

was acceptable with a CV of 4.08 % (6.91 %) after 48 h (96 h).

Chapter I

39

B A

Fig. 1.24: Combined graphs of three independent runs with triethylene glycol after 48 h (A)


axis.

1.4.3.13 Results for dimethyl sulfoxide

Pretests with dimethyl sulfoxide

Two pretests were performed with dimethyl sulfoxide. The test concentrations of the first pre-

test were 4, 8, 16, 32 and 80 g/L (after 96 h no mortality was observed at 4, 8 and 16 g/L,

15 % at 32 g/L and 100 % at 80 g/L. For the second pretest 10, 17, 28.9, 49.13 and

83.521 mg/L were chosen as test concentrations (spacing factor 1.7, after 96 h no significant

mortality occurred at 10 to 28.9 g/L, at 49.13 g/L and higher 100 % mortality was achieved).

The test concentrations of the second pretest were also used in the validation study.

Three independent runs with dimethyl sulfoxide

The mean LC50 of all three independent runs with dimethyl sulfoxide (Fig. 1.25) resulted in

36.77 ± 0.67 g/L both after 48 h and 96 h. The reproducibility of the runs was acceptable with

a CV of 1.82 %.

Chapter I

40

Fig. 1.25: Combined graphs of three independent runs with dimethyl sulfoxide after 48 h

(A) and 96 h of exposure (B) including negative (NC) and positive (PC) controls on the right

x-axis.

A B

1.5 Discussion

The reproducibility of the ZFET within the laboratory Heidelberg was very high (CV below

30 %) for all 20 chemicals; a maximum CV of 25.15 % was calculated for dinitro-o-cresol

after 48 h, whereas the majority of the CVs ranged between 0 and 15 % both after 48 and

96 h. The test concentrations determined in the pretests were appropriate, in the lowest test

concentrations no significant mortality occurred, whereas at the highest concentrations

enough embryos were dead to be able to calculate LC50 values. Both after 48 and 96 h, LC50

values were calculated for every chemical to assess time dependent changes in toxicity. Four

different patterns were observed: (A) Toxicity occurs very early in the development, generally

within the first 24 h, (B) toxicity was a continuous process all over the time, (C) toxicity in-

creased very fast after 24 h, and (D) toxicity was heavily increased immediately after hatch.

Furthermore, a distinct pattern of the chemical category, the use and the mode of action re-

garding the grouping of the chemicals using a temporal pattern as LC50, was not possible.

Chemicals of group A had equal or at least very similar LC50 values both after 48 and 96 h.

Hence, the most prominent lethal endpoint was coagulation already within 24 h with no fur-

ther changes in the following days. Members of that group are copper(II)sulfate pentahydrate,

tetradecyl sulfate sodium salt, prochloraz, 1-octanol, ethanol, 2,3,6-trimethylphenol and sodi-

um chloride. Chemicals of group B with continuous increasing toxicity over the test period

are carbamazepine, malathion, dimethylsulfoxide, 3,4-dichloroaniline, triethylene glycol and

Chapter I

41

6-methyl-5-hepten-2-one. Chemicals of group C, a more extreme version of group B, can be

characterized by a strong increase of toxicity between 24 and 48 h. Members of this group are

methyl(II)mercury chloride, 2,4-dinitrophenol, dinitro-o-cresol, dibutyl maleate and triclosan.

Group D comprises chemicals exerting their toxicity immediately after hatch (72 h), because

they were not able to cross the chorion. These chemicals are the cationic polymers merquat

100 and luviquat HM 552. A clear correlation between chemical class, functional use, mode

of action and level of toxicity could not be performed.

Discussion of the results of all laboratories participating in the ZFET validation

First of all, based on the overall results it was concluded that the intra-laboratory (data not

shown, but described in detail in the validation reports of phase 1 and 2) as well as the inter-

laboratory transferability of the ZFET were acceptable in general with most CV < 30 %

(Tab. 1.1). Nevertheless, for several chemicals inter-laboratory CVs > 30 % were calculated.

These were methyl(II)mercury chloride, copper(II)sulfate pentahydrate, merquat 100,

prochloraz and 6-methyl-5-hepten-2-one. The main fact contributing to these elevated CVs,

except the 6-methyl-5-hepten-2-one and prochloraz, was the very high acute toxicity, since

even very small differences in the LC50s were magnified resulting in larger CVs. Exposure to

6-methyl-5-heptene-2-one resulted in the highest CV determined in the whole validation

study, most probably because of handling difficulties (e.g. preparing of the stock solutions or

test concentrations) with the chemical itself. 6-Methyl-5-heptene-2-one was highly volatile

which could explain why 2 of the 4 laboratories had much lower toxicity compared to the 2

others. The CV for prochloraz after 96 h was about 30.4 %, but similar to 6-methyl-5-

heptene-2-one, only 2 of the 4 laboratories delivered runs with significant mortality allowing

the calculation of LC50 values. In contrast to 6-methyl-5-heptene-2-one, prochloraz was not

Tab. 1.3: Toxicity patterns of all 20 test chemicals.

A B C D

Copper(II)sulfate pentahydrate Carbamazepine Methyl(II)mercury chloride Merquat 100

Tetradecyl sulfate sodium salt Malathion 2,4-Dinitrophenol Luviquat HM 552

Prochloraz Dimethylsulfoxide Dinitro-o -cresol

1-Octanol 3,4-Dichloroaniline Dibutyl maleate

Ethanol Triethylene glycol Triclosan

2,3,6-Trimethylphenol 6-Methyl-5-hepten-2-one

Sodium chloride

Chapter I

42

Tab. 1.4: Summary of the ZFET results of all laboratories. Mean LC50 values after 48 and 96 h

of all 20 chemicals tested in phase 1 and 2, including the coefficients of variation (CV), the

laboratory number (N) each with 3 runs and literature values of fish acute LC50s. (Adapted

from the final OECD reports of phase 1 and 2).

* Note that all data for methyl(II)mercury chloride, copper(II)sulfate pentahydrate and

tetradecyl sulfate sodium salt are corrected to indicate exposure only to the respective

cations or anions

Numbers in brackets represent the number of studies.

Fish acute LC50 values were retrieved from literature and the OECD QSAR toolbox.

NA: LC50 values could not be calculated

Fish acute mean

Chemicals Phase 1 and 2 LC50 [mg/L]

48 h 96 h 48 h 96 h 96 h (min - mean - max)

Methylmercury* 0.042 0.028 46.9 50.2 3 0.031 - 0.145 - 0.46 (6)

Copper* 0.308 0.291 41.7 33.6 4 0.008 - 0.224 - 0.749 (11)

Tetradecyl sulfate* 0.337 0.339 25 25.8 4 2.5 - 3.031 - 3.55 (3)

Triclosan 0.42 0.3 9.2 11.8 4 0.5 (2)

Merquat 100 NA 0.496 NA 40.8 4 0.3 - 1.82 - 6.51 (5)

4,6-Dinitro-o-cresol 0.723 0.567 2.8 7.5 4 0.066 - 0.863 - 2.2 (7)

Luviquat HM 552 NA 0.876 NA 24.8 4 0.748 (1)

Dibutyl maleate 1.38 0.7 17.6 13.3 5 1.2 (1)

3,4-Dichloroaniline 3.2 2.7 22.1 23.6 5 8.59 (1)

2,4-Dinitrophenol 4.123 3 23.5 22.7 4 0.39 - 6.843 - 27.1 (19)

Prochloraz 4.461 5.6 5.8 30.4 4 0.53 - 0.583 - 0.68 (3)

Malathion 6.123 4.56 25.8 13 4 0.003 - 0.289 - 25 (47)

2,3,6-Trimethylphenol 10.9 10.8 16.4 15.8 4 8.2 (1)

1-Octanol 20.7 20.675 5.9 5.9 4 13 - 15.68 - 24 (10)

Carbamazepine 177 153 6.4 3.8 4 43 (1)

6-Methyl-5-hepten-2-one 279 243 65.9 56.3 4 85.7 (1)

Sodium chloride 5340 5140 16.9 18.9 4 7700 (14)

Ethanol 13200 12000 7.1 4.8 5 14200 (1)

Dimethyl sulfoxide 40200 34100 14.9 6.6 4 59900 - 71251 - 92500 (5)

Triethylene glycol 71300 54800 8.4 6.3 4 34000 - 40429 - 52000 (6)

[mg/L]

Mean LC50 CV [%] N

volatile but had a high log Kow resulting in very low water solubility. These two laboratories

did not succeed in properly solving the prochloraz in the stock solution and/or test concentra-

tions resulting in too low mortalities.

Chapter I

43

The mean LC50 values and the interlaboratory reproducibility of all chemicals and laboratories

are shown in Tab 1.4., whereas all LC50 values for methyl(II)mercury chloride, cop-

per(II)sulfate pentahydrate and tetradecyl sulfate sodium salt were corrected to indicate expo-

sure only to the respective cations or anions, namely methylmercury, copper and tetradecyl

sulfate.

Keeping in mind the fish toxicity levels from very toxic, toxic, moderately toxic and non-

toxic, the comparison of the ZFET results of the validation study with literature acute fish

data revealed that the non-fish-toxic chemicals triethylene glycol, dimethyl sulfoxide, ethanol

and sodium chloride were non-embryo toxic, too. The moderately fish toxic chemicals

6-methyl-5-heptene-2-one and carbamazepine were non-toxic in the ZFET, but it should be

noted that only very few acute fish data were available for most of the chemicals tested. The

ZFET results of 1-octanol and 2,3,6-trimethylphenol were very similar, deviating to a very

low extent. Malathion and prochloraz were moderately toxic in the ZFET but very toxic to

adult fish, whereas for malathion the span of literature adult LC50 was very broad from 0.003

to 25 mg/L; the significance of these values should be questioned. 2,4-Dinitrophenol, 3,4-

dichloroaniline, dibutylmaleate, Luviquat HM 552, 4,6-dinitro-o-cresol, triclosan, copper and

methylmercury showed very similar toxicities in the same order of magnitude. The toxicities

of merquat 100 and tetradecyl sulfate sodium salt were higher, if compared to adult fish.

Regarding the comparison between 48 and 96 h of exposures, the results indicate that the

chorion acts as a barrier for chemicals with a high molecular weight such as merquat 100 and

luviquat HM 552. In both cases mortality only occurred after hatch; hence mortality was not

observed at 48 h. Most of the other substances were slightly more toxic after 96 h if compared

to 48 h; however the resulting LC50s were still at the same toxicity level. Just to be on the safe

side, the ZFET should be performed for 96 h.

1.6 Conclusions

For all of the 20 substances tested in the validation study, the capacity of the ZFET to predict

acute fish toxicity is very promising, especially regarding that only a few, partly strongly dis-

persive acute fish data are available for comparison (Tab. 1.4). First LC50 correlations be-

tween zebrafish embryos and adults were already performed in 2003 by Ratte and Hammers-

Wirtz, resulting in an r² of 0.85 with a total of 60 different chemicals. In 2010, Lammer et al.

provided an update of the correlation with a slightly elevated r² of 0.9 and 77 chemicals in

Chapter I

44

total. Furthermore, correlations between (adult) acute LC50 of different fish species were per-

formed, resulting in r² values of 0.83 (Pimephales promelas vs. Oryzias latipes) up to 0.96

(Oryzias latipes vs. Oncorhynchus mykiss/ Pimephales promelas vs. Oncorhynchus mykiss).

Only the correlation between Oryzias latipes and Danio rerio was lower (r² = 0.72), but this is

most probably caused by insufficient data (only 12 chemicals were used for this calculation).

Hence, the ZFET with its correlation of r² > 0.9 to acute fish tests with any other fish species

(S. Belanger, to be published) is neither better nor worse than acute fish toxicity testing and

provides a suitable alternative to the increasing demands of fish in the context of the European

Chemicals Regulation REACH.

Chapter II

45

Chapter II

Zebrafish (Danio rerio) embryos as a model for testing

proteratogens

Chapter II

46

2 Zebrafish (Danio rerio) embryos as a model for testing

proteratogens

Chapter I demonstrated that the ZFET is a suitable alternative method to acute fish toxicity

testing, independent of the fish species. However, the assessment of teratogenicity requires

the usage of additional endpoints, e.g. malformation of the head, the eyes, the

otholiths/sacculi, the corda and the tail, deformation of the yolk sac, growth retardation and

scoliosis. Regarding teratogenicity, the question about bioactivation of the test chemicals via

cytochrome P 450 is of major importance. The zebrafish embryo teratogenicity test delivers

reliable information if a test compound has been bioactivated, since the immediate teratogenic

effects in the embryos can be observed directly. This is an advantage to mammalian terato-

genicity tests, not only regarding animal welfare considerations.

2.1 Abstract

Zebrafish embryos have been shown to be a useful model for the detection of direct acting

teratogens. This study presents a protocol for a 3-day in vitro zebrafish embryo teratogenicity

assay and describes results obtained for 10 proteratogens: 2-acetylaminofluorene,

benzo[a]pyrene, aflatoxin B1, carbamazepine, phenytoin, trimethadione, cyclophosphamide,

ifosfamide, tegafur and thio-TEPA. The selection of the test substances accounts for differ-

ences in structure, origin, metabolism and water solubility. Apart from 2-acetylaminofluorene,

which mainly produces lethal effects, all proteratogens tested were teratogenic in zebrafish

embryos exposed for 3 days. The test substances and/or the substance class produced charac-

teristic patterns of fingerprint endpoints. Several substances produced effects that could be

identified already at 1 dpf (days post fertilization), whereas the effects of others could only be

identified unambiguously after hatching at ≥ 3 dpf. The LC50 and EC50 values were used to

calculate the teratogenicity index (TI) for the different substances, and the EC20 values were

related to human plasma concentrations. Results lead to the conclusion that zebrafish embryos

are able to activate proteratogenic substances without addition of an exogenous metabolic

activation system. Moreover, the teratogenic effects were observed at concentrations relevant

to human exposure data. Along with other findings, our results indicate that zebrafish embry-

os are a useful alternative method for traditional teratogenicity testing with mammalian spe-

cies.

Chapter II

47

2.2 Introduction

In recent years, zebrafish have become an intensively used model in ecotoxicology

(Braunbeck et al., 2005; Lammer et al., 2009a; Scholz et al., 2008; Seok et al., 2008), genetics

(Amsterdam, 2006; Amsterdam et al., 2004; Haffter et al., 1996) and safety pharmacology

(Pugsley et al., 2008; Redfern et al., 2008; Valentin et al., 2009), and many strains and mu-

tants are available to fit specific purposes (Guryev et al., 2006; Kishi et al., 2008; Nechiporuk

et al., 1999; Nissen et al., 2006; Sadler et al., 2005; Streisinger et al., 1981). Since zebrafish

embryo development is very similar to embryogenesis in higher vertebrates including hu-

mans, this species is ideally suited to study the fundamental processes underlying embryonic

development (Bachmann, 2002; Brannen et al., 2010; Busquet et al., 2008; Chakraborty et al.,

2009; Hill et al., 2005; Loucks and Carvan, 2004; McGrath and Li, 2008; Nagel, 2002;

Selderslaghs et al., 2010; Selderslaghs et al., 2009; Ton et al., 2006; Weigt et al., 2010; Yang

et al., 2009). Another advantage of the zebrafish model is related to animal welfare, because

experiments with zebrafish embryos are defined as in vitro tests (Belanger et al., 2010; EU,

2010; TSO, 2000 (revised edition).

Embryotoxic (embryolethal or teratogenic) activity is not always due to parent compounds,

but may be caused by metabolites formed by maternal and/or embryonic metabolism (Fantel

et al., 1979; Webster et al., 1997). Parent compounds, termed proteratogens, can be

bioactivated via oxidation to reactive metabolites, for example, electrophiles or free radical

intermediates (Wells et al., 2005). Cytochrome P450 enzymes (CYPs) represent the most im-

portant enzyme family involved in the oxidation of xenobiotics, which finally can result in the

toxification of chemicals, e.g. in embryo toxicity (Guengerich, 2001).

From first experiments with thalidomide, it was concluded that zebrafish embryos are not able

to activate substances by their own metabolism (Bachmann, 2002). However, on the other

hand, CYP activity and CYP mRNA has been detected in zebrafish embryos at different de-

velopmental stages: Otte et al. (2010) showed CYP1 activity and induction as early as 8 hours

post fertilization (hpf) using a 7-ethoxyresorufin-O-deethylase-based in situ assay. Wang et al.

(2004) first detected CYP1A mRNA in the integument at 24 hpf. CYP2K6 mRNA first ap-

peared at 120 hpf, and gene analysis showed 42 % sequence homology with human CYP

2C19 (Wang-Buhler et al., 2005). Tseng et al. (2005) were able to detect a weak level of

CYP3A65 transcription as early as at 24 hpf by RT-PCR, while CYP3A65 mRNA was not

detectable before 72 hpf by in situ hybridization. Jones et al. (2010) demonstrated CYP1A,

CYP2J26, CYP3 and UGT1 expression from 24 hpf onwards by RT-PCR. These data clearly

Chapter II

48

illustrate that it is difficult to demonstrate the explicit presence or absence of CYPs in

zebrafish embryos, especially by mRNA detection. However, the presence of basic CYP ac-

tivities in zebrafish embryos may be concluded. Therefore, it is hypothesized that zebrafish

embryos are able to CYP-activate proteratogens.

In literature, no other proteratogens, apart from thalidomide, have been tested in zebrafish

embryos without the addition of an exogenous metabolic activation system. Therefore,

zebrafish embryos were exposed to a selection of 10 substances (Tab. 2.1) known to require

metabolic activation prior to causing embryotoxic effects. The list comprises one aromatic

amine (2-acetylaminofluorene), one polycyclic aromatic hydrocarbone (benzo[a]pyrene), one

mycotoxin (aflatoxin B1) and 7 pharmaceuticals (carbamazepine, cyclophosphamide,

ifosfamide, phenytoin, tegafur, thio-TEPA and trimethadione).

2-Acetylaminofluorene (AAF) and benzo[a]pyrene (B[a]P) are carcinogenic (Buening et al.,

1978; Irigaray et al., 2006; Wörner and Schrenk, 1996), mutagenic (Ioannides et al., 1993;

Pillai et al., 1999) and teratogenic (Faustman-Watts et al., 1986; Shum et al., 1979). The poly-

cyclic aromatic hydrocarbon (PAH) B[a]P is formed by incomplete combustion of organic

matter (Brown et al., 2007; Parman and Wells, 2002) and can be found in ambient air, ciga-

Tab. 2.1: Test substances - overview

Test substance Abbreviation Reactive Metabolite(s) CAS No. Characterization

2-Acetylaminofluorene AAF N-Hydroxy-2-

acetylaminofluorene,

corresponding sulfat conjugate

53-96-3 Aromatic amine,

environmental

contaminant

Benzo[a]pyrene B[a]P Benzo[a]pyrene 7,8 diol- 9,10

epoxid

50-32-8 PAH,

environmental

contaminant

Aflatoxin B1 AFB1 Aflatoxin B1-8,9-epoxide 1162-65-8 Mycotoxin,

food contaminant

Carbamazepine CBZ Carbamazepine-l0,ll-epoxide 298-46-4 Antiepileptic drug

Phenytoin PHE 5-(p -hydroxyphenyl)-5-diphenyl-

hydantoin

57-41-0 Antiepileptic drug

Trimethadione TMO Dimethadione 127-48-0 Antiepileptic drug

Cyclophosphamide CPA Phosphoramide mustard,Acrolein,

(Dechloro-ethyl-

Cyclophosphamide,

Chloroacetaldehyde)

6055-19-2 Cytostatic drug

Ifosfamide IFO Ifosfamide mustard, Acrolein,

Dechloro-ethyl-Ifosfamide,

Chloroacetaldehyde

3778-73-2 Cytostatic drug

Tegafur TEG 5-Fluorouracil 17902-23-7 Cytostatic drug

Thio-TEPA TT TEPA 52-24-4 Cytostatic drug

Chapter II

49

rette smoke and water (Brown et al., 2007; Irigaray et al., 2006; Thompson et al., 2010). AAF,

an aromatic amine, was originally developed as insecticide, but it was never used as such after

early studies revealed its strong carcinogenic activity (Zaliznyak et al., 2006). Aflatoxin B1

(AFB1) is a carcinogenic mycotoxin produced by Aspergillus parasiticus and A. flavus and

occurs as a food contaminant mainly in subtropic and tropic countries (Shuaib et al., 2010).

AFB1 is teratogenic in rodents and rabbits (Raisuddin, 1993; Roll et al., 1990; Schmidt and

Panciera, 1980; Wangikar et al., 2004; Wangikar et al., 2005) and suspected to produce re-

duced birth weight and stillbirth in humans (Shuaib et al., 2010).

The pharmaceuticals can be divided in two groups. The first group consists of the three an-

tiepileptic drugs carbamazepine (CBZ), phenytoin (PHE) and trimethadione (TMO), which

are known to be teratogenic in humans and animals (Brown et al., 1979; Diav-Citrin et al.,

2001; Fradkin et al., 1981; German et al., 1970; Kasapinovic et al., 2004; McClain and

Langhoff, 1980; Novartis, 2000; Nulman et al., 1997). It is also worth noting that maternal

epilepsy itself has been associated with higher rates of birth defects in the offspring (Nulman

et al., 1997; Yerby et al., 2004). The second group of teratogenic pharmaceuticals comprises

the four anticancer drugs cyclophosphamide (CPA), ifosfamide (IFO), tegafur (TEG) and

thio-TEPA (TT) (Bus et al., 1973; Gerlinger and Clavert, 1964; Gililland and Weinstein,

1983; Hales, 1982; Nagaoka et al., 1982; Schardein and Macina, 2007; Shuey et al., 1995;

Stephens et al., 1980; Tanimura, 1968; Yukiyama et al., 1996). The alkylating properties of

the activated metabolites of CPA, IFO and TT are responsible for their teratogenic activity;

inter- or intra-strand cross linking of DNA and alkylation of other essential macromolecules

causes inhibition of DNA replication (de Jonge et al., 2005; Harbison, 1978; Lin et al., 2007).

5-Flourouracil, the activated metabolite of TEG, is an anti-metabolite which inhibits nucleic

acid replication by incorporation as a base-analogue (Harbison, 1978). All substances tested

in this study produced embryo lethality and/or teratogenicity in zebrafish embryos, and the

main enzymes involved in the bioactivation of these substances are CYPs and epoxide hydro-

lases.

To the best of our knowledge, this is the first report to document that zebrafish embryos were

able to activate proteratogenic/procarcinogenic substances without any addition of exogenous

metabolic activation systems. Results indicate that zebrafish embryos show phase I enzyme

activity at very early stages of development.

Chapter II

50

2.3 Materials and Methods

2.3.1 Materials

Tris (Tris(hydroxymethyl)-aminomethan) and HCl were obtained from Merck KGaA (Darm-

stadt, Germany). Aflatoxin B1, benzo[a]pyrene, carbamazepine, cyclophosphamide,

ifosfamide, phenytoin, thio-TEPA, trimethadione, 3-aminobenzoic acid ethyl ester

methanesulfonate (MS-222), and dimethyl sulfoxide (DMSO) were purchased from Sigma

(Deisenhofen, Germany). Tegafur and 2-acetylaminofluorene were delivered from Tokyo

Chemical Industry (Tokyo, Japan) and ABCR (Karlsruhe, Germany), respectively. 12.1 g Tris

was dissolved in fish medium to prepare a 100 mM buffer solution and 2 molar HCl was used

to adjust the pH value to 7.4 (26°C). The fish medium (reconstitued water consisting of 2 mM

CaCl2, 0.5 mM MgSO4, 0.7 mM NaHCO3, and 0.07 mM KCl) was prepared in the facility

according to OECD 2006 (OECD, 2006). All solutions were freshly prepared, and chemicals

were dissolved two hours before incubation.

2.3.2 Methods

2.3.2.1 Animal care and egg production

A breeding stock of unexposed and healthy mature wild type Tübingen strain zebrafish Danio

rerio, (original supplier: Max Planck Institute for Developmental Biology Tübingen, Germa-

ny; breeder: Institute of Toxicology, Merck KGaA) older than 6 months was used for egg

production. Spawners were maintained in a recirculating zebtec housing system (Tecniplast,

Hohenpeißenberg, Germany) at 26°C with a loading capacity of a minimum of 1 L water per

gram fish. The housing system is equipped with a mechanical, a biological, a UV light and an

activated carbon filter; pH value is automatically kept at 7.8 and the conductivity at 800 µS.

The fish medium in the housing system was prepared by the system itself from stock solutions

according to OECD 2006 (OECD, 2006).The automatic water exchange was adjusted to 10 %

of the system total volume per day. Lighting was controlled by a timer to provide a 12/12 h

light/dark cycle. Females and males were continuously kept together in a ratio of 1:2 or 1:1.

Dry flake food was fed twice daily and frozen food (Cyclops and Artemia) was fed once a

day, occasionally supplemented with Artemia nauplii (OECD, 2006; Westerfield, 2000). The

concentration of nitrate and nitrite were checked once a week, but were consistently

< 100 mg/L and 1.0 mg/L respectively.

Chapter II

51

Mating and spawning took place within 30 minutes after turning on the lights in the morning.

To prevent adult zebrafish from egg predation, egg trays were covered with a 2 mm plastic

mesh. Plastic plant imitations fixed to the mesh served as spawning substrate. About 30 min

after the onset of light, egg trays were removed and eggs were collected (OECD, 2006). Un-

der the culture conditions described above, fertilized eggs undergo the first cleavage after

approximately 15 min. Based on their transparency, the 4- to 32-cell stages, eggs can clearly

be identified as fertilized.

2.3.2.2 Embryo exposure

CPA, IFO, TEG, TT, TMO were dissolved in Tris-buffer. AAF, B[a]P, PHE, AFB1 and CMP

were dissolved in DMSO; the final solvent concentration was 0.5 %. For all substances, a

concentration range-finding experiment was conducted with a constant spacing factor of 2.

Concentration selections were based on the solubility of the test compound and the number of

affected embryos in the concentration range finding experiments (details not shown). Each

substance was tested in 4 - 7 concentrations and Tris-buffer or Tris-buffer with 0.5 % DMSO

was used as control; twenty embryos were used per group. Tris-buffer was chosen as vehicle

to keep the pH value at a physiological level. On the one hand, a controlled pH in the test me-

dia is relevant, since zebrafish embryos are very sensitive to pH changes (Augustine-Rauch et

al., 2010); on the other hand, the test substance is available at a pH similar to that in human

blood.

Eggs were rinsed twice in glass Petri dishes with fish medium. Within 2 hpf, fertilized eggs

(from 4- to 64-cell blastomeres) were selected under a CKX41 stereomicroscope (Zeiss,

Göttingen, Germany) into in a plastic Petri dish containing Tris-buffer.

At latest 2.5 hpf, the incubation was started by addition of the fertilized fish eggs to the test

substance. The embryos were exposed individually in 24-well plates (Nunc, Wiesbaden,

Germany) containing a final volume of 2 mL per well at 26°C with a 12:12-h light/dark cycle

in a precision incubator (Memmert, Schwabach, Germany). The well plates were sealed with

self-adhesive foil (MicroAmp® optical adhesive film, Applied Biosystems, Darmstadt, Ger-

many) to prevent evaporation. Before the embryos were added to the test solutions, the well

plates were pre-warmed to 26°C.

Chapter II

52

2.3.2.3 Evaluation (scoring) of lethality and teratogenic effects

At 8 hpf as well as 1, 2 and 3 dpf (days post fertilization), the embryos were evaluated and

scored for lethal or teratogenic effects using a Zeiss CKX41 inverted microscope with phase

contrast optics, a mounted time-lapse recorder and the analySIS software (Olympus, Ham-

burg, Germany). The 8 hpf time point served as a control step to identify unfertilized eggs,

which entered the test accidentally. Scoring for effects at 1 and 2 dpf was performed to track

the fate of the treated embryos and to give information about the time point when the different

endpoints were detectable (data not shown). The final scoring at 3 dpf was performed on em-

bryos anesthetized by addition of MS-222 (ethyl 3-aminobenzoate methanesulfonate, tricaine,

Sigma-Aldrich) solution (concentration about 0.1 %); after final scoring, the anesthetized em-

bryos were killed by freezing for ≥ 24 hours at -20°C.

CategoryPhysiological/

dysmorphogenic effect8 hpf 1 dpf 2 dpf 3 dpf

Lethal effect Coagulated egga + + + +

No heart beat +

Teratogenic effect Malformation of head + + +

Malformation of eyes + + +

Malformation of sacculi/otolithsb + +

Malformation of chordac + + +

Malformation of taild + + +

Malformation of tail tipe + + +

Scoliosis + + +

Deformity of yolk + + +

Growth retardationf + + +

Tab. 2.2: Lethal and teratogenic effects observed in zebrafish (Danio rerio) embryos depend-

ing on the observation time.

a No clear organ structures are recognized.

b Malformation of sacculi/otoliths cover formation of no, one or more than two otoliths per

sacculus as well as include absence or abnormally shaped sacculi (vesicles).

c Malformation of chorda often entail malformation of the spinal cord.

d Malformation of tail was assessed when the tail was bent.

e Malformation of the tail tip was assessed when the spike was bent or twisted.

f At 3 dpf growth retardation was assessed when an embryo shows a body length below 2.8

mm (using analySIS software; Olympus, Hamburg, Germany).

Chapter II

53

All embryos were staged as described by Kimmel et al. (1995), and lethal or teratogenic ef-

fects were recorded according to Bachmann and Nagel (Bachmann, 2002; Nagel, 2002)

(Tab. 2.2). Teratogenic effects were considered as fingerprint endpoints, if the following crite-

ria were fulfilled: (i) concentration-response relationship and (ii) the endpoint must be ob-

served in ≥ 50 % of all embryos showing teratogenic effects in all test groups of a test sub-

stance.

2.3.2.4 Calculation of LC50, EC20, EC50 and teratogenicity index (TI)

For the calculation of EC and LC values, ToxRatPro (ToxRat®, Software for the Statistical

Analysis of Biotests, ToxRat Solutions GmbH, Alsdorf, Germany, Version 2.10), using probit

analysis with linear maximum likelihood regression or moving average computation after

Thompson, was used. In order to characterize the teratogenic potential of a test substance, the

teratogenicity index (TI), which is defined as the quotient of LC50 and EC50, was calculated. If

the TI of a given substance is greater than 1, the substance is considered to be teratogenic and

if the TI is below 1, the substance produces mainly embryolethal effects.

2.3.2.5 Validity criteria and statistics

Egg batches were only used, if fertilization rates were ≥ 80 %. An assay was considered valid

if the controls did not show > 10 % teratogenic plus lethal effects at 3 dpf.

A Student’s t-test (one-tailed) was performed to identify statistically significant differences

between treatment and controls groups. Statistics were done on the basis of affected embryos

(embryos with lethal or teratogenic effects).

2.4 Results

All controls fulfilled the ac-

ceptance criteria of ≤ 10 % affect-

ed embryos at 3 dpf. The inci-

dence of affected embryos in the

control groups with Tris-buffer

plus 0.5 % DMSO was about

twice as high as in the controls

with Tris-buffer only (Tab. 2.3

and 2.4). Fig. 2.1 shows a control embryo at 3 dpf.

Fig. 2.1: Inverted microscope image of a control zebrafish

embryo at 3 dpf. H: head; E: eye; S/O: sacculi/otoliths;

YS: yolk sac; C: chord; T: tail; TT: tail tip; dpf: days

post-fertilization.

Chapter II

54

Tab. 2.3: Overview of the lethal and teratogenic effects of 2-acetylaminofluorene, ben-

zo[a]pyrene, aflatoxin B1, carbamazepine and phenytoin on zebrafish embryos at 3 dpf.

a Mean percentage ± SD

** Significantly different from controls at p < 0.05

Normally

developed

embryos [%]

Embryos with

teratogenic

effects [%]

Embryos with

lethal effects

[%]

Affected

embryos [%]

Number of

replicates

Tris-buffer with

0.5 % DMSO95.4 + 4.4

a 3.7 + 4.4 1.0 + 2.1 4.6 + 4.4 15

2-Acetylaminofluorene

5 µM 76.7 ± 7.6 8.3 ± 2.9 15.0 ± 5.0 23.3 ± 7.6**

6 µM 25.0 ± 35.0 18.3 ± 16.1 56.7 ± 20.2 75.0 ± 35.0**

7 µM 23.3 ± 20.8 25.0 ± 21.8 51.7 ± 2.9 76.7 ± 20.8**

8 µM 26.7 ± 5.8 23.3 ± 7.6 50.0 ± 5.0 73.3 ± 5.8**

9 µM 10.0 ± 5.0 25.0 ± 15.0 65.0 ± 10.0 90.0 ± 5.0**

10 µM 3.3 ± 5.8 11.7 ± 2.9 85.0 ± 5.0 96.7 ± 5.8**

Benzo[a]pyrene

0.25 µM 80.0 ± 13.2 20.0 ± 13.2 0.0 ± 0.0 20.0 ± 13.2

0.5 µM 45.0 ± 15.0 50.0 ± 17.3 5.0 ± 8.7 55.0 ± 15.0**

1 µM 13.3 ± 12.6 75.0 ± 21.8 11.7 ± 12.6 86.7 ± 12.6**

5 µM 3.3 ± 5.8 31.7 ± 28.4 65.0 ± 30.4 96.7 ± 5.8**

10 µM 0.0 ± 0.0 43.3 ± 45.1 56.7 ± 45.1 100.0 ± 0.0**

Aflatoxin B1

0.5 µM 86.7 ± 2.9 13.3 ± 2.9 0.0 ± 0.0 13.3 ± 2.9**

1 µM 85.0 ± 8.7 15.0 ± 8.7 0.0 ± 0.0 15.0 ± 8.7

2 µM 26.7 ± 7.6 51.7 ± 2.9 21.7 ± 5.8 73.3 ± 7.6**

3 µM 0.0 ± 0.0 11.7 ± 7.6 88.3 ± 7.6 100.0 ± 0.0**

4 µM 0.0 ± 0.0 0.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0**

Carbamazepine

31.25 µM 90.0 ± 5.0 10.0 ± 5.0 0.0 ± 0.0 10.0 ± 5.0

62.5 µM 81.7 ± 2.9 16.7 ± 2.9 1.7 ± 2.9 18.3 ± 2.9**

125 µM 71.7 ± 12.6 28.3 ± 12.6 0.0 ± 0.0 28.3 ± 12.6**

250 µM 51.7 ± 11.5 48.3 ± 11.5 0.0 ± 0.0 48.3 ± 11.5**

500 µM 0.0 ± 0.0 100.0 ± 0.0 0.0 ± 0.0 100.0 ± 0.0**

Phenytoin

31.25 µM 81.7 ± 7.6 16.7 ± 5.8 1.7 ± 2.9 18.3 ± 7.6**

62.5 µM 71.7 ± 25.7 26.7 ± 25.2 1.7 ± 2.9 28.3 ± 25.7

125 µM 66.7 ± 7.6 33.3 ± 7.6 0.0 ± 0.0 33.3 ± 7.6**

250 µM 55.0 ± 21.8 43.3 ± 18.9 1.7 ± 2.9 45.0 ± 21.8**

3

3

3

3

3

Chapter II

55

2.4.1 Exposure of zebrafish embryos to 2-acetylaminofluorene, benzo[a]pyrene

and aflatoxin B1

AAF exposure mainly resulted in embryo lethality (Tab. 2.3). However, it should be noted

that many AAF-treated embryos, which were dead at 3 dpf, showed teratogenic effects at 1

and 2 dpf. A unique observation was made at 1 dpf: Several embryos showed a reduction in

size combined with 2 rather small yolk sacs per embryo (Fig. 2.2 A; video see supplemental

data). One yolk sac was connected to the embryo and another separate yolk sac was present in

the liquid of the chorion. Despite this abnormal development, nearly all embryos showed

spontaneous movement at 1 dpf (supplemental data); however, in general, these embryos died

during the following 24 hours. Statistical significance was already reached at a concentration

of 5 µM. Since a clear concentration-response relationship (concerning teratogenicity) could

not be observed for AAF treatment, no fingerprint endpoints were determined.

In contrast to AAF, B[a]P, clearly pro-

duced a higher incidence of teratogenic

than lethal effects, and statistical signif-

icance was reached already at a concen-

tration of 0.5 µM (Tab. 2.3). The

teratogenic effects were most prominent

at 3 dpf, when the hatching process is

normally complete in all embryos, and

malformation of head (54.5 %), tail

(84.1 %), tail tip (100 %) and growth

retardation (75.0 %) were identified as

fingerprint endpoints (Tab. 2.5; Fig.

2.2 B).

Teratogenic effects in zebrafish embry-

os exposed to 0.5, 1, and 2 µM AFB1

were observed at 3 dpf; however, higher

concentrations of AFB1 (3 and 4 µM)

resulted in teratogenic effects already at

2 dpf. Treatment with 0.5, but not with

1 µM AFB1 resulted in a statistically

significant increase in the percentage of

Fig. 2.2: Zebrafish embryos exposed to 2-acetyl-

aminofluorene (AAF) and Benzo[a]pyrene. (A) 1

dpf, 6 µM AAF: embryo with an additional yolk

sac. (B) 3 dpf, 1 µM B[a]P: Malformation of

head, tail, tail tip and growth retardation. H:

head; T: tail; TT: tail tip; aYS: additional yolk

sac; dpf: days post-fertilization.

Chapter II

56

affected embryos compared to controls; the reason may be due to the low standard deviation

of the three replicates of 0.5 µM AFB1. Lethal effects started at 2 µM and reached 100 % at

4 µM AFB1 (Tab. 2.3). At 2 and 3 µM AFB1, lethality manifested mainly in a lack of heart

beat, whereas at 4 µM all embryos were coagulated. Similar to AAF, AFB1, which is also a

strong mutagen, produced an extra yolk sac in many embryos, but this effect appeared be-

tween 1 and 2 dpf in the AFB1-treated embryos. The fingerprint endpoints of AFB1 were

malformation of tail (69.1 %), tail tip (70.9 %) and growth retardation (56.4 %; Tab. 2.5).

2.4.2 Exposure of zebrafish embryos to the antiepileptic drugs carbamazepine,

phenytoin and trimethadione

The very poor water solubility of CBZ and PHE made these substances difficult to test. Even

when using 0.5 % DMSO as a solvent, it was not possible to reach CBZ and PHE concentra-

tions that produced embryo lethality clearly above control levels. There was an increase in the

percentage of embryos with teratogenic effects in the experiments with PHE, despite precipi-

tation of test substance in some test groups of the two highest concentrations. The water solu-

bility of PHE was extremely low, and, thus, the maximum teratogenic effect level of PHE was

only 43.3 ± 18.9 % at 250 µM, nominally. Contrary to PHE, the highest CBZ concentration

produced 100 % teratogenicity. In the treatment groups with PHE, statistical significance was

reached at all concentrations tested, except 62.5 µM, maybe due to the relatively high stand-

ard deviation in the replicates of this concentration. Regarding the percentage of affected em-

bryos in the experiments with CBZ, differences from controls were statistically significant

from 62.5 µM (Tab. 2.3).

Malformation of the tail represents the only fingerprint endpoint of PHE and CBZ (51.4 %

and 95.9 %, respectively; Tab. 2.5). However, the appearance of the malformed embryos was

different between PHE and CBZ: Whereas the CBZ-treated embryos preferentially showed a

strong, mainly upward bending (Fig. 2.3), the PHE-treated embryos displayed a slight down-

ward bending of the tail. In case of CBZ, it was not possible to clearly identify the tail mal-

formations in the 500 µM test groups, because not a single embryo hatched. In order to enable

correct scoring of effects, the embryos were manually dechorionated and scored again 6 hours

later. The additional period of 6 hours was included to ensure that the embryos would have

enough time to stretch and only real tail malformations were scored. As all dechorionated

embryos showed strong (upward) bending of the tail and also moderate bending of the tail

Chapter II

57

was found in lower concentrations, it could be concluded that prevented hatching is a result of

this malformation and not the cause.

a Mean percentage ± SD

** Significantly different from controls at p < 0.05

Tab. 2.4: Overview of the lethal and teratogenic effects of cyclophosphamide, ifosfamide,

tegafur, thio-TEPA and trimethadione on zebrafish embryos at 3 dpf.

Normally

developed

embryos [%]

Embryos with

teratogenic

effects [%]

Embryos with

lethal effects

[%]

Affected

embryos

[%]

n

Cyclophosphamide

1 mM 90.0 ± 0.0 10.0 ± 0.0 0.0 ± 0.0 10.0 ± 0.0**

3 mM 71.7 ± 2.9 25.0 ± 5.5 3.3 ± 2.9 28.3 ± 2.9**

5 mM 11.7 ± 10.4 58.3 ± 7.6 30.0 ± 13.2 88.3 ± 10.4**

8 mM 8.3 ± 7.6 56.7 ± 10.4 35.0 ± 8.7 91.7 ± 7.6**

10 mM 0.0 ± 0.0 33.3 ± 10.4 66.7 ± 10.4 100.0 ± 0.0**

Ifosfamide

0.25 mM 95.0 ± 5.0 5.0 ± 5.0 0.0 ± 0.0 5.0 ± 5.0

1 mM 73.3 ± 11.5 18.3 ± 7.6 8.3 ± 7.6 26.7 ± 11.5**

2 mM 48.3 ± 2.9 40.0 ± 10.0 11.7 ± 12.6 51.7 ± 2.9**

3 mM 13.8 ± 10.4 50.0 ± 10.0 36.7 ± 12.6 86.7 ± 10.4**

4 mM 1.7 ± 2.9 21.7 ± 20.2 76.7 ± 22.5 98.3 ± 2.9**

Tegafur

1.25 mM 90.0 ± 5.0 10.0 ± 5.0 0.0 ± 0.0 10.0 ± 5.0

2.5 mM 68.3 ± 5.8 30.0 ± 5.0 1.7 ± 2.9 31.7 ± 5.8**

5 mM 28.3 ± 10.4 68.3 ± 10.4 3.3 ± 5.8 71.7 ± 10.4**

10 mM 1.7 ± 2.9 96.7 ± 2.9 1.7 ± 2.9 98.3 ± 2.9**

20 mM 1.7 ± 2.9 96.7 ± 2.9 1.7 ± 2.9 98.3 ± 2.9**

30 mM 1.7 ± 2.9 55.0 ± 5.0 43.3 ± 7.6 98.3 ± 2.9**

40 mM 0.0 ± 0.0 3.3 ± 5.8 96.7 ± 5.8 100.0 ± 0.0**

Thio-TEPA

15.6 µM 91.7 ± 5.8 8.3 ± 5.8 0.0 ± 0.0 8.3 ± 5.8

31.25 µM 85.0 ± 5.0 15.0 ± 5.0 0.0 ± 0.0 15.5 ± 5.0**

62.5 µM 18.3 ± 7.6 81.7 ± 7.6 0.0 ± 0.0 81.7 ± 7.6**

250 µM 0.0 ± 0.0 88.3 ± 7.6 11.7 ± 7.6 100.0 ± 0.0**

500 µM 0.0 ± 0.0 0.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0**

Trimethadione

10 mM 91.7 ± 5.8 5.0 ± 0.0 3.3 ± 5.8 8.3 ± 5.8

20 mM 66.7 ± 2.9 33.3 ± 2.9 0.0 ± 0.0 33.3 ± 2.9**

40 mM 0.0 ± 0.0 88.3 ± 20.2 11.7 ± 20.2 100.0 ± 0.0**

50 mM 1.7 ± 2.9 26.7 ± 2.9 71.7 ± 2.9 98.3 ± 2.9**

60 mM 0.0 ± 0.0 0.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0**

3

3

3

Tris-buffer 15

3

98.0 ± 2.5a 1.3 ± 2.3 0.7 ± 1.8 2.0 ± 2.5

3

Chapter II

58

The majority of teratogenic effects of CBZ and PHE were detectable only at 3 dpf with the

exception of CBZ-induced sacculi/otoliths malformations, which were observable already at 2

dpf. In contrast to malformations of the tail and tail tip, sacculi/otoliths malformations can be

easily assessed even when the zebrafish embryo is still non-hatched. In contrast to PHE and

CBZ, TMO is an example of a perfectly water soluble antiepileptic drug. The teratogenic ef-

fects found in the embryos exposed to the 2 lowest TMO concentrations (10 and 20 mM)

could be mainly identified at 3 dpf. In the test groups exposed to 40, 50 and 60 mM TMO,

many malformations were detected at ≥ 1 dpf. Many of these early malformations were very

strong and resulted in embryo lethality within the next 2 days, reaching 100% lethality at 60

mM. Except for 10 mM, all concentrations tested caused statistically significant effects com-

pared to the controls (Tab. 2.4). In addition to the fingerprint endpoint of the 2 other antiepi-

leptic drugs, PHE and CBZ (tail malformation), additional malformations of head and tail tip

were found for TMO (Tab. 2.5 and 2.6).

Tab. 2.5: Frequency of endpoints of benzo[a]pyrene, aflatoxin B1, carbamazepine and

phenytoin at 3 dpf.

a Sum of embryos with teratogenic effects displaying this endpoint (in all experiments).

b Sum of embryos with teratogenic effects displaying this endpoint/sum of embryos with

teratogenic effects detected in all concentrations of the given test substance.

Major endpoints given in bold.

Head 72a

54.5b

24 43.6 26 21.3 31 43.1

Eyes 0 0.0 0 0.0 2 1.6 0 0.0

Sacculi/otoliths 0 0.0 2 3.6 30 24.6 0 0.0

Chorda 13 9.8 17 30.9 17 13.9 6 8.3

Tail 111 84.1 38 69.1 117 95.9 37 51.4

Tail tip 132 100.0 39 70.9 43 35.2 16 22.2

Scoliosis 0 0.0 1 1.8 1 0.8 0 0.0

Yolk deformity 0 0.0 0 0.0 1 0.8 0 0.0

Growth retardation 99 75.0 24 56.4 35 28.7 25 34.7

Malformation [%] [%]

AFB 1

∑t [%]

CBZ

∑t

B[a]P

∑t

PHE

∑t[%]

Chapter II

59

2.4.3 Exposure of zebrafish embryos to the anticancer drugs cyclophosphamide,

ifosfamide, tegafur and thio-TEPA

All 4 anticancer drugs tested show good water

solubility. At 1, 3 and 5 mM CPA, teratogenic

effects were usually detectable at 3 dpf, but at

the two higher concentrations effects could al-

ready be identified at 2 dpf. However, a high

variability with regard to the onset of teratogenic

effects was observed between the 3 replicates.

Especially the chorda malformations were diffi-

cult to detect as long as the embryos had not

hatched. At lower concentrations, the chorda

malformations appeared as isolated lesions; in

the high dose groups, the chorda structure very often displayed complete disintegration. Sta-

tistical significance was reached at 1 mM (10 % affected embryos). At ≥ 5 mM CPA, lethal

effects became also important (Table 2.4).

Fig. 2.3: Zebrafish embryos exposed to

125µM carbamazepine (CBZ) at 3 dpf:

Malformation of tail. T: tail; dpf: days

post-fertilization.


b Sum of embryos with teratogenic effects displaying this endpoint/sum of embryos with

teratogenic effects detected in all concentrations of the given test substance.


Tab. 2.6: Frequency of endpoints of cyclophosphamide, ifosfamide, tegafur, thio-TEPA

and trimethadione at 3 dpf.

Head 47a

42.7b

30 37.0 110 52.4 111 95.7 70 76.1

Eyes 0 0.0 0 0.0 6 2.9 54 46.6 3 3.3

Sacculi/otoliths 1 0.9 9 11.1 35 16.7 56 48.3 29 31.5

Chorda 90 81.8 66 81.5 188 89.5 48 41.4 30 32.6

Tail 26 23.6 9 11.1 153 72.9 54 46.6 86 93.5

Tail tip 19 17.3 5 6.2 25 11.9 57 49.1 53 57.6

Scoliosis 1 0.9 0 0.0 1 0.5 37 31.9 10 10.9

Yolk deformity 0 0.0 0 0.0 1 0.5 1 0.9 1 1.1

Growth retardation 42 38.2 20 24.7 160 76.2 71 61.2 26 28.3

∑t[%]∑t [%]Malformation [%] [%]

TEG

∑t [%]

TT

∑t

CPA IFO

∑t

TMO

Chapter II

60

IFO, which is structurally almost identical to CPA, produced similar effects in zebrafish em-

bryos, but lower concentrations were needed to reach the same effect levels (i.e. statistically

different to controls at 1 mM). In the majority of experiments with ≥ 2 mM IFO, the detection

of several teratogenic effects was possible from 2 dpf. As with CPA, the variation with regard

to the onset of teratogenic effects between the replicates was relatively high (Tab. 2.4). The

incidence of all endpoints, especially of chorda malformation, was also very similar between

IFO and CPA (81.8 and 81.5 % respectively; Tab. 2.6). However, IFO treatment resulted in

embryo lethality and teratogenicity at slightly lower concentrations, which is also reflected in

the different LC50 and EC50 values between the two alkylating substances (Tab. 2.7).

At 1.25, 2.5, 5 and 10 mM TEG, teratogenic effects were nearly exclusively detectable at 3

dpf in zebrafish embryos, however, at ≥ 30 mM malformations were already identified after 1

or 2 dpf. Many of the embryos showing strong malformations within the first 2 days of

exposure died between 2 and 3 dpf, reaching nearly 100 % embryo lethality at 40 mM. Apart

from 1.25 mM, all TEG concentrations tested were significantly different from controls (Tab.

2.4). The fingerprint endpoints of TEG were malformation of head, chorda, tail and growth

retardation (Tab. 2.6; Figs. 2.4A and B).

Tab. 2.7: LC50, EC50 and teratogenicity index (TI) of all test substances at 3 dpf.

* Calculated with ToxRat®.

Substance LC50* EC50* TI (LC50/EC50)

2-Acetylaminofluorene 6.9 µM – < 1

Benzo[a]pyrene 5.1 µM 0.52 µM 9.81

Aflatoxin B1 2.3 µM 2.2 µM 1.05

Carbamazepine > 500 µM 222 µM >1

Phenytoin > 250 µM 386 µM >1

Trimethadione 45.7 mM 23.5 mM 1.95

Cyclophosphamide 8.4 mM 4.7 mM 1.79

Ifosfamide 3.2 mM 3.1 mM 1.03

Tegafur 30.3 mM 3.4 mM 8.91

Thio-TEPA 306 µM 53.2 µM 5.75

Chapter II

61

In the experiments with 15.6, 31.25

and 62.5 µM TT, teratogenicity was

observed at 3 dpf only. At higher test

concentrations, malformations were

identified already at 1 and 2 dpf. After

250 µM treatment, only a few of these

early malformations resulted in em-

bryo lethality within the next 2 days,

but 100% embryo lethality was

reached in all replicates of the 500 µM

treatment groups. The percentage of

affected embryos was statistically dif-

ferent from controls from 31.25 µM

(Table 2.4). Malformation of the head

(95.7 %) and growth retardation

(61.2 %) could clearly be recognized

as fingerprint endpoints of TT, but 5

other endpoints were also found fre-

quently: malformation of eyes

(46.6 %), sacculi/otoliths (48.3 %),

chorda (41.4 %), tail (46.6 %) and tail

tip (49.1 %; Tab. 2.6; Fig. 2.5). This

accumulation of teratogenic effects

was characteristic for TT.

Fig. 2.5: Zebrafish embryos exposed to 200 µM thio-

TEPA (TT) at 3 dpf: malformation of head, eye,

chorda, tail, tail tip and growth retardation. H: head;

E: eye; C: chorda; T: tail; TT: tail tip; dpf: days post-

fertilization.

Fig. 2.4: Zebrafish embryos exposed to tegafur

(TEG) at 3 dpf. (A) 3 dpf, 5mM TEG: malformation

of head, chorda, tail and growth retardation. (B)

Detail, completely destructed chorda structure. H:

head; C: chorda; T: tail; dpf: days postfertilization.

Chapter II

62

2.4.4 LC50, EC50 and teratogenicity index (TI)

The LC50 and EC50 values and corresponding TIs of all substances tested are listed in

Tab. 2.7. On the basis of the TI values, all test substances except for AAF were considered

teratogenic in zebrafish embryos. For AAF, no EC50 and corresponding TI could be calculat-

ed, thus demonstrating that AAF mainly produced lethal effects in zebrafish embryos. B[a]P,

TEG and TT showed TI values ≥ 1 (9.81, 8.91 and 5.75 respectively), indicating a strong

teratogenic potential of these substances. Since the TIs for IFO, AFB1, CPA and TMO ranged

between 1 and 2 (1.03, 1.05, 1.79 and 1.95 respectively), these substances were categorized as

teratogenic, but embryo lethality is almost as important as teratogenicity, especially in the

cases of IFO and AFB1. Due to the low water solubility of CBZ and PHE, concentrations

causing embryo lethality above control levels could not be reached and, thus, the correspond-

ing LC50 values could not be calculated. Therefore, the TI values of these substances are >1.

2.5 Discussion

The aim of this study was to investigate if zebrafish embryos are able to activate

proteratogens to their active metabolites without the addition of an exogenous metabolic acti-

vation system such as S9 mixes or microsomes. For this purpose, we developed a 3-day

zebrafish embryo teratogenicity assay and tested 10 proteratogens, including environmental

contaminants, pharmaceuticals and a mycotoxin. Both easily (CPA, IFO, TEG, TT and TMO)

and poorly (AAF, B[a]P, AFB1, CBZ and PHE) water soluble substances produced effects in

zebrafish embryos. It is important to note that poorly water soluble substances are also detect-

ed by this in vitro system, as the majority of the new pharmaceuticals being developed nowa-

days show very low water solubility, and one important application of this assay should be the

use as screening method in the drug discovery process. The selection of test substances also

accounts for different metabolic pathways. CYPs are some of the most important drug metab-

olizing enzymes; however, out of the 57 CYPs listed for humans only 15 are involved in the

metabolism of xenobiotics and only 5 account for 95 % of phase I metabolism of all drugs on

the market (Guengerich, 2001, 2003, 2006, 2008). Apart from CYP2D6, all CYPs described

by Guengerich (Guengerich, 2001, 2003, 2006, 2008) to be important for drug metabolism in

humans, were also involved in the activation of the substances tested in this study (Tab. 2.8).

Furthermore, epoxide hydrolases and sulfotransferases are known to contribute to the

bioactivation of B[a]P, PHE and AAF (Tab. 2.8). However, it is not known to which extent

Chapter II

63

these reactions contribute to the formation of reactive metabolites in zebrafish embryos, be-

cause the metabolites produced by initial CYP metabolism of these substances are also

reactive (Goldstein and Faletto, 1993; Jiang et al., 2007; Parman et al., 1998).

Rubinstein described parallels between human and zebrafish CYP metabolism and suggested

that the zebrafish, in some cases, may be a better model for human toxicity than some com-

monly used mammalian models (Rubinstein, 2006). The zebrafish embryo data presented

herein point in the same direction. Given their external development and the fact that

zebrafish embryos cannot rely on maternal metabolism for protection, it is not surprising that

zebrafish embryos are capable of xenobiotic metabolism.

Tab. 2.8: Human Phase I and II enzymes involved in the metabolism of the test substances.

SubstanceHuman Phase I and II

enzymesLiterature

2-Acetylaminofluorene CYP1, 2A6; sulfotransferases Goldstein and Faletto (1993), Hodgeson

(2004), Ioannides et al. (1993)

Benzo[a]pyrene CYP1A1, 1B1; epoxide

hydrolases

Goldstein and Faletto (1993), Ioannides

and Lewis (2004), Jiang et al. (2007), Par-

man and Wells (2002), Willett et al. (2000)

Aflatoxin B1 CYP1A, 1B, 2A6, 2B6, 3A4;

epoxide hydrolases

Goldstein and Faletto (1993), Van Vleet et

al. (2002), Ioannides and Lewis (2004),

Jain and Iyer (2004), Guengerich (2006,

2008)Carbamazepine CYP1A2, 2A6, 2B6, 2C8,

2C19,

Pearce et al. (2008, 2005, 2002)

Phenytoin CYP2C8, 2C9; epoxide

hydrolases

Edeki and Brase (1995), Finnell and

Chernoff (1987), Goldstein and Faletto

(1993), Parman et al. (1998)

Trimethadione CYP2C9, 2E1, 3A4 Kurata et al. (1998), Nakamura et al.

(1998), Tanaka et al. (1996)

Cyclophosphamide CYP2A6, 2B6, 2C8/9,

2C18/19,

2E1, 3A4, 3A5

de Jonge et al. (2005), Dirven et al. (1994,

1996), Griskevicius et al. (2003), Gut et al.

(2000), Lin et al. (2007)

Ifosfamide CYP2A6, 2B6, 2C8/9,

2C18/19,

2E1, 3A4, 3A5

de Jonge et al. (2005), Dirven et al. (1994,

1995, 1996), Griskevicius et al. (2003), Gut

et al. (2000), Lin et al. (2007)

Tegafur CYP1A2, 2A6, 2C8 Fukami et al. (2005), Ikeda et al. (2000),

Komatsu et al. (2000)

Thio-TEPA CYP 2B6, 3A4 Ekhart et al. (2009), Jacobson et al. (2002)

Chapter II

64

In order to make a 1:1 comparison between the effect levels of the zebrafish assay and the in

vivo situation, it would be important to know the concentrations of a given substance in the

maternal or umbilical cord blood of a developmental toxicity study. Since the doses from

these studies have traditionally been given in mg per kg body weight, it is not possible to

make a direct comparison to this study. However, it is possible to compare human (therapeu-

tic) plasma concentrations with the zebrafish embryo data (Tab. 2.9). The EC20 was used for

this purpose, because experience has shown that statistical significance is generally reached

around the 20 % effect level (provided that the variation between the replicates is not too

high). Of course, there are no therapeutic plasma concentrations for the environmental con-

taminants AAF and B[a]P as well as for the food contaminant AFB1. However, it was possi-

ble to find plasma concentration data for AFB1 and B[a]P, even if the plasma concentration of

B[a]P derives from an inhalation study with Fischer rats (Ramesh et al., 2001). The EC20 val-

ue for B[a]P was only 2.4- to 6.1-fold higher than the plasma concentrations found in Fischer

rats. The EC20 value of AFB1 was 23- to 2843-fold higher than the human plasma concentra-

tions described by De Vries et al. (De Vries et al., 1989), who detected significantly lower

birth weights in newborns from AFB1 positive mothers, two stillbirths, but no malformations.

In contrast to humans, but similar to rodents and rabbits (Raisuddin, 1993; Roll et al., 1990;

Schmidt and Panciera, 1980; Wangikar et al., 2004; Wangikar et al., 2005), zebrafish embryos

also showed structural abnormalities in addition to growth retardation.

The analysis of the ratios between EC20 and human therapeutic plasma concentrations of the

pharmaceuticals reveal that the EC20 values are maximally 7.6-fold (TEG) the concentrations

of high-dose therapy and in the case of PHE and TT they are even below 1. Regarding the

ratios of the EC20 and low-dose therapy concentrations, the values of PHE, TT, TMO and

CBZ (3.3, 4.8, 5.2 and 6.2 respectively) showed a very good concordance between zebrafish

and human data. In contrast, the values of IFO (120-fold), CPA (171-fold) and TEG (472-

fold) low-dose therapy fall out of alignment. In oral therapy, TEG is administered together

with uracil or leucovarin (1:4 molar ratio) to enhance therapeutic efficacy (Engel et al., 2008;

Ikeda et al., 2000; Shepard and Lemire, 2004). It is possible that the addition of uracil or

leucovarin would potentiate the teratogenicity in zebrafish embryos. The reason for the ab-

sence of teratogenic effects in zebrafish embryos at low IFO and CPA concentrations remains

unclear; maybe, zebrafish embryos are less sensitive to IFO and CPA than mammals. Another

possibility could be the instability of these two anticancer drugs in the fish medium. Since

chemical analyses were not included in this study, the difference between the initial (nominal)

Chapter II

65

concentrations and the real concentrations over the 3 days of exposure is unknown. A renewal

system as described by Lammer et al. (Lammer et al., 2009b) could lead to lower effect lev-

els, perhaps not only for IFO and CPA. Apart from this, two aspects have to be considered: (i)

even under therapeutic conditions with teratogenic drugs, the incidence of newborns with

malformations is a relatively rare event (Banhidy et al., 2005) and (ii) it has been described

that sometimes only very high doses of a potent human teratogen are able to cause

malformations in experimental animals (Nau, 1986; Schardein et al., 1985).

The data presented in Tab. 2.9 clearly demonstrate that it is not reasonable to classify a sub-

stance as “not teratogenic”, when malformations are exclusively detectable at millimolar con-

centrations. Especially the example of TMO illustrates that the therapeutic concentrations can

sometimes achieve values in the upper millimolar concentration range. The evaluation of the

* calculated with ToxRat®

.

** Fischer 344 rats exposed via inhalation.

Tab. 2.9: Comparison between EC20 of the zebrafish teratogenicity assay at 3 dpf and human

(therapeutic) plasma concentrations.

SubstanceEC20*

[µM]

Human plasma

concentration

[µM]

Human plasma

concentration

literature

EC20/human plasma

concentration

2-Acetylaminofluorene 6.4 - - -

Benzo[a]pyrene 0.24 0.04 - 0.1** Ramesh et al. (2001) 2.4 - 6.1

Aflatoxin B1 0.85 0.0003 - 0.037 De Vries et al. (1989) 23.1 - 2843.0

Carbamazepine 74.2 12 - 50 Bertilsson and Tomson

(1986), O'Dougherty et al.

(1987)

1.5 - 6.2

Phenytoin 39.8 12 - 178 Cicurel and Schmid (1988) 0.2 - 3.3

Trimethadione 15691 3000 - 11000 Azarbayjani and

Danielsson (2002)

1.4 - 5.2

Cyclophosphamide 1885 ~11 - 700 Cicurel and Schmid

(1988), Ekhart et al. (2009)

2.7 - 171.4

Ifosfamide 916 ~7.6 - 490 Kerbusch et al. (2000,

2001 a,b)

1.9 - 120.5

Tegafur 1891 4 - 250 Cicurel and Schmid (1988)

Jeung et al. (2009)

7.6 - 472.8

Thio-TEPA 24.0 5 - 80.1 Ackland et al. (1988),

Ekhart et al. (2009),

Przepiorka et al. (1995)

0.3 - 4.8

Chapter II

66

teratogenic potential of a substance should ideally be assessed in comparison to relevant or

expected exposure concentrations.

In this study, IFO was shown to be more potent than CPA. However, it is known from Dirven

et al. (Dirven et al., 1995) that the minor structural difference of these 2 alkylating agents may

result in remarkable changes in their pharmacokinetics and pharmacodynamics. Especially the

increased half-life time of IFO mustard, an important metabolite of IFO, can be assumed to be

responsible for the increased sensitivity of zebrafish embryos to IFO.

In contrast to the results of the zebrafish embryo assay, AAF produced statistically significant

teratogenic effects in rat whole embryo culture at concentrations greater than 100 µM

(Faustman-Watts et al., 1986; Harris et al., 1989; Stark et al., 1989b, a). It is very likely that

AAF did not result in lethality in the rat embryos, because in whole embryo cultures the expo-

sure is limited to a short period during organogenesis (Shepard et al., 1983); in contrast, in the

zebrafish assay, the exposure starts not later than early blastula stage and covers the complete

embryonic development. In addition, the AAF-exposed zebrafish embryos also showed

teratogenic effects before they died. Apart from the experiments with cultured rat embryos,

“The catalogue of teratogenic agents” (Shepard and Lemire, 2004) does not give any infor-

mation on teratogenicity in mammals and the citations for teratogenicity of AAF in birds and

rodents mentioned in Stark et al. (Stark et al., 1989a) are not available. Thus, it remains un-

clear if zebrafish embryos or cultured rat embryos better mimic the in vivo situation for AAF.

As already mentioned many AAF and also AFB1 treated embryos showed an extra yolk sac

before they died. Further investigation revealed that the additional yolk sac originated from

either a single cell or a small cell population, which was separated from the initial yolk sac

and underwent accelerated cell proliferation. The reason for this uncontrolled cell prolifera-

tion in the developing zebrafish embryo is not known. However, as AAF is a strong mutagen,

it is not surprising that this substance causes uncontrolled cell proliferation in the developing

zebrafish embryo. Henn and Braunbeck also described a similar effect after pronase treatment

(Henn and Braunbeck, 2011).

The variation of teratogenic effects observed between the replicates for AAF and PHE (see

standard deviation values in Tab. 2.3) was sometimes relatively high. This could be due to the

low water solubility or variations in enzyme activity (CYP1, 2C9 and epoxidhydrolases;

Tab. 2.8) from batch to batch. Inter-individual variations in CYP activity, especially for

CYP2C9 and 2C19, are also known from humans (Guengerich, 2006; Rosemary and Adithan,

2007; Sistonen, 2008). In this context, it should be mentioned that one of the pre-tests with

Chapter II

67

PHE did not show any teratogenicity. In the experiments with B[a]P, the percentage of em-

bryos with teratogenic and lethal effects also varied. Only the distribution of embryos with

teratogenic and lethal effects was different between the replicates; the total percentage of af-

fected (teratogenic and lethal together) embryos was very similar (Tab. 2.3).

Several substances produced effects that could be identified already at 1 dpf, whereas the ef-

fects of some others could only be identified, unambiguously, after hatching at 3 dpf. These

results do not directly allow conclusions on the time point of proteratogen activation. When

an effect is detectable depends on the type of effect and/or on the intensity of the effect. Fur-

ther investigations on the time-dependent CYP activity in the developing zebrafish embryo

are required to elucidate these issues. The test substances as well as the drug/substance class

produced characteristic patterns of fingerprint endpoints. For example, the 2 aromatics AAF

and B[a]P caused mainly malformation of head, tail, tail tip and growth retardation, whereas

TT mainly induced head malformations and growth retardation (Tab. 2.5 and 2.6).

The TI is a strict mathematically calculated value and does not consider concentration-

response relationships or the ratio of embryos with teratogenic and lethal effects. AFB1 and

IFO sowed TI values just above 1 (Tab. 2.7) and were, thus, considered weakly teratogenic,

but Tab. 2.3 and 2.4 clearly demonstrate that teratogenicity occurred before lethality. The TI

value is just a first estimation for the teratogenic potential of a substance, and it is essential

that the kind of effect, effect intensity and concentration-response relationships of the

teratogenic and embryolethal effects are taken into account before making a final decision

(Bachmann, 2002). The initial values used to calculate the TI (EC and LC50) also depend on

the program used for calculation.

Originally, zebrafish embryos were thought not to be capable of activating substances, which

need metabolic activation, since exposure to thalidomide, a very popular proteratogen, did not

cause any effects in zebrafish embryos (Bachmann, 2002). This was why Busquet et al.

(2008) developed a zebrafish teratogenicity assay with microsomes as an exogenous metabol-

ic activation system (mDarT). Further development of the mDarT revealed that very often

only high concentrations produced effects in the embryos and that many substances with

known teratogenic potential were in fact false negatives (Weigt, unpublished data). The short

exposure period of 1 h, which is limited by the toxicity of the metabolic activation system,

may be the reason for such problems. In addition, recent data from Ito et al. (2010) showed

that thalidomide is able to produce malformations also in zebrafish embryos.

Chapter II

68

Potentially, the contradicting results by Bachmann (2002) and Ito et al. (2010) are caused by

strain-dependent differences. All of the different zebrafish embryo teratogenicity screening

protocols reported (Bachmann, 2002; Brannen et al., 2010; Selderslaghs et al., 2009) have

certain advantages. E.g., the measurement of jaws and eyes, as described by Brannen et al.

(2010), seems to be a good method to make the scoring of craniofacial effects more objective.

However, for a rapid validation it is essential to reach an agreement concerning temperature,

medium, solvent, fish strain, scoring time points, selection of endpoints, start and duration of

exposure. It would also be helpful to know if dechorionation of the embryos is absolutely

necessary for correct testing or if the use of DMSO is sufficient to allow sufficient transport

rates of the test substance into the embryo. Another important issue, which needs further dis-

cussion within a pre-validation process, is whether a substance should be considered

teratogenic when the malformed embryos die before the end of the test (as observed for, e.g.,

AAF). As already mentioned, the selection of endpoints itself is a most important aspect in

these considerations: in this study, we excluded, for example, heart malformations from our

list of teratogenic endpoints, since pre-tests with other substances (data not shown) had shown

heart malformations in embryos exposed both to teratogenic and to non-teratogenic sub-

stances. It is known that a heart malformation can be secondary to pericardial edema for-

mation (Antkiewicz et al., 2005).

2.6 Conclusions

Combined with data from studies with non-teratogens and direct acting teratogens (Bach-

mann, 2002; Brannen et al., 2010; Selderslaghs et al., 2009), our results clearly demonstrate

that zebrafish embryos are a suitable model to predict human/mammalian teratogenicity. This

is due to the facts that the incidence of false positive and false negative results is low and that

zebrafish embryos are capable of metabolic activation of proteratogens. All 10 test sub-

stances, which cover 4 important properties (structure, origin, water solubility and metabolic

pathways), produced embryolethal and/or teratogenic effects in zebrafish embryos without

any addition of an exogenous metabolic activation system. The comparison of EC20 values

and human (therapeutic) plasma concentrations revealed that the zebrafish embryo data identi-

fy teratogenic activity at concentrations close to the in vivo situation. The in vitro zebrafish

embryo teratogenicity assay has, thus, the potential to serve as a rapid, cost effective and pre-

dictive alternative to in vivo testing and can be implemented as a very early screening assay in

Chapter II

69

the drug development process. For economic reasons and animal welfare aspects, testing for

teratogenicity is traditionally done at the end of the preclinical safety studies or during clinical

phase I. The application of a 3-day in vitro zebrafish embryo teratogenicity test as a screening

assay could dramatically reduce the number of in vivo reproduction toxicity studies required.

Chapter II

70

Chapter III

71

Chapter III

Developmental effects of coumarin and the anticoagulant coumarin

derivative warfarin on zebrafish (Danio rerio) embryos

Chapter III

72

3 Developmental effects of coumarin and the anticoagulant

coumarin derivative warfarin on zebrafish (Danio rerio) embryos

Chaper II described the ability of zebrafish embryos to serve as a useful alternative test

method for the identification of proteratogenic substances. In this chapter, two highly

teratogenic and in humans strongly metabolized compounds were investigated. A comparison

of teratogenic effects between vertebrate test animals and humans has been performed.

3.1 Abstract

Coumarin and warfarin, two substances which are intensively metabolized in animals and

humans, were tested for teratogenicity and embryo lethality in a 3-day in vitro assay using

zebrafish embryos. Warfarin is a coumarin derivative, but in contrast to the mother substance

warfarin has anticoagulant properties. Both substances produced teratogenic and lethal effects

in zebrafish embryos. The LC50 and EC50 values for coumarin are 855 µM and 314 µM, re-

spectively; the corresponding values for warfarin are 988 µM and 194 µM. For coumarin,

three main or fingerprint endpoints (malformation of head, tail and growth retardation) were

identified, whereas malformation of tail was the only fingerprint endpoint of warfarin. The

analysis of the ratios between the zebrafish embryo effect concentrations of both substances

and human therapeutic plasma concentrations confirmed the teratogenic potential of warfarin,

as well as the equivocal status of coumarin.

3.2 Introduction

Over the last years, zebrafish embryos have been discussed as a promising alternative model

for teratogenicity testing (Bachmann, 2002; Brannen et al., 2010; Chakraborty et al., 2009;

Hill et al., 2005; Louks and Carvan, 2004; MacGrath and Li, 2008; Nagel, 2002; Selderslaghs

et al, 2009, 2010; Ton et al., 2006; Yang et al., 2009). Originally it was assumed that zebraf-

ish embryos are not able to activate proteratogenic substances by themselves (Busquet et al.,

2008, Weigt et al., 2010). After having shown that zebrafish embryos are also able to metabo-

lize/activate proteratogenic substances without addition of an exogenous metabolic activation

system (Weigt et al., 2011), we have now investigated the effects of coumarin and the cou-

Chapter III

73

marin derivative warfarin in a zebrafish embryo teratogenicity test. Several aspects make

these two substances interesting test compounds in zebrafish embryos: (i) either substance is

intensively metabolized in both humans and animals (Guengerich et al. 1982; Guo et al. 2006;

Ishizuka et al., 2007; Kaminsky and Zhang, 1997; Lake, 1999; Miller et al., 2009); (ii)

bioavailability of warfarin strongly depends on CYP metabolism (Hirsh et al., 2001; Wadelius

and Pirmohamed, 2007; Zielinska et al. 2008); (iii) parallels between human and zebrafish

CYP metabolism have been described and it was suggested that the zebrafish, in some cases,

may be a better model for human toxicity than some commonly used mammalian models

(Rubinstein, 2006); (iv) coumarin derivatives with anticoagulant properties are teratogenic in

humans, but do not show teratogenic effects in any animal developmental toxicity study

(Brent and Beckman, 1990; Nau, 1986; Schardein et al., 1985; Shepard et al., 1983); (v) cur-

rently, there is no clear information available concerning the teratogenic potential of coumarin

in humans (Friedman and Polifka, 2000; Shepard and Lemire, 2004).

In contrast to many of its derivatives, coumarin itself has no anticoagulant properties

(McKnight et al., 1992, Merkel et al., 1994). But it is used in other medical indications, such

as treatment of edemas, cancer and chronic infections (Abraham et al., 2010; Egan et al.,

1990; Jamal and Casley-Smith, 1989; Marshall et al., 1994; Mohler et al., 1994). In humans,

the CYP isoform found to be clearly dominant in coumarin metabolism is CYP2A6

(Guengerich, 1992, 2003; Lewis and Lake, 2002). Warfarin, also known as coumadin, is an

oral anticoagulant drug very common in the prophylaxis and treatment of thromboembolic

diseases, especially in the UK and the USA (Rojas et al., 2005). Warfarin acts as a vitamin K

antagonist, and vitamin K is needed as a cofactor for the carboxylation of glutamate residues

of several clotting factors (Freude et al., 1991; Hirsh, 1991; Hirsh et al., 2001). For oral medi-

cation, a racemic mixture of enantiomers is used, but the (S) enantiomer exhibits approxi-

mately 2 to 5 times more anticoagulant activity than the (R) enantiomer (Breckenridge, 1977;

Chan et al., 1994; Pitsiu et al., 2003; Zielinska et al., 2008). The two enantiomers are also

metabolized by different CYP isoforms in humans. The (R) enantiomer is hydroxylated by

CYPs 1A1, 1A2, 2C8, 2C9, 2C18, 2C19, 3A4 and 3A5 (questionable) with CYP1A2 and

CYP3A4 being most important. (S)-warfarin hydroxylation is mainly catalyzed by CYP2C9,

but CYPs 2A6 (questionable), 2C8, 2C18 and 2C19 also contribute to the metabolism of this

enantiomer (Freeman et al., 2000; Grossman et al., 1993; Guengerich, 2003; Huang et al.,

2004; Kamisky and Zhang, 1997; Miller et al., 2009; Rettie et al., 1992; Wadelius and Pirmo-

hamed, 2007; Zhang et al., 1995; Zielinska et al., 2008). The CYP enzyme which is most im-

Chapter III

74

portant for the therapeutic use of warfarin is CYP2C9. However, since warfarin interacts with

vitamin K, the enzyme vitamin K epoxide reductase (VKORC1) also plays a fundamental role

(Bell and Matschiner, 1972; Bell et al., 1972; Geisen et al., 2005; Li et al., 2004; Rieder et al.,

2005; Rost et al., 2004).

The terms “coumarin embryopathy” and “coumarin syndrome” used in human medicine today

are a little confusing, as these terms are only related to coumarin derivatives with anticoagu-

lant properties and not to coumarin itself. Yet, in order to avoid misunderstandings, the old-

fashioned terms “warfarin embryopathy” and “warfarin syndrome” are still used in this publi-

cation. In 1966, the first case of warfarin embryopathy was described by DiSaia (1966), and

Kerber et al. (1968) were the first to suggest warfarin to be a teratogenic agent. Today the

embryotoxic potential of warfarin and other coumarin anticoagulants is well-accepted (Briggs

et al., 2008; Friedman and Polifka, 2000; Lewis, 1991 Shepard and Lemire, 2004), but no

epidemiological studies of children born to women treated with coumarin during pregnancy

have been reported (Friedman and Polifka, 2000; Shepard and Lemire, 2004). The lack of

teratogenic effects of coumarin in developmental toxicity studies in animals is not sufficient

to conclude a lack of teratogenic potential in humans, because coumarin derivatives with anti-

coagulant properties (and human teratogenicity) also have not produced malformations in any

experimental animal species exposed prenatally (Brent and Beckman, 1990; Nau, 1986;

Schardein et al., 1985, 2007; Shepard et al., 1983). Pauli et al. (1987) suggested that the war-

farin embryopathy resulted from the pharmacological effect of warfarin, i.e., inactivation of

vitamin K. If this is also the case for zebrafish embryos, coumarin, which lacks anticoagulant

properties on the basis of vitamin K antagonism (McKnight et al., 1992; Merkel et al., 1994),

would not be able to produce warfarin embryopathy in this test system.


3.3.1 Materials

Tris (Tris(hydroxymethyl)-aminomethan) and HCl were obtained from Merck KGaA (Darm-

stadt, Germany). Coumarin, warfarin, 3-aminobenzoic acid ethyl ester methanesulfonate (MS-

222), and dimethyl sulfoxide (DMSO) were purchased from Sigma (Deisenhofen, Germany).

12.1 g Tris was dissolved in fish medium to prepare a 100 mM buffer solution and 2 molar

HCl was used to adjust the pH value to 7.4 (at 26°C). The fish medium (reconstitued water

Chapter III

75

consisting of 2 mM CaCl2, 0.5 mM MgSO4, 0.7 mM NaHCO3, and 0.07 mM KCl) was pre-

pared in the facility according to OECD Fish Embryo Test Draft Guideline (OECD, 2011).

All solutions were freshly prepared, and chemicals were dissolved two hours before incubation.

3.3.2 Methods

3.3.2.1 Animal care and egg production

A breeding stock of unexposed and healthy mature wild type Tuebingen strain zebrafish

(Danio rerio) (original supplier: Max Planck Institute for Developmental Biology Tübingen,

Germany; breeder: Institute of Toxicology, Merck KGaA) older than 6 months was used for

egg production. Spawners were maintained in a recirculating zebtec housing system (Tec-

niplast, Hohenpeißenberg, Germany) at 26°C with a loading capacity of a minimum of 1 L

water per gram fish. The housing system is equipped with mechanical, biological, UV light

and activated carbon filter systems; pH value is automatically kept at 7.8 and the conductivity

at 800 µS. The fish medium in the housing system was prepared by the system itself from

stock solutions according to OECD Fish Embryo Test Draft Guideline (OECD, 2011).The

automatic water exchange was adjusted to 10 % of the system total volume per day. Lighting

was controlled by a timer to provide a 12/12 h light/dark cycle. Females and males were con-

tinuously kept together in a ratio of 1:2 or 1:1. Dry flake food was fed twice daily and frozen

food (Cyclops and Artemia) was fed once a day, occasionally supplemented with Artemia

nauplii (OECD, 2011; Westerfield, 2000). The concentration of nitrate and nitrite were

checked once a week, but were consistently < 100 mg/L and 1.0 mg/L, respectively.

Mating and spawning took place within 30 minutes after turning on the lights in the morning.

To prevent adult zebrafish from egg predation, egg trays were covered with a 2 mm plastic

mesh. Plastic plant imitations fixed to the mesh served as spawning substrate. About 30 min

after the onset of light, egg trays were removed and eggs were collected (OECD, 2011). Un-

der the culture conditions described above, fertilized eggs undergo the first cleavage after

approximately 15 min. Based on their transparency, the 4- to 32-cell stage eggs can clearly be

identified as fertilized.

Chapter III

76

3.3.2.2 Embryo exposure

Coumarin and warfarin were dissolved in DMSO; with a final solvent concentration of 0.5 %

in the test media. For both substances, range-finding experiments were conducted with a con-

stant spacing factor of 2. The highest tested concentrations for coumarin and warfarin were

5 mM and 1.5 mM respectively. Concentration selections for the main study were based on

the number of affected embryos (embryos with teratogenic and/or lethal effects) in the range-

finding experiments (data not shown). Coumarin was tested in 5 concentrations and warfarin

in 7 concentrations. Tris-buffer with 0.5 % DMSO was used as control. Twenty embryos were

used per group and each experiment was repeated 3 times. Tris-buffer was chosen as vehicle

to keep the pH value at a physiological level. On the one hand, a controlled pH in the test me-

dia is relevant since zebrafish embryos are very sensitive to pH changes (Augustine-Rauch et

al., 2010); on the other hand, the test substance is made available at a pH similar to that in

human blood.

Eggs were first rinsed twice in glass Petri dishes with fish medium. Within 2 hours post-

fertilization (hpf), fertilized eggs (from 4- to 64- cell blastomeres) were selected under a

CKX41 stereomicroscope (Zeiss, Göttingen, Germany) into a plastic Petri dish containing

Tris-buffer.

At latest 2.5 hpf, the incubation was started by addition of the fertilized fish eggs to the test

solution. The embryos were exposed individually in 24-well plates (Nunc, Wiesbaden, Ger-

many) containing a final volume of 2 mL per well at 26°C with a 12:12-h light/dark cycle in a

precision incubator (Memmert, Schwabach, Germany). The well plates were sealed with self-

adhesive foil (MicroAmp®

optical adhesive film, Applied Biosystems, Darmstadt, Germany)

to prevent evaporation. Before the embryos were added to the test solutions, the well plates

were pre-warmed to 26°C.

3.3.2.3 Evaluation (scoring) of lethality and teratogenic effects

At 8 hpf as well as at 1, 2 and 3 dpf (days post-fertilization), the embryos were evaluated and

scored for lethal or teratogenic effects using a Zeiss CKX41 inverted microscope equipped

with phase contrast optics, a mounted time-lapse recorder and the analySIS software (Olym-

pus, Hamburg, Germany). The 8 hpf time point served as a control step to identify unfertilized

eggs, which had entered the test accidentally. Scoring for effects at 1 and 2 dpf was performed

Chapter III

77

to track the fate of the treated embryos and to give information about the time point when the

different endpoints were detectable. The final scoring at 3 dpf was performed on embryos

anesthetized by addition of MS-222 (ethyl 3-aminobenzoate methanesulfonate, tricaine,

Sigma-Aldrich) solution (concentration about 0.1 %). After final scoring the anesthetized em-

bryos were killed by freezing at -20°C for ≥ 24 hours.

All embryos were staged as described by Kimmel et al. (1995), and lethal or teratogenic ef-

fects were recorded according to Bachmann (2002) and Nagel (2002) (Tab. 3.1). Teratogenic

effects were considered as fingerprint endpoints, if the following criteria were fulfilled: (i)

concentration-response relationship is present, and (ii) the endpoint must be observed in

≥ 50 % of all embryos showing teratogenic effects in all test groups of a test substance.

Tab. 3.1: Lethal and teratogenic effects observed in zebrafish (Danio rerio) embryos depend-

ing on the observation time.

CategoryPhysiological/

dysmorphogenic effect8 hpf 1 dpf 2 dpf 3 dpf

Lethal effect Coagulated egga + + + +

No heartbeat +

Teratogenic effect Malformation of head + + +

Malformation of eyes + + +

Malformation of sacculi/otolithsb + +

Malformation of chordac + + +

Malformation of taild + + +

Malformation of tail tipe + + +

Scoliosis + + +

Deformity of yolk + + +

Growth retardationf + + +

a No clear organ structures are recognized.

b Malformation of sacculi/otoliths cover formation of no, one or more than two otoliths per

sacculus as well as include absence or abnormally shaped sacculi (vesicles). c Malformation of chorda often entail malformation of the spinal cord.

d Malformation of tail was assessed when the tail was bent.

e Malformation of the tail tip was assessed when the spike was bent or twisted.

f At 1 and 2 dpf, when the embryos were not hatched, growth retardation was assessed by

optical comparison to the control embryos (e.g. size, development). At 3 dpf

growth retardation was assessed when an embryo shows a body length below 2.8 mm (using

analySIS software; Olympus, Hamburg, Germany).

Chapter III

78

3.3.2.4 Calculation of LC50, EC20, EC50 and teratogenicity index (TI)

For the calculation of EC and LC values, the program ToxRatPro (ToxRat®, Software for the

Statistical Analysis of Biotests, ToxRat Solutions GmbH, Alsdorf, Germany, Version 2.10),

using probit analysis with linear maximum likelihood regression, was used. In order to char-

acterize the teratogenic potential of a test substance, the teratogenicity index (TI), which is

traditionally defined as the quotient of LC50 and EC50, was calculated. If the TI of a given

substance is > 1, the substance is considered to be teratogenic; if the TI is ≤ 1, the substance

produces mainly embryolethal effects. The EC20 is used for the comparison between zebrafish

embryo and human exposure data.

3.3.2.5 Validity criteria and statistics

Egg batches were only used, if fertilization rates were ≥ 80 %. An assay was considered valid,

if the controls did not show > 10 % teratogenic plus lethal effects at 3 dpf.

A Student’s one-tailed t-test was performed to identify statistically significant differences

between treatment and control groups. Statistics were done on the basis of affected embryos

(embryos with lethal and/or teratogenic effects).

3.4 Results

Lethal and teratogenic effects recorded at 3 dpf are summarized in Tab. 3.2 and 3.3. All con-

trols (Fig. 3.1A) fulfilled the acceptance criteria of ≤ 10 % affected embryos at 3 dpf (Tab. 3.2

and 3.3). Except for yolk deformities, all malformations listed in Tab. 3.1 were also observed

in embryos treated with coumarin and warfarin. In the low-dose treatment groups of coumarin

and warfarin, the chorda malformations appeared as isolated lesions; in the high-dose groups,

the chorda structure often displayed complete disintegration.

Chapter III

79

3.4.1 Exposure of zebrafish embryos to coumarin

The percentage of embryos with teratogenic and lethal effects clearly increased with increas-

ing coumarin concentrations (Tab. 3.2): Teratogenic effects in zebrafish embryos exposed to

200 and 400 µM coumarin were almost exclusively observed at 3 dpf; higher concentrations

of coumarin (600, 800 and 1000 µM), however, resulted in teratogenic effects already at 1 dpf

(Tab. 3.4). At the two highest concentrations, many of these early malformations were very

prominent and resulted in embryo lethality within the next 2 days, reaching 85 % lethality at

1000 µM (Tab. 3.2). The major lethal endpoint in the experiments with coumarin was lack of

heart-beat, and all of these embryos also showed many strong malformations. All coumarin-

treated embryos, which were coagulated at 3 dpf, showed teratogenic effects at 1 and 2 dpf.

The fingerprint endpoints of coumarin were malformations of head and tail (Fig. 3.1B) as well

as growth retardation. However, 3 other endpoints were also frequently found: malformation

of sacculi/otoliths (33.1 %), chorda (36.9 %) and tail tip (29.9 %; Tab. 3.6).

- No effects.

* Significantly different from controls at p < 0.05. a Mean percentage ± SD (three experiments).

Tab. 3.2: Overview of lethal and teratogenic effects of coumarin in zebrafish (Danio rerio)

embryos at 3 dpf.

1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

- - - 6 2 2 12 13 14 20 19 19 12 15 14 3 1 5

- - 1 - - - - - - - - 1 8 5 6 17 19 15

- - 1 6 2 2 12 13 14 20 19 20 20 20 20 20 20 20

20 20 19 14 18 18 8 7 6 - 1 - - - - - - -

Experiment No.

Σ Affected embryos

Σ Normal embryos

% Teratogenic embryos

% Lethal embryos

% Affected embryos

No. of teratogenic embryos

No. of lethal embryos

No. of affected embryos

No. of normal embryos

Σ Teratogenic embryos

Σ Lethal embryos

0.0±0.0

Coumarin test groups

-

15.0±10.0

85.0±10.0

100.0±0.0*

1000 µM

9

51

6039 59 6010

1.7±2.9 0.0±0.0 0.0±0.0

1.7±2.9 16.7±11.5 100.0±0.0*

0.0±0.0a 16.7±11.5 68.3±7.6

98.3±2.9*

65.0±5.0

65.0±5.0*

1.7±2.9

39

-

21

19

-1

58

1

200 µM 800 µM

Control

600 µM400 µMTris-buffer,

0.5 % DMSO

31.7±7.6

- 10

96.7±2.9

1 -

59

1

41

50

0.0±0.098.3±2.9 35.0±5.0 1.7±2.983.3±11.5 % Normal embryos

Chapter III

80

Except for 200 µM, all coumarin concentrations tested caused statistically significant effects

compared to controls (Tab. 3.2). The calculated LC50 and EC50 value of coumarin were 855

and 314 µM, respectively, resulting in a teratogenicity index of 2.7. The corresponding con-

centration-response curves for coumarin are given in Fig. 3.2A and B. The EC20 value, which

is used for comparison to human exposure data in the discussion, was 219 µM.

- No effects.

* Significantly different from controls at p < 0.05. a Mean percentage ± SD (three experiments).

Tab. 3.3: Overview of lethal and teratogenic effects of warfarin in zebrafish (Danio rerio) em-

bryos at 3 dpf.

1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

1 2 - 2 3 2 8 6 3 8 8 3 13 9 12 17 16 12 15 9 8 5 - -

- - - - - - - - - - - - - 1 1 - 1 1 5 11 12 15 20 20

1 2 - 2 3 3 8 6 3 8 8 3 13 10 13 17 17 13 20 20 20 20 20 20

19 18 20 18 17 18 12 14 17 12 12 17 7 10 7 3 3 7 - - - - - -

% Lethal embryos

% Affected embryos

No. teratogenic

embryos

No. lethal embryos

No. affected embryos

No. normal embryos

Σ Teratogenic embryos

% Normal embryos

Σ Lethal embryos

Σ Affected embryos

Σ Normal embryos

% Teratogenic embryos

78.3±11.5*

46.7±18.9

Warfarin test groups

100.0±0.0*

1000 µM

32

28

Experiment No.

-

8.3±14.4

91.7±14.4

1500 µM

0.0±0.0

-

53.3±18.9

100.0±0.0*

0.0±0.0

60

2

60

5

55

3.3±2.9

36 47

56.7±10.4

45

5.0±5.0 28.3±12.6*

62.5 µM

7

-

Control

5.0±5.0a 28.3±12.6

Tris-buffer,

0.5 % DMSO

-

250 µM125 µM31.25 µM

2

0.0±0.0 0.0±0.0 0.0±0.0

75.0±13.2

19

31.7±14.4*

3

17

41

19

500 µM

-

3417

-

11.7±2.9

88.3±2.9

7

53

11.7±2.9

40.0±8.771.7±12.6

0.0±0.0

21.7±11.595.0±5.0 68.3±14.4

57

3

43 1324

3.3±2.9

60.0±8.7*

31.7±14.4

Chapter III

81

- : No effects; ∑t: sum of teratogenic embryos per endpoint out of all teratogenic embryos in

the three test groups at 72 hpf; one embryo can display multiple teratogenic malformations. a Number of embryos displaying this endpoint (%).

b Sum of the teratogenic embryos per group at 3 dpf of three experiments.

Tab. 3.4: Summary of teratogenic effects of coumarin in zebrafish (Danio rerio) embryos.

Malformation Time Control Coumarin test groups

Head 1 dpf - 0 2 20 56 55

2 dpf - 3 6 39 53 41

3 dpf - 6 (10.0)a

19 (31.7) 47 (78.3) 41 (68.3) 9 (15.0) 122

Eyes 1 dpf - 0 0 0 17 33

2 dpf - 0 0 0 22 30

3 dpf - 0 (0.0) 1 (1.7) 1 (1.7) 15 (25.0) 4 (6.6) 21

Sacculi/otoliths 2 dpf - 1 3 11 39 35

3 dpf - 0 (0.0) 3 (5.0) 12 (20.0) 30 (50.0) 7 (11.7) 52

Chorda 1 dpf - 0 0 1 18 38

2 dpf - 0 2 11 41 36

3 dpf - 3 (5.0) 6 (10.0) 13 (21.7) 29 (48.3) 7 (11.7) 58

Tail 1 dpf - 0 2 27 59 55

2 dpf - 0 2 6 47 36

3 dpf - 3 (5.0) 28 (46.7) 57 (95.0) 39 (65.0) 9 (15.0) 136

Tail tip 1 dpf - 0 0 1 21 39

2 dpf - 0 2 4 27 30

3 dpf - 0 (0.0) 5 (8.3) 9 (15.0) 18 (30.0) 6 (10.0) 38

Scoliosis 1 dpf - 0 0 0 5 15

2 dpf - 0 0 0 9 23

3 dpf - 0 (0.0) 0 (0.0) 0 (0.0) 6 (10.0) 4 (6.7) 10

Yolk deformity 1 dpf - 0 0 0 0 0

2 dpf - 0 0 0 0 0

3 dpf - 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0

Growth retardation 1 dpf - 0 0 6 51 54

2 dpf - 0 1 4 22 31

3 dpf - 1 (1.7) 16 (26.7) 59 (98.3) 40 (66.7) 9 (15.0) 125

200 µM 400 µM 600 µM 800 µM 1000 µM ∑tTris-buffer, 0.5

% DMSO

Sum teratogenic

embryosb

- 10 15739 41 958

Chapter III

82

3.4.2 Exposure of zebrafish embryos to warfarin

A clear concentration-response relationship was detectable for the percentage of embryos with

lethal and teratogenic effects after exposure to warfarin (Tab. 3.3). However, it should be not-

ed that there was only a marginal difference between the incidence of teratogenic/affected

embryos between the 62.5 and 125 µM treatment groups (28.3 ± 12.6 % and 31.7 ± 14.4 %,

respectively; Tab. 3.3). There was no increase in the severity of teratogenic effects between

these two concentrations (Tab. 3.5).

At the three lowest concentrations (≤ 125 µM), teratogenic effects were usually detectable at

3 dpf, whereas at 500 and 1000 µM effects were seen from 2 dpf. Only at the highest concen-

tration could effects already be identified at 1 dpf (Tab. 3.5). Many embryos of the 1000 and

1500 µM treatment groups, which showed strong malformations within the first 2 days of

exposure, died between 2 and 3 dpf, reaching nearly 92 % embryo lethality at the highest con-

centration (Tab. 3.2).

Similar to coumarin, there were

also some embryos without

heart-beat and strong malfor-

mations at the two highest con-

centrations, but the majority of

embryos with lethal effects at 3

dpf were coagulated. Again, all

coagulated embryos showed

teratogenic effects, before they

died. It is also worth mention-

ing that some embryos treated

with 1500 µM warfarin showed

delayed development at 1 dpf

(Fig. 3.3) prior to severe mal-

formations at 2 dpf.

In contrast to coumarin, mal-

formation of the tail was identi-

fied as the only fingerprint end-

point of warfarin (Fig. 3.1C).

Fig. 3.1: Inverted microscope images of zebrafish embry-

os at 3 dpf. (A) Embryo of the control group. (B) Embryo

exposed to 200 µM coumarin with malformation of head

and tail. (C) Embryo exposed to 125 µM warfarin with

malformation of head, eye and tail; H: head; E: eye; S/O:

sacculi/otoliths; C: chorda; T: tail; TT: tail tip.

Chapter III

83

However, together with the other frequently found endpoints (malformation of head (32.7 %),

chorda (46.5 %), tail tip (44.7 %) and growth retardation (33.3 %), warfarin revealed an over-

all pattern of teratogenic effects similar to coumarin, which is quiet obvious at higher concen-

trations (Tab. 3.6, Fig. 3.4). Regarding the percentage of affected embryos in the experiments

with warfarin, differences from controls were statistically significant from 62.5 µM (Tab.

3.3). Calculation of LC50 and EC50 led to values of 988 and 194 µM, respectively, with a cor-

responding teratogenicity index of 5.1, which is about twice as high as that one for coumarin.

The corresponding concentration-response curves for warfarin are illustrated in Fig. 3.2C and

D). The EC20 value, which is used for comparison to human exposure data in the discussion,

was 52.4 µM.

Fig. 3.2: Concentration–response curves used for the calculations of the EC and LC values

(ToxRatPro®; probit analysis with linear maximum likelihood regression). (A) EC curve for

coumarin. (B) LC curve for coumarin. (C) EC curve for warfarin. (D) LC curve for warfarin;

95%-CV: 95%-confidence limits.

Chapter III

84

- : No effects; ∑t: sum of teratogenic embryos per endpoint out of all teratogenic embryos in

the three test groups at 72 hpf; one embryo can display multiple teratogenic malformations. a Number of embryos displaying this endpoint (%).

b Sum of the teratogenic embryos per group at 3 dpf of three experiments.

Tab. 3.5: Summary of teratogenic effects of warfarin in zebrafish (Danio rerio) embryos.

Malformation Time Control

Head 1 dpf - 0 1 1 2 2 5 19

2 dpf - 0 3 1 7 6 37 42

3 dpf - 2 (3.3)a

2 (3.3) 3 (5.0) 10 (16.7) 8 (13.3) 23 (38.3) 4 (6.7) 52

Eyes 1 dpf - 0 1 0 2 1 4 4

2 dpf - 0 1 0 1 0 2 7

3 dpf - 0 (0.0) 2 (3.3) 0 (0.0) 1 (1.7) 0 (0.0) 2 (3.3) 1 (1.7) 6

Sacculi/otoliths 2 dpf - 0 3 0 2 2 28 43

3 dpf - 0 (0.0) 2 (3.3) 1 (1.7) 3 (5.0) 2 (3.3) 18 (30.0) 3 (5.0) 29

Chorda 1 dpf - 0 1 0 2 3 8 14

2 dpf - 0 1 0 3 0 25 42

3 dpf - 2 (3.3) 6 (10.0) 3 (5.0) 15 (25.0) 14 (23.3) 29 (48.3) 5 (8.3) 74

Tail 1 dpf - 0 1 0 2 2 5 14

2 dpf - 0 1 0 6 4 31 43

3 dpf 3 (5.0) 6 (10.0) 8 (13.3) 18 (30.0)28 (46.7) 37 (61.7) 36 (60.0) 5 (8.3) 138

Tail tip 1 dpf - 0 1 0 2 2 4 3

2 dpf - 0 1 0 4 3 15 23

3 dpf 1 (1.7) 0 (0.0) 5 (8.3) 3 (5.0) 16 (26.7) 15 (25.0) 27 (45.0) 5 (8.3) 71

Scoliosis 1 dpf - 0 1 0 1 1 1 0

2 dpf - 0 1 0 0 0 1 0

3 dpf - 0 (0.0) 2 (3.3) 0 (0.0) 2 (3.3) 0 (0.0) 0 (0.0) 0 (0.0) 4

Yolk deformity 1 dpf - 0 0 0 0 0 0 0

2 dpf - 0 0 0 0 0 0 0

3 dpf - 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0

Growth retardation1 dpf - 0 1 0 2 1 4 5

2 dpf - 0 1 0 2 0 13 36

3 dpf - 1 (1.7) 2 (3.3) 1 (1.7) 11 (18.3) 9 (15.0) 24 (40.0) 5 (8.3) 53

5

31.25

µM

7

Tris-buffer,

0.5 % DMSO∑t

3 dpfSum teratogenic

embryos3 17 15919 45 3234

62.5 µM 125 µM 250 µM 500 µM 1000 µM1500

µM

Warfarin test groups

Chapter III

85

Fig. 3.3: Inverted micro-

scope images of zebrafish

embryos at 1 dpf. (A) Em-

bryo of the control group.

(B) Embryo with develop-

mental delay after exposure

to 1500 µM warfarin; H:

head; E: eye; S/O:

sacculi/otoliths; YS: yolk

sac; C: chorda; T: tail; TT:

tail tip.

Fig. 3.4: Inverted microscope images of zebrafish embryos at 3 dpf. (A) Embryo exposed to

800 µM coumarin with malformation of head, eye, sacculi/otoliths, chorda, tail, tailtip and

growth retardation. (B) Embryo exposed to 500 µM warfarin with malformation of head,

eye, sacculi/otoliths, chorda, tail, tail tip and growth retardation; H: head; E: eye; S/O:

sacculi/otoliths; C: chorda; T: tail; TT: tail tip.

Chapter III

86

3.5 Discussion

In contrast to all other well-accepted human teratogens, anticoagulant coumarin derivatives

have not produced malformations in any experimental animal species exposed prenatally

(Brent and Beckman, 1990; Nau, 1986; Schardein et al., 1985; Shepard et al., 1983). Howe

and Webster (1992) described a model in the rat, which managed to demonstrate most of the

features of human warfarin teratogenicity, but in these experiments the rats were exposed to

warfarin postnatally. In the experiments with prenatal exposure of warfarin in conjunction

with vitamin K supplementation Howe and Webster (1990) only detected haemorrhages and

some associated changes mainly in the brain, but no other external malformations. Even tha-

lidomide, which is famous for its strong species-specific teratogenicity, produced teratogenic

effects at least in some experimental animal species, such as rabbits and primates

(Schumacher et al., 1968; Szabo and Steelman, 1967; Teo et al., 2004). In this context, the

classification of warfarin as clearly teratogenic in zebrafish embryos is of outstanding impor-

tance. The teratogenic potential of coumarin in humans is unknown/unclear (Shepard et al.,

2004; Friedman and Polifka, 2000), but in the zebrafish embryo assay coumarin also was

shown to be clearly teratogenic. Both warfarin- and coumarin-exposed embryos showed simi-

lar effects, even if extent and frequency of the different endpoints varied (Tab. 3.6).


b Sum of embryos with teratogenic effects displaying this endpoint/sum of embryos

with teratogenic effects detected in all concentrations of the given test substance.


Tab. 3.6: Frequency of endpoints of coumarin and warfarin at 3 dpf.

Head 122a 77.7

b52 32.7

Eyes 21 13.4 6 3.8

Sacculi/otoliths 52 33.1 29 18.2

Chorda 58 36.9 74 46.5

Tail 136 86.6 138 86.8

Tail tip 38 29.9 71 44.7

Scoliosis 10 6.4 4 2.5

Yolk deformity 0 0.0 0 0.0

Growth retardation 125 79.6 53 33.3

Malformation

Warfarin

∑t [%]

Coumarin

∑t [%]

Chapter III

87

In humans, the first trimester, especially the 6th

to 9th

week of gestation, is supposed to be the

critical period of exposure for warfarin embryopathy (Hall et al. 1980), while the critical pe-

riod of exposure for warfarin syndrome seems to be mainly during the 2nd

and 3rd

trimester of

pregnancy (Hall et al., 1980; Holzgreve et al., 1976; Kaplan, 1985). Warfarin embryopathy

refers to bone defects, and the warfarin syndrome to central nervous system anomalies (Van

Driel et al. 2000). The exposure period in the zebrafish embryo assay mainly covers the criti-

cal period of exposure for warfarin embryopathy, but to some extent also for warfarin syn-

drome. The following features of human warfarin embryopathy/syndrome (Brent and Beck-

man, 1990; Briggs et al., 2008; Gärtner et al., 1993; Hetzel et al., 2006; Van Driel et al., 2000)

could also be identified in zebrafish embryos exposed to warfarin (Tab. 3.5 and 3.6): devel-

opmental delay, growth retardation, eye defects, scoliosis and ear defects. Nasal hypoplasia,

which is also characteristic of human foetuses exposed to warfarin, has no clear analogy in the

zebrafish embryo, but malformation of the head can perhaps be considered as a corresponding

effect. The chorda/spinal cord malformations observed in zebrafish embryos partly corre-

spond to the central nervous system anomalies known from human foetuses with warfarin

syndrome. The only endpoint frequently found in humans after warfarin exposure, which is

completely missing in zebrafish, is hypoplasia of the extremities.

The EC50 is traditionally used for calculation of the teratogenicity index, which is just a first

estimation for the teratogenic potential of a substance. For the comparison to human exposure

data the EC20 was used, because experience has shown that statistical significance is generally

reached around the 20 % effect level (provided that the variation between the replicates is not

too high). Furthermore it is known that many popular teratogens have a teratogenic risk be-

tween 15 and 30 % in humans (Banhidy et al., 2005).

Human therapeutic plasma concentrations of warfarin are usually reported to be between 1.2

and 17.8 µM (Sun et al., 2006; Vaz-da-Silva et al., 2010; Zacchigna et al., 2004). The EC20 of

warfarin in the zebrafish embryo assay was 52.4 µM, which is in a similar range of concentra-

tions found under therapeutic conditions (3 to 44-fold higher), showing a very good concor-

dance between zebrafish and human data. In the case of coumarin, the human therapeutic

plasma concentrations range between 3.4 nM and 5.1 µM (Lamiable et al., 1993; Ritschel et

al., 1977; Ritschel, 1984). The corresponding EC20 in the zebrafish embryo assay was 219

µM, which is 43 to 64400-fold the concentrations found under therapeutic conditions. The

ratio of the EC20 and high-dose coumarin therapy concentrations is very close to that of war-

farin low-dose therapy. However, the ratio of the EC20 and low-dose coumarin therapy con-

Chapter III

88

centrations is extremely high, suggesting that, there is a teratogenic potential but under normal

therapeutic conditions, teratogenicity is not an issue for coumarin. In summary, these data

confirm the teratogenic potential of warfarin, but also the questionable status of coumarin.

Finally it should be mentioned that extrapolation of aquatic concentrations of test substances

in the zebrafish assay to human therapeutic plasma concentration is a bit difficult, because not

only the pH value may influence the uptake of a test substance, but also other elements as e.g.

the protein level (Kramer et al., 2007). For this reason the concentration in the embryonic

tissue and the nominal concentration in the medium could be different and measurement of

the test substance in the embryo would be more accurate and will be considered in future

work.

Similar to the data presented here, coumarin was also reported to be teratogenic in the frog

embryo teratogenesis assay using Xenopus (FETAX), with an EC50 value of 547 µM. Addi-

tion of Aroclor-induced rat liver microsomes decreased the FETAX EC50 value 2.7 fold to

205 µM and further experiments with different xenobiotic metabolizing enzyme inhibitors

suggested CYP1A1/2-mediated activation of coumarin to a reactive epoxide metabolite was

critical (Fort et al., 1998). The metabolite, o-hydroxyphenylacetaldehyde, which arises after

CYP1A- and CYP2E-mediated epoxidation of coumarin and subsequent spontaneous break-

down is involved in liver carcinogenicity in the rat (Lake, 1999; Vassallo et al., 2004). How-

ever, since rat offspring did not show malformations after maternal exposure to coumarin

(Friedman and Polifka, 2000), o-hydroxyphenylacetaldehyde does not seem to reach the rat

embryos, perhaps because of its very short half-life (EFSA, 2008). In contrast to the hepato-

toxicity and carcinogenicity, the teratogenic effects of coumarin have not been investigated in

sufficient detail.

It has been assumed that teratogenicity induced by many coumarin derivatives is due to the

anticoagulant properties of this substance class (Pauli et al., 1987). In contrast to warfarin and

other coumarin anticoagulants, standard and low molecular weight heparin, which is used as

an anticoagulant during pregnancy, cannot pass the placental barrier and do not reach the em-

bryo (Andrew et al., 1985; Flessa et al., 1965; Harbison, 1978; Hirsh et al., 1970; Pieper et al.,

2008). Assuming that anticoagulant properties are directly connected with teratogenicity,

coumarin or coumarin derivates without anticoagulant properties should not to be teratogenic

or should produce other teratogenic effects than those known from anticoagulant exposure.

The zebrafish embryo data presented seem to contradict this hypothesis and might have mul-

tiple possible explanations. In this context two main questions have to be considered: (i) Are

Chapter III

89

the teratogenic effects of warfarin and coumarin in zebrafish embryos caused by the same

mechanism and (ii) Are the teratogenic effects of warfarin in zebrafish embryos and humans

caused by one common or two different mechanisms?

However, clarification of these aspects goes beyond the scope of this communication and

should be considered in separate experiments using adequate molecular techniques. As cou-

marin derivatives are used for different therapeutic indications, it is important to clarify if the

teratogenic activity is due to the coumarin moiety or due to the anticoagulant properties alone.

Chapter III

90

Chapter IV

91

Chapter IV

Cartilage and bone malformations in the head of zebrafish (Danio rerio)

embryos following exposure to disulfiram and acetic acid hydrazide

Chapter IV

92

4 Cartilage and bone malformations in the head of zebrafish

(Danio rerio) embryos following exposure to disulfiram and ace-

tic acid hydrazide

As presented in the previous chapters, zebrafish embryos are able to bioactivate

proteratogenic substances and show similar effects as other vertebrates, including mammals.

Therefore, in this chapter it has been shown that even very specific dose dependent

teratogenic effects can easily be assessed, namely cartilage and bone defects in the head of

zebrafish larvae. This chapter should also give a general overview about the (molecular) pro-

cesses how these specific cartilage malformations occur after exposure to dithiocarbamates

and hydrazides.

4.1 Abstract

In order to investigate teratogenic effects, especially on cartilage and bone formation,

zebrafish embryos were exposed for 144 h to the dithiocarbamate pesticide disulfiram (20 -

320 µg/L) and acetic acid hydrazide (0.375 - 12 g/L), a degradation product of isoniazid. Af-

ter fixation and full-mount staining, disulfiram could be shown to induce strong cartilage mal-

formations after exposure to ≥ 80 µg/L, whereas acetic acid hydrazide caused cartilage altera-

tions only from 1.5 g/L. Undulating notochords occurred after exposure to disulfiram even at

the lowest test concentration of 20 µg/L, whereas at the two lowest concentrations of acetic

acid hydrazide (0.375 and 0.75 g/L) mainly fractures of the notochord were observed. Con-

centrations of acetic acid hydrazide ≥ 1.5 g/L resulted in undulated notochords similar to

disulfiram. Cartilages and ossifications of the cranium, including the cleithrum, were individ-

ually analyzed assessing the severity of malformation and the degree of ossification in a semi-

quantitative approach. Cartilages of the neurocranium such as the ethmoid plate proved to be

more stable than cartilages of the pharyngeal skeleton such as Meckel’s cartilage. Hence, ossi-

fication proved significantly more susceptible than cartilage. The alterations induced in the

notochord as well as in the cranium might well be of ecological relevance, since notochord

malformation is likely to result in impaired swimming and cranial malformation might com-

promise regular food uptake.

Chapter IV

93

4.2 Introduction

The cranium of adult zebrafish (Danio rerio) consists of 74 cranial bones, of which the devel-

opment requires at least 70 days for completion (Cubbage and Mabee 1996). There are two

modes of bone formation in most vertebrates: (1) dermal ossification with bones developing

directly within connective tissues and (2) enchondral/perichondral ossification with bones

preformed as cartilages. Visible dermal bones in the head of 144 h old zebrafish larvae (Tab.

1) are the parasphenoid, the opercles, the cleithrum and more or less visible the occipitals,

branchiostegal rays, maxilla and entopterygoids. The fifth ceratobranchial (CB5) is the only

bony element, which is still only of cartilaginous nature at this developmental stage. Only

occasionally, additional cartilaginous elements have already just initiated transformation into

bony structures after 144 hours post fertilization (hpf), yet CB5 is the only cartilage element

ossified to a significant extent. The anterior-most ossified front of the notochordal sheath is

defined as a perichordal bone. The notochord possesses mechanical and signaling functions,

essential for the whole development (Pagnon-Minot et al. 2008).

Dithiocarbamates such as disulfiram are well-known teratogens causing wave-like defor-

mation of the notochord and cartilage malformation in fish embryos (Suzuki et al., 2001;

Tilton et al., 2006; Van Boxtel et al., 2010a) and alter gene expression profiles of several

types of collagen, e.g. collagen type IIα1 (Haendel et al. 2004, Tilton et al. 2006), a compo-

nent of the surrounding sheath of the developing notochord (van Boxtel et al. 2010 a). Several

studies, e.g., on mice have shown that thiram, another dithiocarbamate, induces skeletal mal-

formations, as cleft palate, micrognathy, wavy ribs and distorted, wavy bones (Matthiaschk,

1973, Fishbein, 1976) Since perturbations of cartilage and bone formations represent severe

teratogenic effects in the early development of zebrafish (Danio rerio), an emerging major

model for vertebrate teratogenesis (Berry et al. 2007, Brannen et al. 2010, Busch et al. 2011,

Carney et al. 2006, McGraph and Li 2008, Nelson et al. 2010, Selderslaghs et al. 2009, 2012,

Teraoka et al. 2003, Van den Bulk et al. 2011, Weigt et al. 2011, Yang et al. 2009), the pre-

sent study was designed to elucidate the morphological basis for skeleton malformations. As

model compounds, disulfiram and acetic acid hydrazide were selected. Although different in

chemical class, molecular weight and log KOW, hydrazides, especially acetic acid hydrazide –

a degradation product of isoniazid – generate similar morphological effects.

Especially in the cranium of a few days old larvae, cartilage is predominant, formed through

the interaction of cranial neural crest cells and the surrounding endodermal and epithelial tis-

Chapter IV

94

sues (Piotrowski et al. 1996, Schilling et al. 1996). Almost 60 % of all cranial bones are pre-

formed as cartilaginous models. Since the mid-1990s, numerous studies have been published

studying the effects of specific zebrafish mutants to the cartilage structure in the cranium of

zebrafish larvae (Nissen et al. 2006, Schilling et al. 1996, Piotrowski et al. 1996, Walker et al.

2006, 2007, Yelick and Connolly, 2010). Yet, data on effects of chemicals as potential carti-

lage and bone teratogens are restricted to a handful. Retinoic acid, e.g., is known to act as a

cartilage teratogen (Suzuki et al. 2000, Vandersea et al. 1998, Vieux-Rochas et al. 2007); the

same holds for 17ß-estradiol (Fushimi et al. 2009), dexamethasone and hydrocortisone (glu-

cocorticoids; Hillegrass et al. 2008), pyrethroids (De Micco et al. 2010), 2,2-dipyridyl (Suzuki

et al. 2000) and TCDD (Hill et al. 2004, Teraoka et al. 2002, Xiong et al. 2008).

Dithiocarbamates and some of their degradation products are strong cartilage and notochord

teratogens (Haendel et al. 2004, Suzuki et al. 2002). However, in mammals, high doses of

dithiocarbamates are needed to induce cartilage and bone malformations. Although their tera-

togenicity to vertebrates is well studied (Roll 1971, Simsa et al. 2007, Tilton et al. 2006, van

Boxtel et al. 2010b), the underlying molecular mechanisms are only poorly understood.

Disulfiram (tetraethylthiuram disulfide) is a dithiocarbamate compound produced since 1881

and has widely been used in industries, agriculture and medicine (Haendel et al. 2004,

Eneanya et al. 1981). Overall, a total 25,000 - 35,000 t per year of dithiocarbamates is con-

sumed worldwide (WHO 2008). Dithiocarbamates are chelators, also used for the treatment of

nickel and copper poisoning, in the experimental treatment of AIDS and have both pro- and

antioxidant characteristics (Burkitt et al. 1998, Eneanya et al. 1981, Lang et al. 1988).

Disulfiram is applied for the vulcanization of rubber, as a fungicide, acaricide and vermicide

and is still used for the treatment of chronic alcoholism (WHO 2008).

Both in humans and animals, acetic acid hydrazide is a metabolite of the antibiotic isoniazid

(Fort and Bantle 1990). The major pathway in human is the acetylation to acetyl isoniazid

followed by a hydrolysis to isonicotinic acid and acetic acid hydrazide (Ryan et al. 1985).

Isoniazid is widely used as an anti-tuberculosis drug inhibiting the ß-ketoacyl ACP synthase

and enayl ACP reductase (Aditya et al. 2010, Ryan et al. 1985), although it has been suggest-

ed to be teratogenic in vivo (Fort and Bantle 1990). Acetic acid hydrazide and isoniazid cause

osteolathyrogenic malformations in Xenopus by disrupting the polymerization of connective

tissue required for the development of the axial skeleton (Fort and Bantle 1990, Schultz and

Ranney 1988). Schultz and Ranney (1988) already showed undulated notochords of Xenopus

embryos as a result of exposure to acetic acid hydrazide.

Chapter IV

95


4.3.1 Fish maintenance and egg production

Zebrafish maintenance and egg production has repeatedly been described in detail (Kimmel et

al. 1988, 1995; Nagel 2002; Spence et al. 2005; Wixon 2000) and has recently been updated

for the zebrafish embryo toxicity test (Lammer et al. 2009).


Test chemicals were purchased from Sigma Aldrich (Deisenhofen, Germany). Disulfiram

(CAS: 97-77-8; purity > 97 %) and acetic acid hydrazide (CAS: 1068-57-1; purity > 99 %)

were stored at 8 °C. Dimethylsulfoxid (DMSO, 99 % purity; Grüssing; Filsum, Germany) was

used as a solvent for disulfiram at 0.01 % in each test concentration. For disulfiram, 0.02,

0.08, 0.32, 1.28 and 5.12 mg/L, and for acetic acid hydrazide, 0.375, 0.75, 1.5, 3, 6, 12 and 24

g/L were selected as test concentrations.

4.3.3 Zebrafish Embryo Test (ZFET)

For the ZFET, 24-well plates from Renner (Dannstadt, Germany) pre-saturated with the re-

spective test solutions for at least 24 h were used. For a detailed description of the ZFET pro-

cedure, see Lammer et al. (2009). In brief, not later than 1 hour post-fertilization, zebrafish

eggs were randomly transferred into glass vessels with the test solutions. Following a viability

control, fertilized eggs were individually transferred into the test wells (total volume 2 mL

and one egg per well) using pipette tips with a widened tip opening. For each test concentra-

tion, 20 eggs plus an internal negative control were incubated in a 24-well plate. In addition, a

full 24-well plate was used for an external negative control; in case a solvent was used (disul-

firam), another plate with 24 external solvent controls (0.01 % DMSO) was run. 3,4-Di-

chloroaniline (3.7 mg/L) was tested as a positive control in another separate 24-well plate

(Lammer et al. 2009). All embryos were inspected daily and both lethal and sublethal effects

were recorded. The terminology for zebrafish developmental stages was defined as follows: 0

hpf – egg, 24 to 48 hpf – embryo, 72 to 120 hpf – eleutheroembryo, 144 hpf – larvae.

Chapter IV

96

For incubation and dilution of test solutions, artificial water as specified in ISO 7346-1 and

7346-2 (ISO 1996; 294.0 mg/L CaCl2 × 2 H2O; 123.3 mg/L MgSO4 × 7 H2O; 63.0 mg/L

NaHCO3; 5.5 mg/L KCl) was used. The temperature was adjusted to 26.0 ± 1 °C in a KB115

incubator (Binder, Tuttlingen, Germany). In all test solutions, pH was adjusted between

7.6 and 7.9 using HCl. The light/dark cycle was 14:10 h. Plates were sealed with self-

adhesive, fully oxygen-permeable Sealing Tape SH (Nunc, Wiesbaden). Determination of

lethal and sublethal endpoints was performed with a CKX41 inverted microscope (Olympus,

Hamburg, Germany) equipped with a digital Olympus C5069 camera and the digitizing soft-

ware Analysis 5.0 (Soft Imaging Systems, Münster, Germany).

4.3.4 Whole-mount Alizarin red and Alcian blue skeletal staining

Alizarin red S (1,2-dihydroxyanthraquinone; Sigma Aldrich) and Alcian blue 8 GS (copper

phthalocyanine; Serva, Heidelberg) dyes were used for staining bony and cartilaginous struc-

tures, respectively. Larvae of 144 hpf age were killed with an overdose of benzocaine (ethyl-

4-aminobenzoate; Sigma Aldrich) and stained as described in the protocol by Walker and

Kimmel (2007) with slight modifications. Briefly, larvae were fixed and rocked for 2 h in 4 %

paraformaldehyde, stained using acid-free double stain solution with 120 mM MgCl2 over-

night (as replacement for acid offering better distinction of all cartilages and bones), bleached

using 3 % H202 and 1 % KOH for 25 minutes, and rocked with 20 % glycerol and 0.25 %

KOH overnight; the latter step was repeated with 50 % glycerol and 0.25 % KOH overnight,

and specimens were stored in 50 % glycerol and 0.1 % KOH at 4°C.

4.3.5 Cartilage and bone scoring

After staining, all larvae were examined using a Stemi 2000-C (Zeiss, Göttingen, Germany)

or Olympus CKX41 microscopes equipped with digital imaging hardware (Power Shot G7

(Canon, Krefeld, Germany) or C-5069 Wide Zoom (Olympus, Hamburg, Germany)) as well

as the imaging software (Analysis 5.0). Larval and adult zebrafish cartilage and bone devel-

opment has been described in detail in several studies (Schilling et al. 1996, Piotrowski et al.

1996 and especially Cubbage and Mabee 1996). Cartilaginous and bony elements analyzed

are listed in Table 4.1 and illustrated in Fig. 4.1. Quantification of cartilage and bone malfor-

mations was assessed in a semi-quantitative approach: 0 – normally developed; 1 – minor

Chapter IV

97

malformations; 2 – strong malformations; 3 – severe malformations or no longer detectable

skeletal elements. However, since in a given larva single cartilages and bones do not show

identical grades of malformation severity, it should be noted that this classification is not gen-

eral for every cartilage or bone in the head of the respective larva. For instance, Meckel’s car-

tilage is frequently strongly malformed, whereas all other elements appear normal or only

show slight malformations. Fig. 4.2 illustrates examples of 144 hpf control and treated (mal-

formed) zebrafish larvae in ventral and lateral views.

Only larvae free of any symptoms of lethality (DIN 2001) were used for cartilage and bone

staining. For this end, test concentrations were selected in a way that at least the two lowest

concentrations showed mortality levels ≤ 20 %. At the third concentration, at least 10 out of

20 embryos (≥ 50 %) had to survive.

4.3.6 Statistics and data presentation

Graphs and correlations were performed with SigmaPlot 11.0 (Statsoft-Jandel Scientific,

Erkrath, Germany) or GraphPad Prism 4 (Statcon, Witzenhausen, Germany). LC50 values

were calculated as probit analysis using ToxRat Professional 2.10.3.1 (ToxRat Solutions,

Alsdorf, Germany) with linear maximum likelihood regression. For characterization of the

teratogenic potential, the teratogenic index (TI) was assessed, which is defined as the quotient

of LC50 and EC50.

Chapter IV

98

Fig. 4.1: Drawings of the zebrafish (Danio rerio) neurocranium (A), lateral view of all head

cartilages (B) as well as the pharyngeal skeleton (C) at 144 hpf, modified from Kimmel et al.

(2001). D - F show photomicrographs of stained zebrafish larvae focused to the corresponding

cartilage and bone elements. Cartilages are stained blue (alcian blue) and bones red (alizarin

red). Abbreviations used for cartilages: abc/pbc − anterior and posterior basicranial commis-

sures summarized as basicranial commissure (bc); bb − basibranchial; bh − basihyal; bp −

basal plate; cb 1 - 5 ceratobranchial 1 - 5; ch − ceratohyal; e − ethmoid plate; hb −

hypobranchial; hs − hyosymplectic; ih − interhyal; m − Meckel’s cartilage; oa − occipital

arch; pq − palatoquadrate; t − trabeculae crania. Abbreviations for bones/ossifications: BR −

branchiostegal rays; C − cleithrum; CB5 − ceratobranchial 5; E − entopterygoid; N − noto-

chord; OP − opercle; OC − occipitals; PS − parasphenoid; V − vertebrae; T- teeth. The abbre-

viation for the otoliths is O.

cb 1 - 4

CB5

PS

T

t

e

ih

hb3hb2hb1 bb

bh

hs

ch

pq

m

pbcoa

bp

OC

N

abc

C

OCP

BR

OP

OP CB5 V

A

F

E

D

C

B

E O

Chapter IV

99

Tab. 4.1: List of cartilages and bones detectable in the anterior body of 144 h old zebrafish

(Danio rerio) embryos. Cartilages were classified into two groups, the pharyngeal skeleton

and the neurocranium, whereas for the ossifications their type is listed.

Pharyngeal skeleton Cartilages Abbrev.

Cart

ilages

Pharyngeal arch 1 (Mandibular arch) Meckel´s Cartilage m

Palatoquadrate pq

Pharyngeal arch 2 (Hyoid arch) Hyosymplectic hs

Interhyal ih

Ceratohyal ch

Basihyal bh

Pharyngeal arch 3 (branchial arch 1) Ceratobranchial 1 cb1





Neurocranium Cartilages Abbrev.

Cart

ilages

Anterior Ethmoid plate e

Trabeculae cranii t

Posterior Anterior basicranial commissure/

Posterior basicranial commissure bc

Basal plate bp

Occipital arch oa

Type of ossification Bones Abbrev.

Bon

es

Perichordal Notochord (Front) N

Dermal Parasphenoid PS

Dermal Branchiostegal rays BR

Dermal Opercle OC

Dermal Cleithrum C

Cartilage CB 5 Ossification CB5

Dermal Occipital OCP

Chapter IV

100

Fig. 4.2: Photomicrographs of zebrafish larvae (Danio rerio) head: A - D ventral and E - H

lateral view. A and E show 144 h old larvae from negative controls with normal development

(severity: 0); B and F illustrate larvae with slight malformations (severity: 1); C and G show

examples of strong malformations (severity: 2); and D and H depict very strong malfor-

mations or even lack of detectable skeletal elements (severity: 3). In A and E, all cartilages

and bones appear normally shaped and developed. In B and F, Meckel’s cartilage (m) is

smaller and misshapen. The position of the ceratohyals (ch) and the angle between the two

ceratohyals is modified. The arrow (see also in F) indicates a more ossified notochord front in

B and a less strongly ossified notochord front in F. A more ossified notochord tip is often ob-

served after disulfiram exposure. The embryo in C and F show severe malformation of the

entire pharyngeal skeleton (Meckel’s cartilage and ceratohyals) and a lack of ossification of

the notochord tip (arrow). The embryos shown in D and G are strongly malformed: in D, the

pharyngeal skeleton is completely absent, and the neurocranium is also strongly deformed, but

still detectable (e − ethmoid plate; t − compressed trabeculae cranii). In H, parts of the phar-

yngeal skeleton are visible, but all elements were categorized severity 3.

Chapter IV

101

4.4 Results

4.4.1 Determination of LC50 values and teratogenic index (TI)

Three static and, for reasons of comparison, one semi-static test (daily renewal) were con-

ducted with disulfiram; for acetic acid hydrazide, two semi-static tests (daily renewal) were

run for subsequent calculation of LC50 values. Mean LC50 values (± standard deviation) for

disulfiram for 48 hpf were identical for static (0.48 ± 0.05 mg/L) and semi-static exposures

(0.44 mg/L). The same holds for 96 hpf (0.45 ± 0.08 mg/L versus 0.41 mg/L) and 144 hpf

(0.36 ± 0.13 mg/L versus 0.26 mg/L). Since full mortality could not be achieved at the high-

est test concentrations, reliable LC50 values for acetic acid hydrazide for 48 and 96 hpf could

not be calculated. After 144 hpf, an LC50 of 12.2 ± 1.2 g/L was calculated. To assess concen-

tration-dependent cartilage and bone malformations, 144 h old larvae exposed to 20, 80 and

320 µg/L for disulfiram and to 0.375, 0.75, 1.5, 3, 6 and 12 g/L for acetic acid hydrazide were

stained and analyzed. An exact teratogenic index (TI) could not be calculated, since even at

the lowest test concentrations of both test chemicals more than 50 % of all embryos already

showed teratogenic effects of the notochord, mainly breaks after exposure to acetic acid

hydrazide (0.37 g/L) and faint undulations after exposure to disulfiram (20 µg/L). Therefore,

the TI was estimated in dividing the LC50 through the lowest test concentration. The TI for

disulfiram is > 16.5 and the TI for acetic acid hydrazide is even higher with a coefficient >

32.5.

4.4.2 Cartilage and bone malformations following exposure with disulfiram

(Fig. 4.3)

In all negative and solvent controls, cartilage and bone elements did not show any effect (se-

verity 0); as an exception, the branchiostegal rays showed minor variability reaching an ossi-

fication grade of 0.57.

At a concentration of 20 µg/L, disulfiram (Figs. 4.3 A - C) did not cause any malformations in

cartilages of the neurocranium; only little effects on the pharyngeal skeleton could be record-

ed. Strong alterations in bones were seen in both the static and the semi-static scenarios. Mal-

formations of the pharyngeal skeleton never exceeded 0.89, whereas the mean values of the

parasphenoid, opercle, cleithrum and the ossification of the CB5 ranged between grades 1 and

2. The branchiostegal rays were not observable.

Chapter IV

102

Fig. 4.3: Semi-quantitative evaluation of zebrafish (Danio rerio) skeletal malformations after

exposure to 20 (A - C), 80 (D - F) and 320 µg/L (G - I) disulfiram for 144 h. A, D and G: car-

tilages of the pharyngeal skeleton; B, E and H: cartilages of the neurocranium and C, F and I:

ossifications. Open columns represent mean values of three independent replicate runs under

static test conditions, whereas solid columns indicate results from a single semi-static test with

daily renewal of the test solutions. Grades of malformation are assessed semi-quantitatively:

degree 0 − no malformation; grade 1 – slight malformations; grade 2 − strong malformations;

grade 3 − very strong malformations or structures no longer detectable. For abbreviations, see

Fig. 4.1.

At a concentration of 80 µg/L, disulfiram (Figs. 4.3 D - F) induced stronger effects except for

the parasphenoid and the opercles. Disulfiram concentrations of 320 µg/L (Figs. 4.3 G - H)

caused strong malformations on the pharyngeal skeleton, with most elements strongly mal-

formed or no longer discernible. The severity values were around 2, and in the semi-static test

all ceratobranchials disappeared. Within the neurocranium, the ethmoid plate showed the low-

est malformation, ranging from severity 1 to 1.33 in the static tests to 1.6 in the semi-static

test, whereas the remaining cartilages were about severity grade 2. With 320 µg/L disulfiram,

all bones reached a malformation index close to 3; only in the static tests the cleithrum was

rated about 2.5.

Chapter IV

103

At the two lowest disulfiram concentrations (20 and 80 µg/L), about 75 % of the individuals

showed stronger ossification of the notochordal sheath (compared to the control). At 320 µg/L

disulfiram, however, about 85 % of the individuals showed a lesser ossification of the

notochordal sheath than in controls or even no ossification at all.

4.4.3 Cartilage and bone malformations following exposure with acetic acid

hydrazide (Fig. 4.4)

In the negative controls, the vertebrae, opercles, maxillas and entopterygoids showed ossifica-

tions to a variable extent (data only shown for the opercles). Even after 144 hpf, ossification

was not complete in the negative controls.

Concentrations of 0.375 and 0.75 g/L acetic acid hydrazide caused no malformations in carti-

lage formation, but differences in bone ossification were observed (Fig. 4.4). The

parasphenoid showed normal ossification in the negative controls, but with 0.375 g/L acetic

acid hydrazide the deviation in ossification increased to a severity grade of about 1;

branchiostegals were even more affected with an increase in malformation severity to about

2.5.

All cartilages showed a strong increase in the severity of malformations between 0.75 and

1.5 g/L acetic acid hydrazide. Malformations of cartilage elements from embryos exposed to

≥ 1.5 g/L acetic acid hydrazide were significant in both replicate runs (no normal distribution,

Mann-Whitney Rank-Sum-Test: p < 0.05). In contrast, bony elements showed a continuous

decrease in ossification. A concentration of 0.375 g/L, acetic acid hydrazide induced a signifi-

cantly lower ossification in both replicates (p < 0.05) for the parasphenoid, the branchiostegal

rays and the opercles. At the notochord front, the cleithrum and the ossifications of the CB5,

concentrations of 0.375 g/L acetic acid hydrazide did not cause significantly lower ossifica-

tion rates; exposure to ≥ 0.75 g/L acetic acid hydrazide resulted in a significant reduction of

ossified elements (p ≤ 0.05). At 12 g/L, acetic acid hydrazide, cartilages of the pharyngeal

skeleton were absent or formed at best diffuse cartilage clusters. The same applies to the

neurocranium, which showed fewer cartilaginous malformations than cartilages of the phar-

yngeal skeleton. At ≤ 6 g/L acetic acid hydrazide, the basal plate, the occipital arches and the

basicranial commissures showed only minor malformation (severity value about 1). The

trabeculae cranii displayed the strongest malformation among cartilages of the neurocranium.

Chapter IV

104

Fig. 4.4: Semi-quantitative evaluation of zebrafish (Danio rerio) skeletal malformations fol-

lowing exposure to 0.375 - 12 g/L acetic acid hydrazide for 144 h. A and B show malfor-

mation severity values of the pharyngeal skeleton, C quantifies malformations of the

neurocranium and D rates the degree of ossification of selected bones. Columns represent raw

data of two independent replicates. Note the strong increase of malformation severity between

0.75 and 1.5 g/L characteristic for all cartilages. For abbreviations, see Fig. 4.1.

At 12 g/L acetic acid hydrazide, bone structures could not be identified, and at 6 g/L acetic

acid hydrazide only exceptional cases of very low ossification of the cleithrum could be de-

tected.

Only few larvae showed strong ossification of the notochordal sheath. The same is true for

three larvae exposed to 0.375 g/L acetic acid hydrazide and one individual exposed to

0.75 g/L acetic acid hydrazide (these individuals were excluded from Fig. 4.4).

Chapter IV

105

Fig. 4.5: Measurement of the head length of zebrafish (Danio rerio) larvae exposed to 20, 80

and 320 µg/L disulfiram. Numbers within columns indicate total numbers of individuals

measured, the negative control (NC) and the solvent control (SC, 0.01 % DMSO). Whereas no

difference was evident between negative and solvent controls, all disulfiram concentrations

induced a significant reduction in head length (p < 0.05). Additionally, non-linear regression

was performed and the controls were set zero (not shown in the diagram). The equation model

f = a*exp (-b*x) +c*exp (-d*x) was used while the single parameters were calculated as: a =

168.94; b = 56.40; c = 641.22; d = 0.24. The equation fits the head length data adequately with

r² = 0.91.

Concentration [µg/]

NC SC 20 80 320

Siz

e [

µm

]

0

550

600

650

700

750

800

850

*

**

10

1720

18

19

4.4.4 Head length of larvae exposed to disulfiram (Fig. 4.5)

In addition to the semiquantitative assessment of cartilage and bone malformations, the head

length (front of the head to the beginning of the pectoral fins) of larvae exposed to disulfiram

in semi-static tests was measured (Fig. 4.5). The larvae of the solvent control (SC) did not

differ significantly from the negative controls (NC), whereas the head length was significantly

reduced after exposure to all disulfiram concentrations (p < 0.05).

4.4.5 Head length of larvae exposed to acetic acid hydrazide (Fig. 4.6)

Both 0.375 g/L and 0.75 g/L acetic acid hydrazide did not modify zebrafish head length, alt-

hough there was already a trend to reduction at 0.75 g/L, which only became significant from

1.5 g/L acetic acid hydrazide (Kruskal-Wallis One-Way ANOVA, multiple comparisons ver-

sus controls; Dunn's method: p < 0.05). The highest acetic acid hydrazide concentrations re-

sulted in a reduction of the mean head length from 800 to approx. 515 µm.

Chapter IV

106

4.4.6 Notochord and otolith malformations

Both disulfiram and acetic acid hydrazide caused wave-like distortions of the notochord (Figs.

4.7 A, B). All disulfiram concentrations ≥ 20 µg/L resulted in notochord undulations, whereas

zebrafish larvae exposed 0.375 g/L acetic acid hydrazide only showed fractions of the noto-

chord (Fig. 4.7 B); from 0.75 g/L, undulations could also be seen for acetic acid hydrazide

(not shown). Malformations of the otoliths (Fig. 4.7 C) were common for disulfiram exposure,

but only infrequent for acetic acid hydrazide exposure. Furthermore, disulfiram concentra-

tions ≥ 20 µg/L generated multiple otoliths (Fig. 4.7 C1) with a strong tendency to fuse (Figs.

4.7 C2 - C4); control larvae normally show two otoliths per sacculus (cf. Figs. 4.1 D - F).

Fig. 4.6: Measurement of the head length of zebrafish (Danio rerio) larvae exposed to 0.375,

0.75, 1.5, 3, 6 and 12 g/L acetic acid hydrazide (n = 2: A and B) as well internal (iNC) and

external negative controls (eNC). Numbers within columns indicate total numbers of individu-

als measured; * p < 0.05 for comparison with external negative controls. Additionally, non-

linear regression was performed and the controls were set zero (not shown in the diagram).

The equation model f = a*exp (-b*x) +c*exp (-d*x) was used while the single parameters

were calculated as: a = 653.99; b = 0.02; c = 122.72; d = 0.71 for Fig. 4.6A and a = 221.16; b

= 0.35; c = 591.04; d = 0.01 for Fig. 4.6B. The equation fits the head length data adequately

with r² = 0.82 (Fig. 4.6A) and r² = 0.92 (Fig. 4.6B).

Chapter IV

107

Fig. 4.7: Notochord and otolith mal-

formations in zebrafish (Danio rerio)

caused by exposure to acetic acid

hydrazide (A, B) and disulfiram (C).

Undulated notochords (A, 3 g/L) and

fractures of the notochords (B, 1.5 g/L)

are both common features of acetic

hydrizide exposure. Malformations of

the otoliths (multiplication and gradual

merging; C) are common effects for

disulfiram at any test concentrations

investigated.

4.5 Discussion

4.5.1 Toxicity, teratology as well as ecological relevance of observations

Literature LC50 data for the toxicity of disulfiram to zebrafish embryos are between 0.4 and

4 mg/L (Tilton et al. 2006). The LC50 values revealed in the present study are within this

range (the 48 and 96 hpf values) or a bit lower (at 144 hpf). For acetic acid hydrazide, toxico-

logical data for fish or fish embryos are not available. Both substances show a clear concen-

tration-dependent potential for malformation of cranial cartilages, bones and the notochord;

overall, they both cause a concentration-dependant reduction of larval head length. At

0.375 g/L acetic acid hydrazide, fractures of the notochord were observed (Fig. 4.7 B),

stronger developed with increasing concentrations and at 1.5 g/L accompanied with undulated

notochords with subsequent accumulations of notochord materials around the break

(Fig. 4.7 B). First cartilage malformations were observed at 1.5 g/L, whereas notochord dam-

age already occurred at 0.375 g/L. Thus, the overall TIs (including notochord and carti-

lage/bone malformations) of both chemicals are considerable (> 16.6 for disulfiram and >

32.5 for acetic acid hydrazide); hence, the teratogenic potential is very high. The mean EC50

for cartilage malformations is, e.g., for acetic acid hydrazide 1 g/L (with EC50s for all single

cartilages ranging from 0.9 to 1.1 g/L). Therefore, the TI for cartilages could be calculated at

Chapter IV

108

12.2. The embryos are strongly malformed long before lethal symptoms occur at much higher

concentrations. These findings correlate well with those by van Boxtel et al. (2010 b), who

tested approx. 30 µg/L (100 nM) disulfiram and observed misshaped cartilage elements as

Meckel’s cartilage, the ceratohyal, the ceratobranchial arches, protruding mouths and stunted

neurocrania. At concentrations of 20 µg/L disulfiram, no malformed cartilages were observed,

but indeed a significant reduction of larval head length and less ossified bones were detecta-

ble. Strongly malformed cartilages occurred first at the next higher concentration of 80 µg/L

disulfiram. Based on results of the present study and that of van Boxtel et al. (2010 b), the

change from normal development to malformed skeletal elements probably can be identified

between 20 and 30 µg/L. At 20 µg/L disulfiram, no cartilage malformations were observed,

but the notochord was strongly undulated.

Particularly with regard to the ecological relevance of disulfiram and acetic acid hydrazide,

the cartilage and bone damages appear of only minor importance, because strong notochord

malformations, which impair/prevent normal swimming behavior, occur already at lower con-

centrations, where effects on cartilage and bone formation are still absent. As normal swim-

ming behavior is essential for foraging and flight behavior, effects on the head cartilage or

bone, which would affect, e.g., opening of the mouth, are only of minor importance. With

respect ecological relevance, it should also be mentioned that disulfiram is supposed to de-

grade very fast under normal environmental or laboratory conditions due to its very short half-

life; the European Commission gives a half-life of 2 days at pH 7 and 4 to 7 h at pH 9 for this

drug (IUCLID Year 2000 Edition), whereas EPI Suite (US EPA, EPI Web 4.0) estimates a

half life of 20 minutes. Nevertheless, in the present study, the static as well as the semi-static

disulfiram exposure gave very similar results concerning toxicity and cartilage/bone terato-

genicity. Literature data indicate that at least some degradation products (degradation in the

medium vs. biotransformation) of dithiocarbamates, e.g. methyl isothiocyanate, have a strong

teratogenic potential at similar concentrations (Deguigne et al. 2011, Haendel et al. 2004,

Johnson et al. 1996). The results of Haendel et al. (2004) and Johnson et al., 1996 are im-

portant, because the authors describe the possible degradation processes and intermediates of

dithiocarbamates. E.g., methyl isothiocyanate, a degradation product, causes similar malfor-

mations at similar concentrations. Half-life data for acetic acid hydrazide are not available,

but the Chembase MSDS (version 0.1, www.chembase.com) states that it may biodegrade

within water to a moderate extent and Schultz and Ranney (1988) found no significant abiotic

loss of acetic acid hydrazide over 96 h. Absorption measurements in the range between 210

Chapter IV

109

and 300 nm with a maximum peak at 230 nm revealed no significant changes over 24 h of

incubation at 20 °C (details not shown). Nonetheless, in our semi-static experiments, very

high concentrations of this isoniazid metabolite, which are unlikely to occur in nature, have to

be used to induce notochord or cartilage malformations. It is known that dithiocarbamates

cause effects on the notochord (Haendel et al. 2004, Suzuki et al. 2001, Teraoka et al. 2006,

Tilton et al. 2006), but for acetic acid hydrazide this has been unknown for fish so far.

4.5.2 Osteolathyrism - a potential underlying mechanism for skeletal deformities

In zebrafish, bones appear as the most susceptible structures in the head showing reduced os-

sification even at the lowest concentrations. It is not surprising that these cartilage bones are

the first skeletal elements showing effects. The ossification of the respective cartilages is a

continuous process during development and if the cartilages themselves show retardations in

size and malformations in shape, ossification is also likely to be disturbed. In contrast, the

neurocranium and especially the ethmoid plate proved to be much more stable elements, even

at highest test concentrations (see also van Boxtel 2010 b), whereas all other cartilage ele-

ments were lacking. Stronger ossification of the notochord front at lower concentrations was

mainly observed after disulfiram and only in a few cases after acetic acid hydrazide treatment.

The reduction of bone matrix at the highest concentrations was observed after treatment with

both substances. Despite completely different chemical structures, molecular masses, log KOW

values and toxicities, the morphological effects of disulfiram and acetic acid hydrazide are

almost identical. Schultz and Ranney (1988) found similar effects in Xenopus laevis larvae

notochords following exposure to several acid hydrazides. Disulfiram already induced carti-

lage malformations at 80 µg/L, whereas after exposure to acetic acid hydrazide microscopi-

cally discernible cartilage malformations appeared only at concentrations ≥ 1.5 g/L.

In general, disulfiram and dithiocarbamates are well investigated. Suzuki et al. (2000) ana-

lyzed the effects of disulfiram on cartilage formation in Japanese flounder embryos

(Paralichthys olivaceus), and results were very similar to those found in zebrafish in the pre-

sent study. Marsh-Armstrong et al. (1995) observed that disulfiram inhibits the conversion

from retinal to retinoic acid by retinal dehydrogenase 2 (Aldh1a2) in zebrafish. Since Aldh1a2

is needed for proper trunk formation, it is assumed that the malformations detected in the pre-

sent study are at least partly caused by reduction of retinoic acid levels. However, retinoic

acid is also a very potent cartilage malforming agent if given in excess exogenously. Only a

Chapter IV

110

very well defined concentration of retinoic acid is compatible with normal development (Col-

lins and Mao 1999).

Hox and shh genes possess response elements for retinoic acid (Suzuki et al. 2000) and are

highly regulated by this vitamin A metabolite. Pagnon-Minot et al. (2008) showed that Shh

signaling interacts with collagen XV, which is essential for notochord differentiation and

muscle development in the zebrafish embryo. Tilton et al. (2006) proved that

dithiocarbamates alter the expression of collagen 2a1, an important component of the

notochordal sheath (Stemple 2005) and the pharyngeal cartilages (Yan et al. 1995). Both the

notochord and the cartilages are closely related, and it is assumed that the notochord repre-

sents a primordial form of cartilage (Stemple 2005, van Boxtel et al. 2010 b). Moreover, Bar-

row et al. (1974) described bone damages in pigs, which are supposed to be related to copper

deficiency and biochemical alterations of aortic elastin as well as collagen. Van Boxtel et al.

(2010 a) found (i) that dithiocarbamates inhibit the copper-containing enzyme lysyl oxidase,

(ii) that a knockdown of lox genes sensitizes zebrafish embryos/larvae to dithiocarbamate and

(iii) that a knock-down of zebrafish lysyl oxidase-like genes or an inhibition via chemicals

resulted in a notochord phenotype similar to dithiocarbamate-induced notochord undulation.

Since dithiocarbamates are Fe2+

and Cu2+

chelators, it is likely that this contributes to their

teratogenicity in general (van Boxtel et al. 2010 a, b).

The mode of action of acid hydrazides in the malformation of cartilage, bone and notochord

seems to be very similar to that of disulfiram: In early biochemical studies, it could be shown

that acid hydrazides (e.g. phenylhydrazide, iproniazide and isoniazid) inhibit the copper-

containing enzyme lysyl oxidase, which is needed for cross-linking collagen and elastin, re-

sulting in increased solubility of collagen (Barrow et al. 1974, Riggin and Schultz 1986). In-

hibition of lysyl oxidase can be caused by direct binding to the active site of the enzyme or

via chelation of copper (Riggin and Schultz 1986). Isoniazid, for example, (Antony et al.

1978, Cole et al. 1983, Fallab and Erlenmeyer 1953) binds and forms complexes with metals

and especially with copper. The failure of connective tissue fibers to polymerize properly is

called osteolathyrism (Barrow et al. 1974, Riggin and Schultz 1986, Harris et al. 1974, Kagan

et al. 1974, Anderson et al. 2007, Mendelsohn et al. 2006).

Thus, even though the respective test concentrations and LC50s of disulfiram and acetic acid

hydrazide differ considerably in the present study, the mechanism underlying the notochord,

bone and cartilage malformations are likely to be the same. The combined 144 h LC50 of

disulfiram of three semi-static and one static test is 0.33 mg/L (1.11 µmol/L) whereas the

Chapter IV

111

mean of the two semi-static replicates with acetic acid hydrazide is 12.2 g/L (164.4 mmol/L).

Both chemicals show very high TIs, > 16.6 for disulfiram and > 32.5 for acetic acid hydrazide

respectively, illustrating a very high ability to cause teratogenic effects far below lethal con-

centrations in zebrafish embryos.

4.6 Conclusions

The present study clearly documents that even small teratogenic effects on single cartilagi-

nous and bony elements in the head of 6 day old zebrafish larvae can easily be assessed in a

semi-quantitative manner. Although disulfiram and acetic acid hydrazide induced malfor-

mations in zebrafish larvae at very different concentration levels (µg versus gram range, re-

spectively), both chemicals caused very similar teratogenic effects in the notochord, cartilages

and bones. After exposure either to disulfiram or acetic acid hydrazide, the concentration-

dependent sequence of teratogenic effects is as follows: at the lowest test concentrations, there

are breaks or faint undulations of the notochord; with increasing concentrations, breaks disap-

pear and undulations get more severe and are accompanied by reduction of bone mass. First

malformations can be observed in cartilages of the pharyngeal skeleton, especially Meckel's

cartilage and both ceratohyals, whereas cartilages of the neurocranium are much less sensi-

tive. Finally, at the highest test concentrations, most bones and cartilages are absent or only

formed as diffuse cartilage clusters; exceptions are some cartilages of the neurocranium. At

these stages of teratogenesis, there is already significant mortality, but a significant number of

embryos still survive until the end of the experiment at 144 hpf.

Chapter IV

112

Chapter V

113

Chaper V

Toxicity and teratogenicity to cartilages and bones of zebrafish em-

bryos (Danio rerio) after exposure to hydrazides and hydrazines

Chapter V

114

5 Toxicity and teratogenicity to cartilages and bones of zebrafish

embryos (Danio rerio) after exposure to hydrazides and hydrazines

Whereas the previous chapter summarized the effects of disulfiram and acetic acid hydrazide

on cartilage and bone development in the head of zebrafish larvae, including a general over-

view about the mechanisms which most probably caused these very specific malformations,

this additional chapter gives further information about the teratogenic potential of additional

hydrazides and hydrazines.

5.1 Abstract

In terms of alternatives to animal testing, the zebrafish embryo test (ZFET) has received much

attention as a refinement or even a replacement for the acute fish toxicity test

(OECD TG 203). Using the ZFET, not only acute mortality can be investigated, but also spe-

cific sublethal alterations can easily be observed. Hydrazides and hydrazines are well known

for their diverse biological effects, e.g. they were used as groth regulators, as herbicides, as

additives in fuels, as antioxidants and as radical scavengers (Toth, 1980, Environment Cana-

da, 2011). Fertilized zebrafish embryos were exposed semi-statically to isoniazid (0.625 -

20 g/L), benzhydrazide (5 - 160 mg/L), benzylhydrazine (1.25 - 40 mg/L) and phenyl-

hydrazine (0.3125 - 10 mg/L) for 144 h (spacing factor 2.0), fixed in paraformaldehyde and

stained as whole mounts. The range of toxicity is very broad; after 144 h, the LC50 of isonia-

zid is about 4 g/L, whereas those of benzhydrazide, benzylhydrazine and phenylhydrazine are

about 50, 10 and 1 mg/L, respectively. All four chemicals caused notochord and cartilage

malformations at sublethal concentrations, e.g. fractures of the notochord could already be

observed at the lowest test concentrations, except for benzhydrazide, where the second lowest

test concentration (10 mg/L) showed a significant amount of notochord fractures. The amount

of fractures diminished with increasing concentrations, whereas the rate of undulations of the

notochord became more pronounced. Despite the different variable extent of acute toxicity,

the dose-dependent malformation of cartilage and decrease of ossification were comparable

between all test substances. The cartilages of the neurocranium, e.g. the ethmoid and basal

plate, proved to be more stable than cartilages of the pharyngeal skeleton such as, e.g.,

Meckel’s cartilage and the ceratohyal. Ossifications seem much more susceptible to the test

compounds than cartilages, with reduction of bone mass as the most prominent alteration.

Chapter V

115

5.2 Introduction

The toxicological and medical potential of both chemical classes on animals and humans was

already identified at the beginning of the 20th century (Gohil et al., 2009), but acute toxicity

and teratogenicity, especially to fish, has barely been investigated. Several compounds, e.g.

isoniazid, phenelzine, isocarboxazid, L-alpha-methyldopahydrazine and iproniazid were used

for the treatment of tuberculosis (Gohil et al., 2009), as antidepressants (Fiedorowicz and

Swartz, 2004, Kauffman, 1979) or for Parkinson treatment (Marsden et al., 1973). Whereas

maleic hydrazide was used as a growth regulator and herbicide (Greulach, 1951), the other

compounds were applied in high energy fuels in the aerospace industry and as antioxidants in

the petroleum industry (Toth, 1980). E.g. in Canada, hydrazines are widely used against cor-

rosion in industrial facilities and as radical scavenger in power plants (Environment Canada,

2011). In 2006, 10 to 100 tons of hydrazines were imported into Canada (Environment Cana-

da, 2011), whereas a considerable part ends up in the aquatic environment. Even though

hydrazides and hydrazines show no attributes of bioaccumulation or persistence, they are

quite toxic especially to aquatic organisms

Nevertheless, both hydrazides and hydrazines can induce cancer in laboratory animals (Gohil

et al., 2009, Toth, 1980). Hydrazines are both of synthetic and natural origin with N2H4 as

functional group with four substituents and are products of the nitrification-denitrification

cycle of various Azobacter strains and Rhizobium (Toth, 1980). Furthermore, hydrazines (e.g.

agaritine) are natural ingredients in cultivated fungi (Toth and Erickson, 1986). Hydrazides

possess the same chemical structure, however at least one of the four substituents must be an

acyl group.

Various studies described the toxicity, teratogenicity, reproductive toxicities, mutagenicity

and carcinogenicity of hydrazides and hydrazines in vivo and in vitro both in vertebrates and

invertebrates (Castellano et al., 1973; Turnherr et al., 1973; Slonim, 1977; Toth, 1975, 1978;

Toth et al., 1980; Malca-Mor und Stark, 1982; Ryan et al., 1984). Mac Ewen et al. (1981)

observed hyperplasia and atrophies of the ovaries in male and female rats after exposure to

hydrazines. However, aquatic organisms and especially fish have barely been investigated.

Only few acute fish data exist, e. g. Proteau et al. (1979; in French) calculated LC50 values

after 24 h hydrazine hydrate exposure of 3 months as well as 5 days old Danio rerio embryos

(3.18 and 1.17 mg/L, respectively) and 3 months old Carassius carassius and Rutilus rutilus

juveniles (1.48 and 0.85 mg/L, respectively). Velte (1984) determined a 96 h LC50 of 5.98 mg

for juvenile fathead minnows (Pimephales promelas). Already Proteau et al. (1979) detected

Chapter V

116

the strong teratogenic effects to the notochord of zebrafish embryos after exposure to hydra-

zine hydrate.



Zebrafish in maintenance and egg production has repeatedly been described in detail (Kimmel

et al., 1988, 1995; Nagel, 2002; Spence et al., 2005; Wixon, 2000, SOP ZFET

OECD_2b_V02.10 17th

January 2011) and has recently been updated for the zebrafish em-

bryo toxicity test (Lammer et al., 2009).


Isoniazid (CAS: 54-85-3), benzydrazide (CAS: 613-94-5), benzylhydrazine dihydrochloride

(20570-96-1) and phenylhydrazine (CAS: 100-63-0) were tested in the FET. The pH of all

chemical stock solutions was adjusted to 7.7 ± 0.2. All tests were conducted according to the

actual OECD draft SOP (SOP ZFET OECD_2b_V02.10; 17th

January 2011). A short sum-

mary of the test procedures is given in chapter IV. The stock solutions of isoniazid and

benzhydrazide were prepared freshly each day, whereas benzylhydrazine and

phenylhydrazine were freshly prepared for each single run the day before starting the test. The

stocks of benzylhydrazine and phenylhydrazine were stored in darkness at 4°C.

5.3.3 Whole-mount alizarin red and alcian blue skeletal staining

Alizarin red S (1,2-dihydroxyanthraquinone; Sigma Aldrich) and Alcian blue 8 GS (copper

phthalocyanine; Serva, Heidelberg) dyes were used for staining bony and cartilaginous struc-

tures, respectively. Larvae of 144 hpf age were killed with an overdose of benzocaine (ethyl-

4-aminobenzoate; Sigma Aldrich) and stained as described in the protocol by Walker and

Kimmel (2007) with slight modifications. Briefly, larvae were fixed and rocked for 2 h in 4 %

paraformaldehyde, stained using acid-free double stain solution with 120 mM MgCl2 over-

night (as replacement for acid offering better distinction of all cartilages and bones), bleached

using 3 % H202 and 1 % KOH for 25 minutes, and rocked with 20 % glycerol and 0.25 %

Chapter V

117

Fig. 5.1: Semi-quantitative staging of embryos exposed to hydrazides and hydrazines.

(A) negative control, (B) severity „1“ after exposure to 5 mg/L benzylhydrazine. Both the

angle of the ceratohyal and Meckel’s cartilage are slightly distorted (arrows). (C) severity

„2“ after exposure to 5 g/L isoniazid especially for cartilages of the pharyngeal skeleton

as Meckel’s cartilage, ceratobranchial and the branchial arches (arrows) (D) severity “3”

for all cartilages of the pharyngeal skeleton after exposure to 10 mg/L benzylhydrazine

(the arrow indicates the remains of the ethmoid plate).

3 2 0 1

KOH overnight; the latter step was repeated with 50 % glycerol and 0.25 % KOH overnight,

and specimens were stored in 50 % glycerol and 0.1 % KOH at 4°C.

5.3.4 Staging of cartilages and bones

After staining, all larvae were examined using a Stemi 2000-C (Zeiss, Göttingen, Germany)

or Olympus CKX41 microscopes equipped with digital imaging hardware (Power Shot G7

(Canon, Krefeld, Germany) or C-5069 Wide Zoom (Olympus, Hamburg, Germany)) as well

as the imaging software (Analysis 5.0). Larval and adult zebrafish cartilage and bone devel-

opment has been described in detail in several studies (Schilling et al. 1996, Piotrowski et al.

1996 and especially Cubbage and Mabee 1996). Cartilaginous and bony elements analyzed

are listed in Chapter IV. Quantification of cartilage and bone malformations was assessed in a

semi-quantitative approach: 0 – normally developed; 1 – minor malformations; 2 – strong

malformations; 3 – severe malformations or no longer detectable skeletal elements. However,

since in a given larva single cartilages and bones do not show identical grades of malfor-

mation severity, it should be noted that this classification is not general for every cartilage or

bone in the head of the respective larva. For instance, Meckel’s cartilage is frequently strong-

ly malformed, whereas all other elements appear normal or only show slight malformations.

Fig. 5.1 illustrates examples of 144 hpf control and treated (malformed) zebrafish larvae in

ventral views.

Chapter V

118

Only larvae free of any symptoms of lethality (DIN 2001) were used for cartilage and bone

staining. For this end, test concentrations were selected in a way that at least the two lowest

concentrations showed mortality levels ≤ 20 %. At the third concentration, at least 10 out of

20 embryos (≥ 50 %) had to survive.

5.3.4.1 Equipment for zebrafish embryo observation

All embryos and later the cartilages and bones were observed using a dissection microscope

(Stemi 2000-C with Canon power Shot G7) in combination with an Olympus CKX41 inverted

microscope equipped with a C-5069 Wide Zoom Camera and Analysis 5.0 software.

5.3.4.2 Statistics

Graphs and correlations were performed with SigmaPlot 11.0 (Statsoft-Jandel Scientific,

Erkrath, Germany) or GraphPad Prism 4 (Statcon, Witzenhausen, Germany). LC50 values

were calculated as probit analysis using ToxRat Professional 2.10.3.1 (ToxRat Solutions,

Alsdorf, Germany) with linear maximum likelihood regression.

5.4 Results

At the lowest test concentrations, both hydrazides and hydrazines caused fractures in the no-

tochord; these effects, however, diminished with increasing concentrations, whereas the noto-

chord got more and more undulated (Fig. 5.2).

5.4.1 Toxicity, general teratology and notochord malformation after exposure to

isoniazid

After 144 h exposure to isoniazid, even the lowest test concentration (0.625 g/L) showed no-

tochord fractures, getting more intense up to 2.5 g/L and 5 g/L whereas at higher concentra-

tions, the notochords were severely undulated. Reduced heartbeat and lack of/reduced blood

Chapter V

119

Fig. 5.2: Notochord malformations

of zebrafish embryos. Micrograph

(A) focuses on a fracture of the no-

tochord (72 h) after exposure to

2.5 g/L isoniazid, whereas (B)

shows a notochord of an 96 h old

embryo exposed to 10 mg/L

benzylhydrazine with accumulation

of collagen matrix around the frac-

ture. Picture (C) indicates a single

kink in the notochord of an 96 h

embryo exposed to 10 mg/L

benzhydrazide whereas (D) shows

an 120 h old embryo with multiple

kinks in the notochord (5 mg/L

benzhydrazide). Micrographs (E)

and (F) illustrate severe bending of

the notochord. Picture (E) displays a

144 h old embryo exposed to

40 mg/L benzhydrazide and (F) a 72

h old embryo exposed to 0.78 mg/L

phenylhydrazine.

circulation was also a very prominent effect at 1.25 g/L and higher. A concentration of

20 (10) g/L became 100 % lethal after 96 (144) hpf (hours post-fertilization). The LC50 values

are 8.8 ± 1.2 g/L after 96 hpf and 4.2 ± 0.9 g/L after 144 h.


benzydrazide

Notochord fractures significantly occurred in the second lowest test concentration (10 mg/L),

whereas at 20 mg/L all embryos were affected. Notochord undulations were mainly observed

at 40 mg/L and higher, accompanying sublethal effects as (pericardial) edema, reduced heart-

beat and lack of/reduced blood circulation. Concentrations of 80 mg/L (160 mg/L) were 90 %

(100 %) lethal after 144 h. The LC50 values are 70.3 ± 18.9 mg/L after 96 hpf and

47.0 ± 17.4 mg/L after 144 h.

Chapter V

120


benzylhydrazine

Fractures of the notochord as the only sublethal effects were observed at 1.25 mg/L; at

2.5 mg/L, notochord undulations were evident, and embryos exposed to 5 mg/L were even

more affected. Benzylhydrazine concentrations of 20 and 40 mg/L were 100 % lethal after

6 days; 10 mg/L caused almost 50 % mortalities. The LC50 values are 11.4 ± 3.2 mg/L after

96 hpf and 9.3 ± 1.4 mg/L after 144 h.


phenylhydrazine

Notochord fractures in up to 50 % of all test embryos (6 days old) occurred in the second

lowest concentration (0.313 mg/L), whereas, at 0.156 mg/L only 5 - 15 % showed this effect.

At 0.626 mg/L, undulated notochords appeared as additional malformation. Up to this concen-

tration other sublethal effects as (pericardial) edema, reduced heartbeat and lack of/reduced

blood circulation were not significant. The average mortality at 1.25 mg/L was approx. 45 %;

2.5, 5 and 10 mg/L were 100 % lethal after 6 days. The LC50 values are 1.5 ± 0.4 mg/L after

96 hpf and 1.3 ± 0.1 mg/L after 6 days.

5.4.5 Head length of exposed embryos after 6 days

The head lengths of 6 days old embryos were measured and compared to the negative controls

(Fig. 5.3). Whereas the mean of the negative controls in each test ranged between 762 and

797 µm, especially the highest test concentrations resulted in significantly (p < 0.05) short-

ened head lengths. After exposure to isoniazid (Fig. 5.3 B), only the lowest test concentration

was not significant, whereas concentrations of 1.25 g/L and higher produced significantly

shorter skulls than the negative controls. Similar observations were made for benzhydrazide

(Fig. 5.3 C): The highest concentration (40 mg/L) resulted in significantly shorter heads in

both runs, whereas at 20 mg/L only the first run gave statistically significant results. The re-

sults of benzylhydrazine and phenylhydrazine both showed the same trend. After exposure to

benzylhydrazine, concentrations of 2.5, 5 and 10 mg/L resulted in significantly shorter head

lengths (725, 695 and 575 µm). Embryos exposed to phenylhydrazine showed a significantly

reduced head size (697 µm) at a concentration of 1.25 mg/L.

Chapter V

121

Fig. 5.3: Measurement of the head lengths

(n = 2) of 6 day old zebrafish embryos after

exposure to isoniazid (B) and

Benzhydrazide (C).

5.4.6 Cartilage and bone malformations

Two replicates with isoniazid and

benzhydrazide, and one for benzylhydrazine

and phenylhydrazine, were stained and as-

sessed for cartilage malformations and bone

reduction. Cartilages of embryos exposed to

0.625 g/L isoniazid showed severity degrees

≤ 0.3 (Meckel’s cartilage); cartilages devel-

oped normally. In addition, the rate of ossifica-

tion was strongly reduced; the bones still ob-

servable were the tip of the notochord and the

teeth on the 5th

ceratobranchial (both with se-

verity values around 1.5); the remaining bones,

e.g. the parasphenoid, the opercles and the

branchiostegal rays, were occasionally slightly

observable as severity 2, but in most cases they

were not present (severity 3). Reduction of

ossification became more prominent with in-

creasing concentrations; at 1.25 g/L, all bones

were almost absent, only very weak hints of

the ossification of the notochord (severity

> 2.5) and the teeth were found (severity 2).

Isoniazid concentrations of 5 g/L proved se-

verely teratogenic to all cartilages (Fig. 5.4 C),

whereas all bones were no longer observable

(severity 3). After exposure to 5 g/L isoniazid,

the severity of cartilage malformation was be-

tween 1 and 2 for all elements of the pharyn-

geal skeleton (arch 1-7); whereas the elements

of the neurocranium showed only slight mal-

formations between 0 and 1.

Chapter V

122

Fig. 5.4: Photomicrographs of 6 day old zebrafish embryos after exposure to various

hydrazides and hydrazines. (A) shows a negative control (side view), whereas embryo (B -

benzhydrazide 20 mg/L) displays slight malformations at the lower jaw, especially Meckel’s

cartilage (arrow). The whole cartilages, especially the lower jaw (arrow, except the ethmoid

plate) of embryo (C) are strongly malformed after exposure to 5 g/L isoniazid. The angles of

the lower jaws of embryos (D – 40 mg/L benzylhydrazide) and (E – 10 mg/L benzylhydra-

zine) are strongly distorted. Embryo (F – 1.25 mg/L phenylhydrazine) shows cartilage re-

mains, but it remains unclear to which cartilage structure they belong to.

Results for benzhydrazide were very similar, but less intense. Embryos exposed to 5 and 10

mg/L benzhydrazide developed normally regarding the cartilages and showed only slight re-

ductions of bone ossification. 20 mg/L benzhydrazide generally caused slight malformation

on the cartilages, not exceeding severity 1, whereas the elements of the neurocranium again

showed less severe malformations, compared to the pharyngel skeleton; e.g. the ethmoid

plate, the trabeculae cranii and the basal plate were normal (0). Cartilage malformation values

after exposure to 40 mg/L were around 1.5 (mean of all cartilage elements of the pharyngeal

skeleton); whereas the malformations of the neurocranium were slightly lower.

Cartilage malformation and bone reduction patterns after exposure to benzylhydrazine and

phenylhydrazine were very similar. Embryos treated with 0.625 mg/L benzylhydrazine or

0.3125 mg/L phenylhydrazine showed no differences to negative controls, whereas those at

10 mg/L benzylhydrazine (1.25 mg/L phenylhydrazine) had severe (moderate) cartilage mal-

formations. The mean severity of all elements of the lower jaw was approx. 2 (1.5); (Fig. 5.4

F) and bones were no longer present (severity 3).

Chapter V

123

5.5 Discussion

This chapter was designed to provide details about zebrafish embryo teratogenesis following

exposure to selected hydrazides and hydrazines, which are well-known inhibitors of various

enzymes (Green, 1963; McCormick und Snell, 1959; Williamson et al., 1986). Van Boxtel et

al. (2010 a+b) described the role of lysyl oxidase (LOX), which is inhibited by hydrazides or

hydrazines, and its role in zebrafish embryo teratogenesis (see chapter IV). LOX, an amine

oxidase, is involved in the cross-linking of collagen and elastin needed for the stability of col-

lagen fibers and, therefore, for the normal development of cartilages and the notochord. Lysyl

oxidases are Cu(II)-dependent and need pyridoxal phosphate as co-factor. Levene and Car-

rington (1985) further investigated the mechanisms of inhibiting LOX through various

lathyrogenic substances and concluded that LOX can be blocked at least in two ways: The

first possibility is the replacement of pyridoxal-5-phosphate and (irreversible) blocking of an

active center of LOX. The second possibility is an indirect blockade of LOX through interfer-

ing with another enzyme, e.g. pyridoxal kinase. Pyridoxal is one of three natural forms of vit-

amin B6 (pyridoxine, pyridoxal and pyridoxamine). Pyridoxal kinase catalyzes the transfor-

mation of each of these three inactive forms to the active pyridoxal-5-phosphate. McCormick

and Snell (1959) observed that the formation of pyridoxal-5-phosphate is blocked by

hydrazines, e.g. the sensitivity of the pyridoxal kinase to isoniazid is several times higher than

to the natural substrate. Since pyridoxal-5-phosphate is an essential co-factor of LOX (Bird

and Levene, 1982), LOX can be indirectly blocked by lack of this co-factor. LOX is also es-

sential for the normal development of the notochord with collagen as an integral component.

Without LOX, the notochord looses much of its stability due to decreased cross-linking of the

collagen with possible subsequent undulation and malformation (Gansner et al., 2007; Van

Boxtel et al., 2010a).

Extensive studies with model organisms as chicken, mouse or rat revealed that chemical clas-

ses containing at least one nitrogen bond have the ability to act as lathyrogens (causing carti-

lage and bone malformations) in vertebrate embryos (Levene and Groß, 1959; Bird and

Levene, 1982; Schultz and Ranney, 1988) Schultz and Ranney (1988) reported teratogenic

and toxic effects of various hydrazides e.g. benzylhydrazide or acetic acid hydrazide in frog

embryos (Xenopus laevis) and found histopathological lesions in the notochord, the collagen-

ous microfibrils linked to the notochord decreased with increasing concentrations of

benzylhydrazide.

Chapter V

124

5.6 Conclusions

Among the hydrazines, the most toxic chemical tested in the ZFET is phenylhydrazine, fol-

lowed by benzylhydrazine, benzhydrazide and isoniazid, which is the by far most non-toxic

substance with an LC50 value in the gram range, whereas the LC50s of the remaining sub-

stances are in the lower mg range. Nevertheless, the teratogenic effects of both, the hydrazines

and the hydrazides, are comparable; notochord fractures occur in the lowest test concentra-

tions followed by notochord undulations with increasing concentrations, cartilage malfor-

mations and reduction of ossification. The most susceptible elements in the head are the

bones. Even at low test concentrations, the rate of ossification is clearly reduced, whereas the

first cartilage malformations occur at higher concentrations. Furthermore, sublethal effects as

reduced heartbeat, pigmentation and blood circulation as well as edema were recorded after

exposure to all of the tested substances. The reduction of blood circulation (anemia) is de-

scribed in literature after exposure e.g. to isoniazid (Castellano et al., 1973) and

phenylhydrazine (Mori et al., 1988; Berger, 2007; Nakanishi et al., 2003).

In general, the teratogenic effects observed in zebrafish embryos correlate well with those,

observed in other vertebrate species, e.g. Castellano et al. (1973) and Levene (1960) found

growth retardations and skeletal malformations in chicken embryos following exposure to

isoniazid, whereas Schultz and Ranney (1988) and Fort and Bantle (1990) described noto-

chord undulations in frog embryos after treatment with various hydrazides, including acetic

acid hydrazide. Hence, zebrafish embryos seem to be a suitable alternative method to conven-

tional skeletal teratology testing. Since the fetuses of rodents (especially rats) are often used

for determination of skeletal malformations (Menegola et al., 2002), further studies on the

correlation between rat and zebrafish embryos need to be performed.

Chapter VI

125

Chapter VI

Oxygen requirements of zebrafish (Danio rerio) embryos in embryo

toxicity tests with environmental samples

Chapter VI

126

6 Oxygen requirements of zebrafish (Danio rerio) embryos in em-

bryo toxicity tests with environmental samples

The ZFET is a relatively new approach for routine toxicity and teratogenicity testing both of

chemicals and environmental samples e.g. effluents and sediments. Therefore, the basic para-

meters, e.g. the oxygen consumption of single zebrafish embryos in relation to the oxygen

availability in the test medium, have to be clarified. Since environmental samples often cause

hypoxic conditions in the test system, in the first part of this study, the amount of oxygen

zebrafish embryos needs for normal development, has been determined. In the second part,

specific native sediments were analyzed and spatial-temporal oxygen patterns were recorded.

6.1 Abstract

The zebrafish embryo test is a widely used bioassay for the testing of chemicals, effluents and

other types of environmental samples. Oxygen depletion in the testing of sediments and efflu-

ents is especially important and may be a confounding factor in the interpretation of apparent

toxicity. In order to identify oxygen levels critical to early developmental stages of zebrafish,

oxygen consumption of zebrafish embryos between 0 and 96 h post-fertilization, minimum

oxygen levels required by the embryos for survival as well as the effects of oxygen depletion

following exposure to model sediments were determined. No significant effects on zebrafish

embryo development were observed for oxygen concentrations between 7.15 and 3.33 mg/L,

whereas at concentrations between 3.0 and 2.0 mg/L minor developmental retardations were

observed, yet without any pathological consequences. Oxygen concentrations lower than 0.88

mg/L were 100 % lethal. In the sediment contact tests with zebrafish embryos, native sedi-

ments rich in organic materials rapidly developed strongly hypoxic conditions, particularly at

the sediment-water interface (0 to 500 μm distance to the sediment).

6.2 Introduction

In search of alternatives to animal experiments (3Rs principle; Russell and Burch, 1959), the

embryo test with zebrafish (Danio rerio) has received increasing attention as a refinement or

even a replacement for the acute fish toxicity test in both whole effluent and chemical testing

Chapter VI

127

(Nagel, 2002; Braunbeck et al., 2005; Lammer et al., 2009). In September 2010, the status of

the FET as a replacement was confirmed in the revision of the EU Animal Protection Di-

rective 2010/63/EU (EU, 2010). At the national level, the 48 h fish embryo test was standar-

dized in Germany for whole effluent testing in 2001 (DIN, 2001), and an international version

was published as an ISO standard 15088 (ISO, 2007). Since January 2005, the fish embryo

test has thus replaced the routine fish test for whole effluent toxicity with golden ide

(Leuciscus idus melanotus) in Germany (OECD, 1992). A modified protocol for the testing of

chemicals was submitted to the OECD test guideline program in 2005 (Braunbeck and

Lammer, 2006), and it could be demonstrated that, after appropriate species-specific adapta-

tion, the zebrafish protocol can also be applied to other OECD test fish species such as

medaka (Oryzias latipes) or fathead minnow (Pimephales promelas; Braunbeck et al., 2005).

Most importantly, an in-depth statistical analysis revealed an excellent correlation (r = 0.89

for 77 chemicals) between the conventional acute fish test and the “alternative” fish embryo

test (Lammer et al., 2009). Yet, the fish embryo test as an alternative to the acute fish test

(OECD TG 203; OECD, 1992) is still being discussed controversially causing an ongoing

debate within the OECD and between experts. On September 8, 2010, however, the European

Parliament has taken a clear position in that fish depending on the yolk as a source of nutri-

tion, are not regarded as laboratory animals and are, thus, under state of protected organisms

(EU, 2010). Low oxygen supply in an embryo test vessel may be a confounding factor in the

testing of whole effluents, surface water or sediments. However, low oxygen supply has not

been adequately addressed and still needs more detailed exploration. Preliminary studies had

documented that zebrafish embryos can adapt to a broad range of dissolved oxygen concentra-

tions and that zebrafish embryos develop normally at oxygen concentrations as low as 2 mg/L

(Braunbeck et al., 2005). Padilla and Roth (2001) reported that zebrafish embryos younger

than 25 h post-fertilization (hpf) can survive for up to 24 h under completely anoxic condi-

tions, however, without any further cell divisions or discernible body movement (develop-

mental arrest). Embryos, which had already developed normal heart function, reduced their

heartbeat rate under low oxygen supplies, but recovered under normoxic conditions. At the

age of 29 hpf, embryos exposed to anoxia for less than 8 h, heartbeat returned to normal with-

in few minutes (Padilla and Roth, 2001). Lack of oxygen for more than 19 h, however, re-

quired a period of 6 h for restoration of normal heartbeat rates. During subsequent further

development, embryos gradually develop an increasing sensitivity to anoxia.

At least early zebrafish embryos thus seem to be quite tolerant (in terms of acute lethality) to

low oxygen conditions over limited periods of time; for periods longer than 24 h, however,

Chapter VI

128

adequate oxygen supplies seem essential. Oxygen depletion or even exhaustion may typically

occur during the testing of oxygen-consuming whole effluents or environmental samples, e.g.

in the sediment contact modification of the zebrafish embryo test (Hollert et al., 2003; Küster

and Altenburger, 2008). Since prolonged very low oxygen conditions may be expected to be

detrimental even for zebrafish embryos, a distinction between effects by oxygen deficiency

and effects due to chemical exposure would not be possible. As a consequence, oxygen depri-

vation by sewage or field samples might be a limiting factor for the practicability of zebrafish

embryo toxicity tests. Since precise measurements of oxygen levels and gradients in sediment

contact tests are an indispensable prerequisite for the evaluation of effects by low oxygen

conditions, the present study was designed (1) to utilize methodologies for the accurate meas-

urement of temporal and spatial oxygen distribution profiles at ranges between 0 and 2000 μm

distance from the sediment surface, (2) to provide more detailed information on minimum

oxygen demands of zebrafish embryos as revealed by mortality and developmental retarda-

tion, and (3) to analyze the reaction of zebrafish embryos to lowest oxygen concentrations in

terms of mortality and developmental retardation.


6.3.1 Oxygen measurements

This investigation has become possible due to significant progress in the development of

physico-optical oxygen sensors and the introduction of new sensor materials, which allow

measurement scenarios previously deemed impossible (Deshpande and Heinzle, 2004; John et

al., 2003; Jorge et al., 2005). The sensors used for the present investigation were provided by

PreSens, Regensburg (Germany) and are based on luminescence quenching by molecular oxy-

gen: Dye molecules (luminophores) are excited by light of a certain wavelength and are lifted

from the ground state to an energized state. After a specific decay period, dye molecules fall

back to the ground state and light of specific wavelength is emitted, which can then be detect-

ed as luminescence. If energized luminophores collide with molecular oxygen, however, the

energy is transferred to the oxygen rather than converted into light emission. In consequence,

the more oxygen is present, the less light will be emitted by the sensor, and the shorter the

decay time is (for details, see, e.g., Trettnak et al., 1998; Trübel and Barnikol, 1998; instruc-

tion manual OXY-4 micro, PreSens, 2004, 2008). In addition to high spatial and temporal

resolution, the advantages of this new technology include signal independence of flow veloci-

Chapter VI

129

ty, covering of a broad oxygen concentration range, and the fact that the sensors do not con-

sume any oxygen during measurement. Hence, in contrast to conventional oxygen sensor

types, there is no need to create a constant flow against the sensor.


For zebrafish, fish maintenance and egg production have repeatedly been described in detail

(Kimmel et al., 1988, 1995; Nagel, 2002; Spence et al., 2006; Wixon, 2000) and have recently

been updated for the zebrafish embryo toxicity test (Lammer et al., 2009).

6.3.3 Oxygen consumption of Danio rerio under normoxic conditions

Clear glass vials with integrated

sensor spots (2 or 5 mL total vol-

ume; Scherf Precision, Meiningen,

Germany), each containing one

zebrafish embryo, were put on the

SensorDish Reader (Figs. 6.1 B

and C), and oxygen consumption

rates were determined. Analogous

test series were performed with the

OXY-4 microsensor (Fig. 6.1 A)

in glass vials without sensor spots

(Scherf Precision) in total volumes

of 5 or 9 mL. Oxygen concentra-

tions from 0 to 24 h, 24 to 48 h

and 48 to 72 h were recorded in

test vials both at the beginning of

each experiments as well as upon

its termination after a test duration

of 24 h, using the OXY-4 micro

microsensor. Mean oxygen consumption values (differences between initial and terminal val-

ues) were calculated from10 individual vials and compared to 2 control replicates containing

Fig. 6.1: (A) Schematic construction of a needle type

sensor from the OXY-4 micro oxygen meter. (B) Sen-

sor Dish Reader: top and lateral view with 24-well

OxoDish plate. (C) Detailed view on a well filled with a

sample and an integrated sensor spot with light path

during measurement.With permission by PreSens

GmbH (Regensburg, Germany).

Chapter VI

130

only artificial water and no embryo. Prior to each measurement, vials were agitated for 30 s

on a REAX 1 vortex (Heidolph) to guarantee even oxygen distribution. Water temperature

was maintained at 26.0 ± 0.5 °C in an ICE 500 incubator (Memmert). In order to avoid for-

mation of gas bubbles, the vials were filled until an upward meniscus had formed; during sub-

sequent sealing with screw cabs, excess water was drained off.

6.3.4 Sediment samples

Within the framework of an

interlaboratory calibration

project on sediment contact

tests (SEKT) funded by the

German Ministry of Educa-

tion and Technology

(BMBF; Feiler et al., 2005;

Wölz et al., 2009), sedi-

ments were sampled from

locations in Germany

(Hunte, Teltow-Canal) and

Belgium (Zenne), which

were selected for differential

toxicity as shown in preli-

minary screening tests (Feiler et al., 2009, details not shown; for the composition of the sedi-

ments Hunte and Teltow see Tab. 6.1). In addition, sediments from different sites in Monte-

negro (Kamenik, Petrovo (Middle Lake), Vranjina and Plavnica (Lake Skadar) were also used

for oxygen deprivation measurements. In any case, sampling of sediments was performed in

accordance with sediment sampling protocols standardized by the German Federal Institute of

Hydrology (BfG, 2000, 2009). After collection, 1 L sediment samples were transferred to the

laboratory with the best cooling possible (maximum: 5 °C over a period of at maximum of

1 day). To avoid water loss and disintegration processes of the organic pollutants, native sed-

iments were sealed and stored at 4 °C.

Tab. 6.1: Physicochemical parameters and grain-size distri-

bution of the sediment collected at the locations Hunte and

Teltow Canal.

Chapter VI

131

6.3.5 Oxygen measurements of sediment samples

Oxygen measurements

were carried out both

directly above the se-

diment/water surface

and water/air surface

using the 4-channel

OXY- 4micro oxygen

meter equipped with

needle-type microsen-

sors (Fig. 6.1). Before

starting the experi-

ments, one microsen-

sor was calibrated against a standard membrane-covered Clark type electrode (Tab. 6.2) as

described in the manufacturer manuals. Oxygen readings were compared over the entire pos-

sible range between 0 and 8 mg/L O2. Throughout the experiments, values determined by the

microsensor were slightly higher than those detected by the standard electrode; however, dif-

ferences were very low, with the greatest deviation being 0.13 mg/L O2. As a rule, the stand-

ard deviations of oxygen measurements with the microsensor were 0.1 - 0.5 %, i.e., the meas-

urements proved highly reproducible. Standard deviations for the standard electrode were not

calculated; however, oxygen concentrations were only read, when the instrument did no long-

er fluctuate by more than ± 0.02 mg/L O2 within 20 s. The exact positioning of the sensors

was accomplished by means of a micromanipulator (Micro Bio-Tech Brand, Wertheim, Ger-

many), allowing the sensors to be moved in 10 μm steps in any direction, while the velocity of

the movement could be adjusted in a stepless fashion.

6.3.6 Temporal and spatial oxygen deprivation profiles of sediments

Three different series of experiments were conducted with the sediment Hunte (Fig. 6.3) in

order to record the time-course of oxygen deprivation (120 h) at given positions inside the

well: (a) directly at the sediment–water interface under conditions of illumination; (b) approx.

500 μm above the sediment surface; and (c) similar to (a), but in darkness. Test series (a) and

(b) were considered to cover the fraction of the water body relevant for zebrafish development

Tab. 6.2: Calibration of the oxygen microsensors versus a standard

oxygen electrode (WTW 350i, WTW, Weilheim, Germany) in the

range between 0 and 8 mg/L oxygen

Data for oxygen measurements with the microsensor are given as

means ± SD from n = 20 measuring points.

Chapter VI

132

on two assumptions: (1) the diameter of D. rerio eggs averages 700 μm (Kimmel et al., 1995;

Westerfield, 2000), and (2) it was assumed that embryos would partially sink into the sedi-

ment. The third test series (complete darkness) was carried out to find out whether the ab-

sence of light would result in reduced oxygen consumption and, thus, in higher final oxygen

concentrations. Due to time-dependent deposition and compression of the sediment, the posi-

tion of the sensor was re-adjusted after approx. 16 - 19 h in order to restore the desired dis-

tance of 500 μm between sediment surface and sensor tip. For each of the sediments Petrovo,

Vranjina and Plavnica, oxygen profiles (144 h) immediately above the sediment surface and

at 500 μm distance above the sediment were measured in darkness (Fig. 6.4). In order to ob-

tain a spatial oxygen deprivation profile within a range between 0 and 2000 μm distance

above the sediment surface, the sensor was moved in a step-wise fashion at intervals of 100

μm every 70 s, using a micro manipulator. After reaching the desired distance to the sediment

the sensor was stopped and after further 2 min oxygen measurements were initiated. Oxygen

concentration values were recorded for a total of 5.5 min at each position at 15 s intervals, and

the first 30 s was excluded from subsequent calculations

6.3.7 Sediment contact test with zebrafish (Danio rerio) embryos

For the sediment contact test, 6-well plates fromRenner (Dannstadt, Germany) were used.

Sediment samples from Hunte, Teltow-Canal and Zenne were prepared first in order to facili-

tate early and simultaneous exposure of eggs as well as to allow for settling of the sediments,

a strongly time-dependent process, which is important for redistribution of the particulates

(depending on the sediment). This also avoids excessive (and artificial) sinking of the eggs

into the sediment. Each well was filled with 3 g sediment and 5 mL artificial water as speci-

fied in ISO 7346-1 and 7346-2 (ISO, 1996a, b, c; 294.0 mg/L CaCl2×2 H2O; 123.3 mg/L

MgSO4×7 H2O; 63.0 mg/L NaHCO3; and 5.5 mg/L KCl). Temperature was adjusted to 26.0 ±

0.5 °C in an ICE 500 incubator (Memmert); pH ranged between at 7.6 and 8.0. Plates were

sealed with self-adhesive, fully oxygen-permeable Sealing Tape SH (cat. no. 236269; Nunc,

Wiesbaden, Germany). Quartz sand as specified by DIN 38412-L48 for the bacteria contact

test with Arthrobacter globiformis (DIN, 2004) served as an inert negative control as well as

for dilution of sediment samples; in addition, quartz powder (Quarzwerke Frechen, Germany)

and pure artificial water were used as additional negative controls. Five mL 3,4-

dichloroaniline per well at a concentration of 3.7 mg/L served as a positive control (Lammer

Chapter VI

133

et al., 2009) and was run in parallel to the test series with the sediments from the locations

Hunte, Teltow-Canal and Zenne. Analyses were performed with a CKX41 inverted micro-

scope (Olympus, Hamburg, Germany) equipped with a digital Olympus C5060 camera and

the digitizing software Analysis 5.0 (Soft Imaging Systems, Olympus).

Eggs were exposed in situ on the sediment and could, therefore, not be observed directly in

the well; thus, all embryos were transferred to a new well-plate and scored for acute mortality

(coagulation of the embryo, non-detachment of the tail, non-formation of somites and non-

detection of the heartbeat) according to DIN 38415-6 (DIN, 2001); in addition, reduced blood

circulation, reduced heartbeat rate, formation of edemata, gross morphological deformation

and retarded development were recorded as sublethal criteria of toxicity (Hollert et al., 2003;

Kosmehl et al., 2006). Subsequently all embryos were carefully transferred back into the re-

spective wells with the smallest volume of water possible. Given oxygen shortage being the

major focus of this study, most attention was given to retarded development, which was de-

fined as follows: the actual developmental stage of the embryo at a given point of time is less

or equal to half of the stage expected for this age according to Kimmel (1995), Nagel (2002)

and Westerfield (2000). After 48 h, e.g., all embryos were categorized as underdeveloped, if

the actual developmental stage was less or equal to the stage expected after 24 h.

For the sediments Hunte, Teltow-Canal and Zenne, a total of 30 eggs were used for the con-

ventional embryo sediment contact test (without additional agitation; Hollert et al., 2003);

experiments with additional agitation were carried out in triplicate with 15 eggs (5 eggs per

well). Two types of shakers were compared: a Laboshake RO 300/11 orbital shaker (Ger-

hardt; Königswinter, Germany) at 55 rpm and a REAX 3 tilt-shaker (Heidolph, Schwabach,

Germany) at 26 rpm.

6.3.8 Statistics

Data were analyzed for normal distribution and homogeneity of variance. Unless stated oth-

erwise, statistical significance of differences were analyzed by Student's paired t-test at level

of p < 0.05.

Chapter VI

134

6.4 Results

6.4.1 Oxygen consumption of D. rerio embryos at different developmental stages

Total daily oxygen consumption of D.

rerio embryos showed a gradual increase

from 0.165 (0 - 24 h) over 0.244 (24 - 48

h) to 0.265 μmol O2 (48 - 72 h; Tab. 6.3;

Fig. 6.2). For the first 72 hpf, the average

total oxygen consumption of zebrafish

embryos amounts to 0.674 μmol corre-

sponding to 0.0216 mg oxygen.

Fig. 6.2: Daily oxygen consumption of zebrafish

(Danio rerio) embryos. Mean values of all repli-

cas of the 3 development stages 0 - 24 hpf, 24 -

48 hpf and 48 - 72 hpf. Temperature during

incubation: 26 ± 0.5 °C, temperature during

measurement: 25.5 ± 1.5 °C.

Tab. 6.3: Oxygen consumption of zebrafish (Danio rerio) em-

bryos

Data for oxygen consumption are given as means ± SD. MS =

PreSens OXY-4 micro microsensor; SDR = PreSens SensorDish

Reader

Chapter VI

135

6.4.2 Survival and performance of D. rerio embryos under low oxygen concen-

trations

Effects of different oxygen concentrations (0 - 7.1 mg/L) on the development of embryos

were studied over 96 h in a total volume of 5660 mL with 10 embryos per selected oxygen

level (Tab. 6.4). From the oxygen consumption studies, Section 3.1 oxygen requirements for

zebrafish embryos were 0.674 μmol for the first 72 hpf of development corresponding to a

total 0.0216 mg oxygen. In a completely filled 5 L bottle (5660 mL) with an overall oxygen

concentration of 8 mg/L, a total of 45.28 mg O2 was dissolved; at O2 concentrations of 2.0

and 0.5 mg/L, a total of 11.32 mg and 2.83 mg oxygen would be dissolved, respectively.

Therefore, even at lowest oxygen concentrations of 0.5 mg/L, the amount taken up by the

embryo was approx. 0.76 % of the totally available O2 amounts. Hence, oxygen consumption

of all ten embryos corresponded to approx. 7.6 % of the total oxygen amount dissolved in

5660 mL artificial water at an overall oxygen concentration of 0.5 mg/L during the first

72 hpf. Extrapolation to a test duration of 96 h would then result in a maximum of 10 % of the

total amount of oxygen available in the tests. Since oxygen consumption rates were calculated

from measurements under normoxic conditions, since this calculation was based on very low

oxygen concentrations, and since zebrafish embryos had been shown to be able to adapt to a

broad range of oxygen levels (Braunbeck et al., 2005) and to reduce their oxygen uptake un-

der hypoxic conditions, it may be assumed that oxygen consumption by the embryos in the

present experiments is negligible. Küster and Altenburger (2008) performed a similar study

for the first 48 hpf of development. At least for higher oxygen concentrations, the differences

between the initial (0 hpf) and final (96 hpf) oxygen concentrations were below 5 %

(Tab. 6.4). No significant differences were seen between oxygen concentrations at the begin-

ning (0 hpf) and at the end of the tests (96 hpf), i.e. oxygen consumption of the embryos could

not be measured. The embryos did not show significant effects between 7.15 and 3.33 mg/L.

At concentrations < 3 mg/L, retardations were observed; however, up to 2.24 mg/L all em-

bryos reached an effective developmental stage beyond 72 hpf. With further diminishing oxy-

gen concentrations, effective developmental stage of the embryos further declined. At oxygen

concentrations ≤ 1.64 mg/L, pericardial edemata could be seen as first sublethal effects; these,

however, disappeared during a 48 h recovery period in oxygen-saturated artificial water. Peri-

cardial edemata became severe with declining oxygen levels. At 1.47 mg/L, 20 % of the em-

bryos showed slight bent tails, embryos lay on their sides when resting and were not able to

swim straight; the effective developmental stage corresponded to 48 hpf.

Chapter VI

136

Tab

. 6

.4:

Eff

ects

of

low

oxygen

conce

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on z

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per

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Chapter VI

137

However, at oxygen concentrations ≥ 1.47 mg/L, a 48 h recovery period under normoxic con-

ditions allowed full recovery of the embryos. At 1.09 mg/L, all embryos showed strongly re-

duced heartbeat, lack of blood circulation and pericardial edemata. The effective developmen-

tal stage was equivalent to approx. 36 hpf, and exposure for 96 h under normoxic conditions

resulted in a partial recovery of only 50 % of the embryos to a stage between 48 and 60 h.

Most survivors showed bent tails and were not capable of normal swimming. The remaining

50 % of the embryos coagulated. After 192 h (96 h exposure + 96 h recovery at normoxic

conditions), oxygen concentrations of 0.88 and 0.52 mg/L were 70 and 90 % lethal, respec-

tively (Tab. 6.4). At 0.52 mg/L O2, the embryos developed to a stage equivalent to 30 h, but

had severe pericardial edemata, extremely low heartbeat rates and no blood circulation. At 0.3

mg/L O2, embryos reached a stage of 16 - 19 hpf and died thereafter. Oxygen concentrations

≤ 0.3 mg/L caused 100 % coagulation during 24 to 48 hpf.

6.4.3 Temporal oxygen deprivation during exposure to selected sediments

In all 3 test series with the sediment Hunte, oxygen concentrations at the beginning (0 h) were

approx. 2 mg/L (Fig. 6.3). The strongest decrease in oxygen concentrations was found directly

above the sediment surface (test scenario a), particularly after recalibrating the distance be-

tween sensor tip and sediment. Changing the position of the sensor towards the sediment

(400 μm) after 19 h resulted in a decrease of oxygen concentration by 1 mg/L (Fig. 6.3). Simi-

lar observations were made with test scenario c. Oxygen concentrations immediately above

the sediment surface were clearly lower than in the two other test scenarios b and c. After

26 h, the oxygen concentration dropped below1 mg/L for the first time at scenario a. This

trend was comparable 500 μm above the sediment surface (scenario b); however, overall oxy-

gen concentrations were considerably higher until they leveled off after 96 h. At the begin-

ning, the test series run in permanent darkness showed a maximum of 2.7 mg/L O2, but

dropped below 1 mg/L after 68 h. Overall, however, trends were similar for the three different

scenarios: A continuous decline over the first 96 h, a plateau phase from 80 to 100 h and an

O2 recovery beginning at approx. 108 h.

Chapter VI

138

For the sediments Petrovo, Vranjina and Plavnica (Fig. 6.4), oxygen concentrations were de-

termined over 144 h both immediately above the sediment surface and 500 μm above in com-

plete darkness. The O2 gradients zebrafish eggs are exposed to (due to their size; shaded in

grey) are basically similar, but slightly differ depending on the specific sediment characteris-

tics: At the beginning of the experiments, oxygen concentrations were always < 0.6 and 1.8

mg/L at 0 and 500 μm above the sediment surface, respectively. After 144 h, O2 concentra-

tions directly at the sediment surface ranged between 5 and 3 mg/L, whereas O2 concentra-

tions 500 μm above the sediment were between 6.6 and 5 mg/L (Petrovo and Plavnica, re-

spectively).

Fig. 6.3: Oxygen deprivation and restoration profile of the sediment Hunte from 0 - 120 h.

The temporary decline in oxygen concentrations is most likely due to very high contents of

DOC and TOC, which had to be oxidized prior to restoration of higher oxygen concentra-

tions above the sediment surface. The solid line (a) shows oxygen concentrations immediate-

ly above the sediment surface (natural day and night rhythm, 25 °C), the dotted line (b) indi-

cates oxygen values at 500 µm distance to the sediment (natural day and night rhythm,

25 °C) and the dashed line (c) illustrates oxygen concentrations under complete darkness

(22 °C). Note sharp decline in oxygen concentrations after repositioning the sensor after 16 -

19 h and O2 recovery at approx. 108 h. All measurements were conducted on a heavy con-

crete weighing table (Köttermann; Uetze/Hänigsen, Germany) to minimize vibrations.

Chapter VI

139

6.4.4 Spatial oxygen deprivation profile above sediments

As shown for the sediment sample collected at Hunte, after 24 h the oxygen concentration

immediately above the sediment surface was 1.7 mg/L (Fig. 6.5); at distances of 500 and 2000

μm distance, O2 concentrations were 2.9 and 6.4 mg/L, respectively. After 48 h, O2 values

were lower, especially immediately above the sediment surface, where 0.6 mg/L O2 was

measured, but O2 concentrations converged with increasing distance to the sediment surface

up to 6.2 mg/L at 2000 μm distance. For the other sediment samples, similar data were re-

corded.

Fig. 6.4: Oxygen deprivation profiles of the

sediments Petrovo (Middle Lake) (A),

Vranjina (B) and Plavnica (C) from 0 to

144 h. The lower (solid) lines show oxygen

concentrations immediately above the sedi-

ment surface, whereas the upper (broken)

lines oxygen represent values at 500 µm

distance above the sediment surface. Mea-

surements were conducted at darkness in a

Heraeus T-6 incubator at 26.5 ± 0.5 °C.

Shaded area indicates the water layer from 0

to 500 µm above the sediment surface, in

which a zebrafish embryo would be deposit-

ed after spawning.

A

Petrovo

B

Vranjina

C

Plavnica

Chapter VI

140

6.4.5 Developmental retardation of zebrafish embryos in consequence of sedi-

ment exposure

Since developmental retardations of zebrafish embryos due to oxygen shortage are frequent

observations in sediment contact tests (Küster and Altenburger 2008), the three sediments

Hunte, Teltow- Canal and Zenne (cf. Feiler et al., 2005) were used as models to compare the

effect of gentle agitation via orbital and tilt shakers to improve oxygen supplies. The sedi-

ments Hunte and Zenne caused mortality ≤ 10 % in all experiments (Figs. 6.6 A and E); the

sediment Teltow-Canal, however, exerted mortality ≥ 25 % in shaken and non-shaken sedi-

ments (Fig. 6.6 C). Both after 48 h and 96 h, almost all embryos of non-shaken Hunte sedi-

ments showed developmental retardations. The same applied to embryos exposed to the sedi-

ment from Zenne (non-shaken sediments: ≥ 60 % developmental retardation). At the location

Teltow-Canal, all surviving embryos in non-shaken sediments were underdeveloped. A signif-

icant reduction of developmental retardations on both shakers was found for the sediments

Hunte and Zenne (Figs. 6.6 A, E); whereas the sediment Teltow-Canal showed only minor

reductions in the rates of developmental retardations (Fig. 6.6 C). There were no statistical

differences between both shaker types. Concentrations of dissolved oxygen at the water/air

boundary were constant between 6.5 and 8 mg/L, i.e. close to 100% saturation, whereas at the

sediment/water boundary, oxygen concentrations were much lower (2 - 5 mg/L) than in glass

sand and quartz powder controls (5.5 - 7.5 mg/L). Significant differences between oxygen

concentrations of shaken and non-shaken plates were not observed, albeit strong reductions of

developmental retardations were recorded on shaken sediments in comparison to non-shaken

samples.

Fig. 6.5: Spatial oxygen depri-

vation profile of the sediment

Hunte from 0 to 2000 µm dis-

tance above the sediment sur-

face after 24 (squares) and 48 h

(circles). Measurements at

100 µm steps distance relative

to the sediment surface. Mea-

surements were conducted in a

T-6, Heraeus incubator at

26.5 ± 0.5 °C.

Chapter VI

141

Fig. 6.6: Oxygen availability at 26 ± 0.5 °C during exposure to the sediment collected at Hunte

(A, B), Teltow-Canal (C, D) and Zenne (E, F). Left panels: Percentage of mortality and devel-

opmental retardations of Danio rerio embryos [%] after 48 h and 96 h (* p < 0.05; n = 3 repli-

cates for Hunte and Teltow-Canal, n = 2 replica for Zenne due to sediment limitations). For a

detailed definition of retardation, see materials and methods. Right panels: Oxygen concentra-

tions in single wells close to the water/air interface (upper two graphs; –●–●– 48 h; ●●●● 96 h)

and sediment/water interface (lower two graphs; – – – – 48 h, –––––––- 96 h). Data are given as

means from 6 (5) measurements of the sediments Hunte and Teltow-Canal (Zenne) and 2 (4)

measurements of QS and QP after 48 h (96 h). N: Non-shaken, O: Orbital-shaker, T: Tilt shaker,

QS: Quartz sand, QP: Quartz powder. Mortalities of QS and QP were below 10 % (p > 0.05).

Chapter VI

142

6.5 Discussion

6.5.1 Oxygen requirements of zebrafish (Danio rerio) embryos

Lowest oxygen requirements of zebrafish embryos were determined for the first day after fer-

tilization (0.165 μmol). On day 2, oxygen consumption increased by 0.079 μmol to 0.244

μmol, whereas on day 3 the increase was minor (0.021 to 0.265 μmol). The overall oxygen

requirements amounted to 0.674 μmol for the first 72 h of development at 26.0 ± 0.5 °C. Oxy-

gen consumption rates of zebrafish embryos in the present study were, thus, slightly higher

than in previous studies. Grillitsch et al. (2005) pooled 10 - 15 eggs and calculated the mean

consumption for a single embryo at 3.8 ± 0.14 and 15.0 ± 0.46 nmol/h for days 1 and 5, re-

spectively (at 28 °C). Unfortunately, no exact data were given for days 2 and 3; however,

from Fig. 7 in Grillitsch et al. (2005), oxygen consumption rates can be estimated at approx. 5

and 9 nmol/h for days 2 and 3, respectively. From these data, daily oxygen consumption can

be calculated as 0.091, 0.120 and 0.216 μmol for days 1, 2 and 3, respectively. The consump-

tion rates measured by Grillitsch et al. (2005) are, thus, lower than those found in the present

study, especially on days 1 and 2, whereas rates for day 3 are comparable. A possible explana-

tion for the deviation for days 1 and 2 might be the fact that Grillitsch et al. (2005) pooled the

eggs and consumption rates for single eggs were calculated from multiple data and the tem-

perature was 28 °C instead of 26 ± 0.5 °C in this study. According to Braunbeck et al. (2005),

it may be assumed that individual embryos are capable of adjusting their oxygen uptake ac-

cording to the simultaneous consumption by other embryos in the same vessel.

Bang et al. (2004) calculated mean oxygen consumption rates of zebrafish embryos to vary

between 0.261 and 0.462 μmol at 25 ± 0.1 °C with a mean value of 0.379 μmol for the period

from 24 to 75 hpf, which corresponds to 0.509 μmol (24 - 72 hpf) in this study. Differences

might be explained by the way oxygen consumption was determined: Whereas in the present

study consumption rates were directly measured inside the vials, Bang et al. (2004) calculated

oxygen consumption via simple diffusion across the chorion as the only possibility for oxygen

to enter the perivitelline space. In literature, there are indications that diffusion is not the only

way for the uptake of gases into the embryo, and there is some evidence that the chorion is not

a strong barrier for gases. Berezovsky et al. (1979; cited from in Rombough et al., 1988) used

microelectrodes to record the oxygen concentrations on the chorion surface, on the

perivitelline membrane and in the perivitelline fluid. Immediately above the chorion, a slight

drop of oxygen concentrations was observed in contrast to a sharp decline across the

Chapter VI

143

perivitelline membrane. One possible explanation is given by Alderdice et al. (1984), who

observed a hydrostatic pressure exerted on the chorion of steelhead trout (Salmo gairdneri)

which was definitely lower than the osmotic pressure of the perivitelline fluid. Calculations

revealed an effective filtration pressure of about -62 mm Hg indicating that water is driven

into the perivitelline space. Since the egg has to maintain a constant volume, compensation by

an equivalent outflow has to be postulated. Pores within the chorion certainly play an im-

portant role in volume regulation. An increased internal pressure is accompanied by volume

expansion; increased internal tension on the chorion might result in enlargement of the pores

and, subsequently, release of water across the pores of the chorion. Such net flux of fluids

across the chorion could considerably improve the oxygen transfer to the embryo.

The flux of fluids across the chorion is equivalent to a 100 % exchange of the entire volume

of the perivitelline fluid every 1 - 4 min (Alderdice et al., 1984; Loeffler and Lovtrup, 1970;

Loeffler, 1971; Potts and Rudy, 1969; Rombough et al., 1988). Movements of the embryo

will further improve oxygen supplies, since trunk movements will also inevitably result in

increased agitation of water inside the chorion. In fact, Rombough et al. (1988) discuss

whether these movements have a primarily respiratory nature. As a consequence, we definite-

ly expect oxygen consumption rates higher than what should be expected for simple diffusion

as assumed by Bang et al. (2004). In contrast to Grillitsch et al. (2005), Bang et al. (2004)

found only a minor increase of oxygenconsumptionbetween 42 and 72 hpf, which is in line

with results from the present study (+0.021 μmol O2 between days 2 and 3). In contrast to

other former studies, e.g. Barrionuevo and Burggren (1999), the consumption rates calculated

by Bang et al. (2004) as well as in this study are definitely higher. Ploug and Grossart (1999)

give a possible explanation for these differences. They found that pooling of five bacterial

aggregates (≥ 0.7 mm) in small vials resulted in reduced exchange of oxygen between the

aggregates and the surrounding water, if compared to single aggregates. In fact, older studies

consistently used clusters of eggs instead of measurements of oxygen consumption by indi-

vidual embryos (present study; Bang et al. (2004).

6.5.2 Oxygen consumption of zebrafish embryos in the fish embryo test for

chemicals

Oxygen consumption of zebrafish embryos seems to be high if related to the amounts of O2

dissolved in the surrounding water. During the first 72 hpf, one embryo consumes 0.674 μmol

Chapter VI

144

(0.022 mg) O2. From the current protocol for the fish embryo test (26 °C; 8.11 mg/L O2; total

test volume 2 mL), total O2 contents at the beginning of the test can be calculated as 0.016 mg

O2, which would definitely not be enough to guarantee for normal development. However, the

fish embryo test is performed in 24-well plates with a headspace of approx. 1.4 mL air, and

the wells are covered with fully oxygen-permeable foil. In fact, there is strong diffusion of

oxygen between test solution and headspace as well as across the cover foil (Strecker, 2008).

In an additional experiment (unpublished data), an increase in oxygen concentrations from

0.34 to 6.59 mg/L was measured in single wells over periods of 5 h. Taken together, it may be

concluded that embryos will not suffer from oxygen deficiency in the fish embryo test for

chemicals. Even in reduced sample volumes (≤ 100 μl), e.g. in 96- or 384-well plates, the fish

embryo test could be performed without any difficulties regarding oxygen availability

(Braunbeck et al., 2005; Strecker, 2008).

6.5.3 Compensation of low oxygen supplies to zebrafish embryos in the fish em-

bryo test for whole effluents and sediments

If compared to tests with pure chemicals, oxygen conditions may be very different during

tests with whole effluents and native samples collected in the field, which may per se be oxy-

gen-consuming due to microbial activities. Results of the present study clearly document that

steep oxygen gradients can be established above sediments in a time dependent fashion. In

some cases, the O2 gradients are linear. During settling of the particles, O2 levels gradually

decrease due to microbial activity, but recover as soon as the sediments solidify and build up

internal O2 gradients within the sediments. Comparison between differentially polluted sedi-

ments shows that effective O2 concentrations may vary considerably depending on the nature

and specific contamination of the sediment. In fact, the Hunte sediment consisted to a great

extent of silt (57.2 %) and clay (38.6 %) with about 65.8 g/kg TOC, whereas larger particles

were basically lacking (Tab. 6.1). As a consequence, the time the sediment required to settle

was longer than required for sediments with bigger particles. For the sediments from Monte-

negro, precise data on grain size fractions do not exist, but water contents were higher and

grain sizes were definitely higher. Hence, settling and solidification also occurred for these

sediments, but to a smaller extent.

Chapter VI

145

6.5.4 Oxygen deprivation of sediment samples

With all sediment samples used in this study, at least initial O2 concentrations were very low;

all sediment samples from Lake Skadar, e.g., showed concentrations lower than 2 mg/L after

48 h. Since zebrafish embryos younger than 24 hpf can withstand anoxic conditions for up to

24 h (Padilla and Roth, 2001) without taking damage by arresting development until

normoxic conditions are restored, such low concentrations can lead to false positive results in

tests with whole effluents and sediments with zebrafish embryos, since oxygen-related devel-

opmental retardation may overlay with chemical-induced effects. The problem becomes even

more critical, when embryos start dying due to hypoxia/anoxia longer than 24 h. Initial oxy-

gen values of all tested sediments were very low (below 2 mg/L) both directly on the sedi-

ment surface and in 500 μm distance to it. In this context it must be pointed out that 3 g more

or less anoxic/hypoxic sediment was transferred to each well. In ventilated artificial water as

used in this study the maximum oxygen solubility is 8.11 mg/L at 26 °C. Hence, in the water

layer (5 mL) not more than 40.55 μg oxygen are dissolved. As the ratio between oxygen to

sediment is approx. 1:75,000 and since the water was mixed with the sediment layer, this ho-

mogenization led to an increased surface area of the sediment rapidly binding the low amount

of water-dissolved oxygen. As a consequence, restoration of appropriate oxygen values was

not detectable before O2 diffusion from outside exceeded O2 deprivation. Since oxygen mea-

surements could only be started after sedimentation of particles, there was an inevitable time

lag of 4 h (Hunte) and 1 h (all other sediments) between mixing of the sediments with water

and the initiation of O2 measurements. As a consequence, the lowest oxygen concentrations

were determined for the sediment Hunte; correspondingly, developmental retardations were

high for this sample. The majority of oxygen concentrations determined for the other sedi-

ments were below 2 mg/L in the first 48 h, whereas concentrations in 500 μm distance to the

sediments were clearly higher. Depending on the individual sediment sample, oxygen condi-

tions on the sediment surface can vary enormously. In particular at the beginning of a sedi-

ment contact test oxygen concentrations may be very low (Fig. 6.4, 0 μm above all three sed-

iments oxygen concentrations below 0.6 mg/L); however, experiments show that zebrafish

embryos can withstand low oxygen concentrations.

Chapter VI

146

6.5.5 Developmental retardation as consequence of oxygen shortage

From an oxygen concentration of ≥ 3.33 mg/L, no significant effect could be observed even

for sublethal effects. At O2 concentrations of 2.2 mg/L, only minor developmental retarda-

tions were observed. In principle, both the fish embryo test and the sediment contact test can

thus be performed without difficulties up to approx. 2 mg/L. This lower threshold for oxygen-

related effects is in contrast to findings by Küster and Altenburger (2008), who performed a

similar study with different oxygen concentrations over 48 h and defined a minimum thresh-

old concentration of 4.5 mg/L O2 to avoid sublethal effects. This discrepancy can partly be

explained by the shorter test duration, partly by different measuring techniques. If oxygen

concentrations are lower than 2 mg/L, oxygen-related underdevelopment has to be expected.

However, 90 % mortality is only reached at O2 concentrations ≤ approx. 0.56 mg/L. This con-

clusion corroborates data by Shang and Wu (2004), who reported approx. 30 and 90 % mor-

tality after 168 hpf for O2 concentrations of 0.8 and 0.5 mg/L, respectively. However, hardly

any data are available on potential interference between low oxygen-related effects and che-

mical-induced damage. For example, zebrafish embryos have been found to be more sensitive

to cadmium when exposed simultaneously to hypoxia (Hattink et al., 2005). Hardly anything

is known about the effects oxygen depletion may have on the uptake and metabolization of

chemicals. Therefore, future experiments will have to clarify additive or even synergistic

effects of low oxygen levels and chemicals especially in relation to contaminated sediments or

whole effluents.

6.5.6 Agitation as a possibility to improve oxygen availability in sediments con-

tact tests

The first symptom of low oxygen-dependent effects in zebrafish embryos is retardation of

development. However, for the sediments Hunte and Zenne, a significant reduction of devel-

opmental retardations could be achieved via gentle shaking on either an orbital-shaker or a

tilt-shaker. No significant mitigation was observed for the sediment Teltow-Canal on both

shakers. The difference was not significant, but retardations were still reduced compared to

the non-shaken test. Overall, the tilt-shaker was thus slightly more effective in reducing sym-

ptoms of oxygen deficiency in the embryos. Interestingly, oxygen measurements run in paral-

lel failed to show any differences between shaken or non-shaken sediments; however, most

likely, this was due to inevitable agitation during the transfer of the well plates from the incu-

Chapter VI

147

bator to the oxygen meter. Tests with quartz powder and quartz sand as positive controls did

not show any effect of agitation on zebrafish embryos, at least at the shaking speeds used in

this study. Since both orbital and tilt shakers successfully reduce the rate of developmental

retardations, agitation is strongly recommended as a modification to the standard protocol of

the zebrafish embryo test.

6.6 Conclusions

The present study clearly documents that minute changes of oxygen concentrations and oxy-

gen gradients can precisely be measured by means of optical oxygen sensors. Zebrafish (D.

rerio) embryos can tolerate low oxygen levels over a wide range, and concentrations

≥ 3 mg/L seem suitable to support normal development. Oxygen concentrations between 3

and 2 mg/L did not induce any teratogenic effect, but resulted in a minor, fully reversible re-

tardation of growth. Between 2 and 0.88 mg/L oxygen, an oxygen-dependent increase of

pathological symptoms could be recorded including (fully reversible) pericardial edemata

(≤ 1.64 mg/L), tail deformations and severe developmental retardation (≤ 1.47 mg/L). Under

normoxic conditions, recovery of 50 % of the individuals was possible even for changes seen

after exposure to 1.47 mg/L oxygen. Extended exposure to oxygen concentrations lower than

0.88 mg/L turned out to be 100 % lethal. At 0.52 mg/L O2, the 96 h old embryos had devel-

oped to a stage equivalent to 30 h under normoxic conditions, but had severe pericardial

edemata, extremely low heartbeat rates and no blood circulation. Recovery under normoxic

conditions for another 96 h resulted in 90 % mortality. At 0.3 mg/L O2, embryos reached a

stage of 16 - 19 hpf and died thereafter. Oxygen concentrations ≤ 0.3 mg/L caused 100 %

coagulation between 24 to 48 hpf. Thus, oxygen depletion should not cause any problems in

chemical testing. In sediment contact tests with zebrafish embryos, however, native sediments

rich in organic materials may rapidly develop strongly hypoxic conditions, particularly at the

sediment– water interface (0–500 μm distance to the sediment), i.e. exactly where the eggs

and embryos would develop. Even mild aeration of the test system, however, will compensate

for this local oxygen depletion.

Chapter VI

148

Chapter VII

149

Chapter VII

Sediment-contact fish embryo toxicity assay with Danio rerio to as-

sess particle-bound pollutants in the Tietê River Basin (São Paulo,

Brazil)

Chapter VII

150

7 Sediment-contact fish embryo toxicity assay with Danio rerio to as-

sess particle-bound pollutants in the Tietê River Basin (São Paulo,

Brazil)

Chapter VI determined both the oxygen consumption of single zebrafish embryos and the

spatial and temporal oxygen concentration profiles of various native sediments. It was demon-

strated, that, depending on the respective sediment, hypoxic conditions can occur and highly

affect the outcome of a test using environmental samples. The recommendation of the previ-

ous chapter, that even mild aeration will compensate the local oxygen depletion, will be im-

plemented in the testing of native sediments from Brazil. Within this cooperation project, the

sediments are tested in an improved sediment-contact test including measurements of oxygen

profiles during the tests.

7.1 Abstract

The Tietê River and its tributary Pinheiros River receive a highly complex organic and inor-

ganic pollutants load from sanitary sewage and industrial sources, as well as agricultural and

agroindustrial activities. The aim of the present study was to evaluate the embryotoxic and

teratogenic effects of sediments from selected locations in the Tietê River Basin by means of

the sediment contact embryo toxicity assay with Danio rerio, in order to provide a compre-

hensive and realistic insight into the bioavailable hazard potential of these sediment samples.

Lethal and sub-lethal effects were recorded, and high embryo toxicity could be found in sam-

ples in the vicinity of the megacity São Paulo (Billings reservoir and Pinheiros River sam-

ples), but also downstream (in the reservoirs Barra Bonita, Promissão and Três Irmãos). Re-

sults confirm that most toxicity is due to the discharges of the metropolitan area of São Paulo.

However, they also indicate additional sources of pollutants along the river course, probably

from industrial, agricultural and agroindustrial residues, which contribute to the degradation

of each area. The sediment contact fish embryo test showed to be powerful tool to detect em-

bryo toxicity in sediments, not only by being a sensitive method, but also for taking into ac-

count bioavailability. This test provides an ecological highly realistic and relevant exposure

scenario, and should therefore be added in ecotoxicological sediment quality assessments.

Chapter VII

151

7.2 Introduction

Contaminated sediments have been recognized not only as a major sink for persistent toxic

substances released into the aquatic environment, but also as a potential source of contami-

nants, since pollutants may be made available under certain environmental conditions (such as

dredging or flood events – Hilscherova et al., 2003; Woelz et al., 2008). These pollutants are

not only linked to organisms in aquatic ecosystems, but also to human health via drinking

water and fish consumption (Chen and White, 2004; Hollert et al., 2005; Keiter et al., 2006;

Maier et al., 2006). In conventional ecotoxicity testing strategies, fish are an indispensable

component of integrated toxicity testing strategies for the aquatic environment (Lammer et al.,

2009a). The use of fish as biomonitor for water and sediment quality assessment provides

specific advantages because fish are especially sensitive to impacts on the aquatic environ-

ment and they respond to toxic agents similarly to higher vertebrates including mammals, thus

allowing an evaluation of the teratogenic, mutagenic and carcinogenic potentials not only to

fish, but also to humans (Lemos et al., 2007). Fish acute toxicity tests thus play an important

role in environmental risk assessment and hazard classification. However, in acute tests with

their exclusive endpoint of mortality, fish have been hypothesized to suffer severe distress and

pain (Braunbeck et al., 2005; Braunbeck and Lammer, 2006; Chandroo et al., 2004; Nagel,

2002), which would be in conflict with current animal welfare legislations in many countries

(Lammer et al., 2009a). Thus, there is an urgent need for the replacement or reduction of in

vivo tests with adult fish by in vitro tests such as cytotoxicity tests, but also tests with early

developmental stages of embryos, since these are also not regarded as experimental animals

(Braunbeck et al., 2005; Lammer et al., 2009a)

Fish embryo toxicity tests have become a promising tool to replace the acute fish test

(Braunbeck et al., 2005; Nagel, 2002). Several toxicological studies comparing different life-

stages of fish concluded that in most cases long-term toxicity could be extrapolated from re-

sults from studies with early life-stages (Chorus, 1987; McKim, 1977; Woltering, 1984). In

Europe, the use of fish embryos is not regulated by current legislations on animal welfare and

is, therefore, considered as a refinement, if not replacement of animal experiments (Scholz et

al., 2008). Fish embryos represent an attractive model for environmental risk assessment of

chemicals, since they offer the possibility to perform small-scale, high-throughput analyses

with an excellent correlation to conventional in vivo testing with an adult fish (Lammer et al.,

2009b). Beyond their application for determining acute toxicity, fish embryos are also excel-

lent models for studies aimed at the understanding of specific toxic mechanisms and the indi-

Chapter VII

152

cation of possible adverse long-term effects (Scholz et al., 2008). Finally, due to its sensitivi-

ty, reproducibility and adaptability, the embryo assay test found its way into the laboratories

not only for testing chemicals, but also for investigations into environmental samples, e.g.

sediments or particulate matters (Hallare et al., 2005a; Hollert et al., 2003; Ulrich et al.,

2002). The present study is part of a weight-of-evidence study aiming at identifying hazard

factors and ecotoxicological risks of sediments in the Tiete River Basin (Rocha et al., 2006,

2009, 2010). The Tietê River was selected as an example for a highly contaminated river sys-

tem. This river and its tributary, the Pinheiros River (São Paulo State, Brazil), are located in

the Tietê River Basin and receive a highly complex organic pollutants load due to the lack of

appropriate treatment of sanitary sewage and industrial effluents in the metropolitan region of

São Paulo, as well as numerous inorganic substances from industrial sources. Moreover, along

the entire course of the Tietê River, it continues to receive considerable pollutant loads from

domestic sewers, agricultural and agroindustrial activities (Calijuri, 1999; CETESB, 1997;

Soares and Mozeto, 2006). Along its course, the Tietê River comprises of several reservoirs,

which are intensively used for providing drinking water, agricultural irrigation and recreation

sites. High mutagenic, genotoxic, as well as aryl hydrocarbon receptor (AhR)-mediated tox-

icity were recorded in some of these reservoirs with a good correlation between in situ/in vivo

and in vitro assays (Rocha et al., 2009, 2010), indicating the high ecological relevance of the

in vitro assays for these endpoints. However, it is well-know that organic extraction of river

sediments usually leads to the transfer of the full spectrum of chemicals adsorbed to the sedi-

ments to the dissolved phase and, thus, provide estimations of the total hazard potential, but

neglect the bioavailability of sediment contaminants (Fent, 2004; Liß and Ahlf, 1997; Seiler et

al., 2006; Wang et al., 2004). Thus, in order to simulate in situ exposure conditions in a more

realistic scenario (Feiler et al., 2005; Triebskorn et al., 1997), a recently developed sediment

contact fish embryo test (Hollert et al., 2003) was applied to sediments collected from loca-

tions along the Tietê River, which had proved to be differentially contaminated in previous

studies (Rocha et al., 2009, 2010). This way, the aim of the present study was to evaluate the

embryotoxic and teratogenic effects of sediments from selected locations in the Tietê River

Basin by means of the sediment contact embryo toxicity assay with Danio rerio in order to

provide a comprehensive and realistic insight into the bioavailable hazard potential of these

sediment samples.

Chapter VII

153


7.3.1 Sediment sampling and treatment

The Tietê River basin is divided in four distinct sub-basins, the Upper Tietê, Superior Middle

Tietê, Inferior Middle Tietê and Lower Tietê. The Upper Tietê River basin corresponds to the

drained area of the Tietê River, from its spring across the metropolitan area of São Paulo city,

which is characterized by high urbanization and dramatic deterioration. The Superior Middle

Tietê River basin is dominated by urban, industrial and agricultural areas; on the other hand,

the Inferior Middle Tietê River and the Low Tietê basins are characterized predominantly by

agricultural areas and pastures and sugar cane culture, respectively (CETESB 1997).

The study area comprised eight areas along these sub-basins. First area is located in

Salesópolis, near the Tietê River’s spring (Upper Tietê). This area was considered as a refer-

ence site, since it is the less impacted area among all others. Second to seventh areas com-

prised the reservoirs: Ponte Nova and Billings (Upper Tietê); Barra Bonita (Superior Middle

Tietê), Bariri and Promissão (Inferior Middle Tietê), Três Irmãos (Lower Tietê). The eighth

area is located at the Pinheiros River, a tributary of Tietê River, located in the Upper Tietê

(Fig. 7.1).

Fig. 7.1: Location of the sampling sites in the Tietê River basin, São Paulo Paulo, Brazil (re-

drawn from Rocha et al., 2010).

Chapter VII

154

From Barra Bonita to Três Irmãos, the reservoirs were built in cascade arrangements for the

generation of electricity. Surface sediments were collected in May and December 2005 in the

Tietê River reservoirs, and in December 2006 in the Pinheiros River, using an Ekman-Birge

dredge, with ten replicates at each site (with a distance of 10 m from sample to sample). Rep-

licates were homogenized, and 1.5 kg of each sediment sample were frozen immediately,

stored at -10 °C and transported to Germany. Transfer of the samples was permitted by the

Brazilian National Department of Mineral Production (DNPM).

Freeze-drying is a process that removes the content water from samples by sublimation, thus

preserving the main characteristics of the samples, allowing them to be used in several assays

for long periods. For this, native sediment samples placed each in 500 ml round bottom flasks

(Schott, Mainz, Germany) were shock-frozen -30 °C over approximately 15 minutes under

rotation in an isopropanol bath (N6, C41, Haake, Karlsruhe, Germany) to favor the freeze-

drying process. Samples were then freeze-dried in an Alfa 1-4 freeze-drier (Christ, Osterode,

Germany) for 72 hours and sieved with a 1.25 mm mesh sieve (Haver and Boecker, Oelde,

Germany) to remove small pieces of vegetation and stones. Samples were then stored at 4 °C

in brown glass bottles.


As a first approach, oxygen levels were measured in the highest concentrations of selected

samples from Promissão, Billings and Barra Bonita to assure that lack of oxygen would not

interact with toxic effects in the embryos. Oxygen sensors were delivered from Precision

Sensing (Regensburg, Germany). The measuring principle is based on the effect of lumines-

cence quenching by molecular oxygen and consume no oxygen itself in contrast to common

sensors (for information on sensor principles see Holst et al. 1997; Klimant and Wolfbeis,

1995 as well as Klimant et al., 1999). The oxygen measurements were performed using the

Oxy-4 micro instrument with needle-type sensors and were carried out immediately (0 to 250

µm) above the respective sediment surface. Mean values of oxygen concentration were calcu-

lated from 10 independent measurements with an interval of 2 seconds between measure-

ments at a constant temperature of 25 °C.

Chapter VII

155

7.3.3 Sediment contact assay with Danio rerio

Sexually mature zebrafish (D. rerio) were obtained from the stocks at the Department of Zo-

ology, University of Heidelberg, Germany, and maintained according to Lammer et al.

(2009a).

Sediment samples were tested in 6 well plates in serial dilutions of 1:1 to 1:32 in quartz pow-

der (grain size W4; Quartzwerke, Frechen, Germany) and artificial water (ISO 7346/3; Tab.

7.1) according to the protocol of Hollert et al. (2003). In brief, 3 g of sediment/quartz mix-

tures were homogenized in a mortar to avoid sediment or quartz powder aggregates. The mix-

tures were placed into the well plates, and 5 ml of artificial water, which had been ventilated

to oxygen saturation for at least 24 h, were added to each well. As negative controls, 3 g of

quartz powder in 5 ml artificial water, as well as only 5 ml of artificial water were used. As

positive controls, 3 g of artificial sediment filled up with 5 ml of a 3.7 µg/ml 3,4-

dichloraniline (3,4-DCA, Fluka, Munich, Germany) solution was used.

According to Strecker et al. (2011), one possibility to avoid (or at least ease) the problems

with oxygen depletion in the sediment contact embryo assay is to incubate the mixture of sed-

iment and artificial water (saturated in oxygen) for 72 h prior to exposure of the embryos, in

order to allow complete oxygen exchanges between sediment and water. Thus, in order to

discriminate between embryotoxic effects by oxygen depletion from effects by chemicals or

to the sample itself, the fish embryo test was carried out as a pre-test after 1h and 72 h of equi-

libration of the sediments for four samples, Billings and Pinheiros, considered the most toxic

samples, and Barra Bonita and Promissão, considered medium or low toxic, according to pre-

vious studies (Rocha et al., 2009; Rocha et al., 2010).

The plates were stored in an incubator at 27°C for 72 h to allow sedimentation and oxygen

exchange between sediment samples and water. After this period, exposure was started by

gentle addition of zebrafish eggs. Three independent assays were run for each sample. For

Tab. 7.1: Concentrations of sediment samples diluted in quartz powder and artificial water.

Dilution 1:1 1:2 1:4 1:8 1:16 1:32

Dried sediment (g) 3 1.5 0.75 0.375 0.1875 0.09375

Quartz powder (g) 0 1.5 2.25 2.625 2.8125 2.90625

Concentrations (mg/ml artificial water) 600 300 150 75 18.75 3.125

Chapter VII

156

each assay, sediment concentrations were assayed in duplicate. Embryo tests were initiated at

latest 3 h after fertilization of the eggs (ca. 128 cell stage). Five fertilized eggs were placed in

each well, two by replicate, filled with the samples, giving a total of 10 eggs for each sedi-

ment concentration, and incubated at 27 ± 0.5 °C.

For recording toxic effects, eggs were carefully transferred into other 6-well plates filled with

artificial water and inspected under an Olympus CK-2 inverted microscope SC-35 camera

(Olympus, Hamburg, Germany). Toxicological endpoints were recorded after 48 and 96 h,

and lethal and sub-lethal effects were estimated according to DIN (2001, Tab. 7.2). In accord-

ance with German national and institutional guidelines for the protection of human subjects

and animal welfare, experiments with zebrafish embryos up to 96 hpf are not considered ani-

mal experiments and do not require specific permission by animal welfare commissions. Le-

thal effects were expressed as LC50 values (concentration inducing 50 % of mortality) and

sub-lethal effects as EC50 values (effective concentration inducing 50 % effects) calculated

using non-linear regres-

sion analyses according to

the endpoints after 48 and

96 h of exposition, using

Prism 4.0 (GraphPad, San

Diego, USA). After this

pre-test, all eight samples

were tested with 72h of

pre-incubation.

7.4 Results


Oxygen levels were measured after 1h and 96 h of incubation with sediments, in the overlay-

ing water of the wells after the samples settled down. For all sediments, oxygen concentra-

tions were very low after 1 h sedimentation (Promissão = 0.02 mg/L, Billings = 0.5 mg/L and

Barra Bonita 1.2 mg/L), but had increased considerably after 96 h (Promissão = 0.9 mg/L,

Billings = 1.4 mg/L and Barra Bonita 1.5 mg/L). Oxygen concentration in the negative con-

trol varied between 5.5 and 7.5 mg/L.

Tab. 7.2: Lethal and sub-lethal effects used in this study for

evaluating the toxicological effects of freeze-dried sediment

samples from Tietê River Basin in Danio rerio embryos (lethal

effects according to DIN, 2001).

Lethal effects Sub-lethal effects

Coagulation of the embryo Lack of blood circulation

Lack of somite formation Edemata formation

Non-detachment of tail Developmental retardation

Lack of heart function Malformation*

Chapter VII

157

7.4.2 Sediment contact assay with Danio rerio

7.4.2.1 Comparison of effects of sediments with and without sediment pre-incubation

Comparing exposure of the embryos to selected samples with and without sample pre-

incubation, effects were clearly lower or even absent in embryos exposed to the samples pre-

incubated for 72 h (Fig. 7.2). Mortality rates of embryos exposed to Billings samples, e.g.,

changed from 100 % in all concentrations without pre-incubation to 100 % only in concentra-

tions 300 to 600 mg/ml after pre-incubation for 72 h. A significant decrease in mortality rates

could also be observed for the other samples (Pinheiros, Promissão and Barra Bonita). There-

fore, for the definite tests, all samples from the Tietê River Basin were pre-incubated with

artificial water for 72 h prior to addition of zebrafish eggs (cf. also for Strecker et al., 2011).

7.4.2.2 Embryo toxicity of the sediment samples of the Tietê River Basin

For all native sediment tests, all three independent investigations were considered valid, since

mortalities in the negative controls were < 10 %, and since the positive controls produced

> 10 % mortality (according to DIN, 2001 and ISO, 2007).

Lethal and sub-lethal recorded effects are represented in Fig. 7.3 as percentage of effects in all

three replicates. Tab. 7.3 presented LC50

and EC50

values.

Chapter VII

158

Fig. 7.2: Relative mortality (lethal effects according to DIN 38415-T6) of Danio rerio in the

sediment contact assay after exposure for 48 and 96 h to sediments from different location in

the Tietê river basin with and without pre-incubation over 72 h. NC w4 = negative control, 600

mg quartz powder/ml artificial water). Note that negative controls from 48 and 96 hours (NC

w4 48h - black inverted triangle and NC w4 98h - white triangle) overlap each other in all

graphs, due to the lack of mortality (mortality 0 %) at the same concentration of 600 mg quartz

powder /ml artificial water.

Chapter VII

159

For the sediment samples collected near the spring, very slight edemata were observed in part

of the embryos after 48 h of exposure to all concentrations except for the lowest one. Since

this effect could only be seen in less than 40 % of the embryos, only an effective concentra-

tion inducing 25 % of effects could be calculated for these samples as EC25= 57 mg/ml. In

fact, after 96 h of exposure, these embryos had recovered, and only a small developmental

retardation of less than 2 h could be observed, if compared to negative controls. No lethal

effects were recorded in embryos exposed to this sample.

After 48 h exposure to the sediment samples taken at Ponte Nova, some embryos showed a

lack of heart function and blood circulation at all concentrations, but an LC50 value could not

be calculated, since mortality was < 50 %. Therefore, an LC25 as the lethal concentration in-

Fig. 7.3: Lethal and sub-lethal effects recorded after 48 and 96 h of D. rerio embryo exposure

to freeze-dried sediment samples from the Tietê River Basin. Percentages of mortality and

sub-lethal effects were calculated as means from 3 independent replicates. Figs. 7.3a to 3h are

arranged from left to right and from top to down according to the flow direction of the river.

Chapter VII

160

ducing 25 % mortality in the embryos was calculated at 46 mg/ml. In addition, slight edemata

were recorded at sediment concentrations between 37.5 mg/ml and 600 mg/ml (EC50= 86

mg/ml). No toxic effects were recorded after 96 h.

After 48 h exposure to the Billings samples, all eggs were coagulated at the highest sediment

concentration. Coagulated eggs were also recorded in concentrations from 75 to 300 mg/ml,

but at lower percentages. Exposure to 300 mg/ml also induced a lack of somite formation,

non-detachment of the tail, lack of heart function and blood circulation (LC50= 203 mg/ml) as

well as developmental retardation, which could be also observed in 150 mg/ml. Edemata were

observed at concentrations from 18.75 to 150 mg/ml (EC50 = 105 mg/ml). After 96 h of expo-

sure, lethal effects were less prominent even at a concentration of 300 mg/ml, but embryos

still showed developmental retardation and lack of blood circulation (Fig. 7.4); almost all of

them failed to hatch (LC50 = 209 mg/ml, EC50 = 110 mg/ml).

a Effective concentration inducing 25 % mortality or 25 % sub-lethal effects.

Tab. 7.3: Lethal and sub-lethal effects recorded after 48 and 96 h of D. rerio embryo expo-

sure to freeze-dried sediment samples from the Tietê River Basin. Lethal effects (LC50 values

- concentration inducing 50 % of mortality) and sub-lethal effects (EC50 values - effective

concentration inducing 50 % effects) were calculated as means from 3 independent replicates.

For the samples Ponte Nova and Barra Bonita, LC50 values could not be calculated, since ef-

fect levels did not reach 50 % mortality of the embryos. For these samples, LC25 values were

extrapolated. For the sample taken from near the spring, EC25 rather than EC50 values are giv-

en, since sub-lethal effects were found in less than 50 % of the individuals. Areas are arranged

from top down according to the flow direction of the river.

Areas

LC50 48 h LC50 96 h EC50 48 h EC50 96 h

Spring Ne Ne 57.22 a

Ne

Ponte Nova 46.6 a

Ne 86.1 Ne

Billings 203.0 209.2 105.7 107.9

Pinheiros 598.6 448.6 48.6 55.0

Barra Bonita 496.0 a

482.0 a

67.0 109.2

Bariri Ne Ne 327.9 461.7

Promissão 280.8 360.6 104.8 109.2

Três Irmãos 439.9 401.2 103.9 107.4

Effects

Chapter VII

161

The sediments collected at Pinheiros induced coagulation at concentrations of 150 to 600

mg/ml after 48 h. In addition, some embryos lacked somites and heart function or the tail was

not detached at the highest concentration (LC50 = 600 mg/ml). In all other embryos exposed to

> 37.5 mg/ml sediments, developmental retardation of 6 to 35 hours was recorded. Some mal-

formation was recorded already at concentrations of 75 mg/ml (Fig. 7.4), and edemata were

recorded at all concentrations (EC50 = 49 mg/ml). After 96 h, all sublethal effects could still

be observed, and developmental retardation could be seen in all surviving embryos between

150 and 600 mg/ml and (at a lower frequency) at 75 mg/ml. In contrast, edemata could only

be recorded at a lower percentage at 600 and 150 mg/ml (EC50 = 55 mg/ml).

Fig. 7.4: Danio rerio embryos after 96 hours post fertilization. Fig. 7.4a: embryo exposed to

artificial water and quartz powder (negative control), showing normal development; Fig.

7.4b: embryo exposed to 3,4-dichloraniline (3.7 µg/ml, positive control), showing edemata

(arrows); Fig. 7.4c: embryo exposed to sediments from Pinheiros River (75 mg/ml), showing

malformation; 7.4d: embryo exposed to sediments from Billings reservoir (300 mg/ml) show-

ing developmental retardation.

Chapter VII

162

After both 48 and 96 h of exposure, Barra Bonita sediment samples only produced low fre-

quencies of lethal effects (coagulation, lack of somites, lack of heart function and non-

detached tail) at the two highest concentrations (LC25 = 496 and 482 mg/ml for 48 and 96 h,

respectively). After 48 h, developmental retardation and edemata were recorded at all concen-

trations (EC50 = 67 mg/ml); after 96h, these were restricted to the two highest concentrations.

With a delay to controls of up to 48 h, developmental retardation was particularly prominent

at the highest concentration; most of these embryos did not hatch.

Only after 48 and 96 h exposure to the highest concentration of Bariri sediments, embryos

showed very minor developmental retardation. Lethal effects were not recorded.

Following exposure to the highest concentration of Promissão sediments, more than half of

the eggs had coagulated. The surviving embryos failed to develop somites, their tails did not

detach, and they lacked hearts function (LC50 = 280 mg/ml). Since lethal effects were also

recorded at 300 mg/ml after 48 h an LC50 of 280 mg/ml could be calculated. All of these em-

bryos suffered from developmental retardation after 48 and 96 h exposure to 150 to 600

mg/ml and completely failed to at the two highest concentrations. After 48 h exposure, slight-

ly edemata were observed at concentrations between 18.75 and 300 mg/ml (EC50 = 105

mg/ml); after 96 h, these were restricted to 150 mg/ml.

Exposure to the Três Irmãos sediments for 48 h resulted in > 50 % coagulation of the eggs in

highest concentration. All surviving showed lack of somites, non-detachment of the tail and

lack of heart function (LC50 = 440 mg/ml). Independent of exposure time, developmental re-

tardation was observed in all survivors exposed to 150 to 600 mg/ml, and edema formation

was evident at any concentration after 48 h (EC50 = 104 mg/ml). However, most embryos re-

covered thereafter, and edemas were recorded after exposure to 300 mg/ml only. At the high-

est concentration of Três Irmãos sediments, none of the embryos hatched.

7.5 Discussion

Since this investigation was done with “native” solid-phase sediments, there was a major con-

cern about insufficient levels of oxygen due to degradation processes. In order to make sure

that toxic effects would not be caused by oxygen depletion, oxygen levels in the test plates

were controlled after 1 and 96 h. Moreover, particular attention was given to an equilibration

period of 72 h, in which the sediment-water interface was allowed to stabilize. As pointed out

Chapter VII

163

by Strecker et al. (2010), a 72 h pre-test incubation of the sediments in the wells can be used

to establish minimum levels of oxygen even above the sediment surface. In fish embryo con-

tact tests with native sediments from Lake Skadar (Montenegro), Strecker et al. (2010)

demonstrated that oxygen concentrations rapidly decreased upon addition of the sediments to

levels as low as 0.8 mg/L, but recovered to 2.5 and 3.5 mg/L after 72 and 144 h, respectively.

Apparently, during the initial mixing procedure, dissolved oxygen is rapidly used up by inor-

ganic and organic redox processes; upon stabilization of the sediment surface, however, albeit

low, but sufficient oxygen conditions are re-established with time.

Therefore, particular care was taken to saturate the medium with oxygen before use and to

guarantee sufficient access of oxygen to the wells in the test systems during the equilibration

phase of 72 h.

After 1 h incubation at 27°C, observed oxygen concentrations were generally very low. For

Promissão sediments, the medium above the sediment surface was anoxic (0.02 mg/L). Oxy-

gen concentrations of Billings and Barra Bonita were higher, but still critical for a normal

development (0.49 and 1.20 mg/L, respectively). After 96 h, oxygen concentrations had in-

creased considerably, and measurements above Promissão sediments revealed oxygen levels

50 times higher than at the beginning of the incubation (0.9 mg/L); conditions above Billings

and Barra Bonita sediments had also improved to 1.4 mg/L. Thus, after 96 h of incubation,

oxygen levels had recovered significantly. Braunbeck and Lammer (2006) stated that even at

oxygen concentrations as low as 2 mg/L, which should be expected to be lethal to adults of

most other cyprinid fish species, D. rerio embryos did not show any symptom of malfor-

mation or even growth retardation. Hollert et al. (2003) reported D. rerio embryos to survive

at oxygen levels of 0.5 mg/ml. Moreover, Barrionuevo et al. (2010), reported that D. rerio

embryos in very early stages did not respond effectively to ambient acute hypoxia, and that

only after the stage corresponding to the age of 30 days, they were able to respond to acute

hypoxia through effective physiological mechanisms involving aerobic and anaerobic metabo-

lism. Thus, based on these studies, it could be expected that oxygen levels should not have

caused adverse effects in the zebrafish embryos. In fact, in a comparison of results from fish

embryo tests performed with samples with and without pre-incubation of 72 h, embryos ex-

posed to pre-incubated sediments displayed much less effects than embryos exposed to non-

pre-incubated samples. This way, toxic effects recorded in this study for experiments with

sediments pre-incubated for 72 h can be assumed to be a consequence of exposure to sedi-

ment-bound contaminants rather than oxygen depletion.

Chapter VII

164

Based on previous studies conducted by Rocha and co-workers (2009a, 2009b) and the fact

that, e.g., the Pinheiros River and Billings reservoir are both located in the São Paulo metro-

politan region, embryo toxicity by sediments from these areas could be expected to be consid-

erable. The Pinheiros River collects all the sewage from the São Paulo Metropolitan region

and has been reported to be free of fish, due to its high pollution load in conjunction with an-

oxic conditions (CONSEMA, 1993). These high levels of water pollution directly influence

Billings reservoir, since from 1952 to 1992, the natural flow of this river had been diverted

into Billings reservoir for electricity generation, and only after 1992 permission for this was

withdrawn except for cases of flood control in São Paulo city (Silva et al., 2002).

As expected, sediments from Billings reservoir turned out to be most toxic with lethal effect

levels (LC50 values) of 200 - 210 mg/ml after 48 and 96 h of exposure, respectively. However,

in a ranking, this was not followed by Pinheiros River, but by Promissão and Três Irmãos

reservoirs (LC50 from 280 mg/ml to 440 mg/ml), which are located downstream by more than

400 and 600 km from São Paulo city, respectively. The Pinheiros River only followed on po-

sition 5 with LC50 values of 600 and 450 mg/ml after 48 and 96 h exposure.

In contrast, sediment samples from Ponte Nova (located only few kilometers from the Tietê

River spring) as well as Barra Bonita reservoir (ca. 270 km downstream São Paulo city) in-

duced low embryo mortalities (LC50 values not calculable); sediments from the reference site

(near Tietê River’s spring) and Bariri reservoir did not induce any toxic effects in zebrafish

embryos.

With respect to sublethal effects, however, Pinheiros River sediment showed to be more toxic

than the other sampling areas with EC50 (48 h) approx. 50 mg/ml. Likewise, sediments from

Barra Bonita reservoir, which induced low acute mortality, also induced sublethal effects with

EC50 values of 70 to 110 mg/ml after 48 and 96 h of exposure. Sublethal toxicities of sedi-

ments from Billings, Promissão, Três Irmãos and Ponte Nova were comparable, with EC50

values between 90 and 110 mg/ml, and only Bariri sediments proved to be less toxic even for

sub-lethal effects (EC50= 330 and 460 mg/ml for 48 and 96 h, respectively. Most importantly,

as for acute toxicity, the reference sites were free of sublethal effects.

Fracácio et al. (2003) investigated the toxicity of sediments from Barra Bonita, Bariri,

Promissão and Três Irmãos in D. rerio larvae at a concentration of 250 mg fresh sediment/ml

artificial water over 7 days from hatching and recorded mortalities of 93.3, 33.3, 43.3 and

10 %, respectively. For samples from Promissão and Três Irmãos, results of the present study

Chapter VII

165

perfectly correlate with those by Fracácio et al. (2003). In contrast, sediments from Barra

Bonita reservoir showed the highest mortality in the study by Fracácio et al. (2003), but did

not even induce 50 % mortality at the highest concentration of 600 mg/ml in the present

study. This is most likely due to different exposure scenarios: Whereas in the present study

embryos were exposed for at maximum 96 h post-fertilization, Fracácio et al. (2003) exposed

larvae for 7 days from the point of hatch. Most interestingly, however, if sublethal effects

were included in the comparison, effect levels were not only similar, but also comparable to

those for Pinheiros.

Seitz (2005) collected sediment samples from the same locations along the Danube River and

obtained EC50 values comparable to those of Pinheiros and Barra Bonita samples (45 to 70

mg/ml) as well as of Billings, Promissão and Três Irmãos (ca. 100 mg/ml). This comparison

illustrates that the embryo toxicity of sediments not only depends on the exact sampling loca-

tion, but also varies considerably with sampling time.

In previous investigations within this weight-of-evidence study, Rocha et al. (2009b) docu-

mented acute cytotoxicity (RTL-W1 cells) for almost all extracts from the Tietê River Basin,

with Billings reservoir and Pinheiros River being the most cytotoxic sites (NR50 approx. 30

mg/ml). As for embryo toxicity, the reference site at the spring showed the lowest cytotoxic

potential in the study (NR50 > 220 mg/ml). However, only few kilometers downstream spring,

in the Ponte Nova reservoir, an abrupt increase in cytotoxicity was recorded (NR50 aprox. 50

mg/ml), suggesting the existence of pollutant sources already at the very beginning of the riv-

er course. In Barra Bonita reservoir, ca. 270 km downstream São Paulo city, a strong decrease

in cytotoxicity was recorded (NR50 >170 mg/ml) followed again by an increase towards the

mouth of the river (NR50 between 40 and 60 mg/ml for Bariri, Promissão and Três Irmãos

reservoirs).

With respect to genotoxicity, sediments from the Pinheiros River and Billings (Rocha et al.,

2009a) showed a higher genotoxic potential than the other areas. The concentration-dependant

induction factors (CDI, Seitz et al. 2008) recorded for Billings sediment extracts were more

than 3 times higher than those from Ponte Nova and Promissão, almost 7 times higher than

those from Bariri and Três Irmãos, and more than 30 times higher than those from Barra

Bonita and the reference site near the Tietê River spring. On the other hand, the genotoxicity

of Pinheiros River sediments was twice as high than that Billings samples.

Chapter VII

166

In order to evaluate the mutagenicity in situ (in vivo), Rocha et al. (2009a) applied the micro-

nucleus test to erythrocytes from Oreochromis niloticus caught in these reservoirs. Fish col-

lected from Billings reservoir revealed by far the highest micronucleus frequencies with a

median of 6.0 ‰. Fish from Ponte Nova and Barra Bonita reservoirs presented frequencies of

3.5 ‰, and Bariri and Promissão reservoirs had the lowest micronucleus frequencies (1.5 and

0.5 ‰, respectively).

For the dioxin-like activity (EROD activity in RTL-W1 cells), Rocha et al. (2009b) found the

following ranking: Pinheiros River >> Bariri reservoir > Billings reservoir >> Três Irmãos

>> Barra Bonita reservoir ≈ Promissão reservoir >> Ponte Nova = reference site (spring).

Comparing all these results (Rocha et al., 2009a, 2009b) with the results obtained with the

sediment fish contact assay, sediments from Billings reservoir and Pinheiros River have to be

classified by far as the most toxic ones. Overall, Billings has to be rated more toxic than

Pinheiros River, since sediments collected at Pinheiros induced mainly sublethal effects in the

embryos, but were less effective with respect to acute toxicity. The lower acute mortality rec-

orded for native Pinheiros samples might be due to reduced bioavailability of specific com-

pounds, which could well have been made available in acetonic extracts, which were used for

the other bioassays. However, bioavailability itself is a poorly understood phenomenon

(Strmac et al., 2002), rather variable and subject to many undefined variables (biological,

physical, and chemical), and thus, difficult to quantify (Hallare et al., 2005b). Likewise, na-

tive sediment samples from Barra Bonita, Promissão and Três Irmãos reservoirs proved to be

quite toxic to the embryos, whereas organic extracts were less effective in the other bioassays.

The fact that acute toxicity data (e.g., cytotoxicity and fish embryo toxicity) for the various

locations along the Tietê River do not necessarily parallel specific effects such as

genotoxicity, mutagenicity and dioxin-like activity, corroborates the view that acute toxicity

data are insufficient to extrapolate to specific toxic mechanisms. Therefore, a thorough as-

sessment of the contamination level of natural ecosystems like a river system definitely re-

quire a battery of different endpoints. Whole sediment exposure scenarios increase ecological

relevance (Feiler et al., 2005; Hollert et al., 2003; Kosmehl et al., 2006) especially in cases

when results are to be extrapolated to field conditions in rivers and lakes (Kosmehl et al.,

2006).

Since the megacity São Paulo is situated close to the Tietê river spring, there is an impact

along the entire course of the Tietê River. The solid-phase embryo test with the whole (native)

Chapter VII

167

sediments, simulating in situ exposure conditions, confirms that surface sediments of the Tietê

River basin, especially from Billings reservoir (considering lethal and sub-lethal effects) and

Pinheiros (considering sub-lethal effects) have very high toxic hazard potentials due to the

discharges of the metropolitan area of São Paulo. However, high embryo toxicity recorded in

locations far downstream São Paulo such as Barra Bonita, Promissão and Três Irmãos indicate

the existence of additional sources of pollutants along the Tietê River course.

7.6 Conclusions

The sediment contact fish embryo test showed to be a powerful tool to detect toxic effects of

sediments not only very sensitively, but also with high ecological relevance, since it takes

bioavailability into account. Significant differences in embryo toxicity could be recorded

along the course of the Tietê River, with high embryo toxicity in samples from the vicinity of

the megacity of São Paulo (Billings and Pinheiros River), but also downstream (Barra Bonita,

Promissão and Três Irmãos). Results confirm then that most toxicity is due to the discharges

of the metropolitan area of São Paulo, but also indicate additional sources of pollutants along

the river course, probably from industrial, agricultural and agroindustrial residues, which con-

tribute to the degradation of the latter reservoirs. Especially for these, the toxicity of whole

(native) sediments was more prominent than expected from results from experiments with

sediment extracts, which usually are interpreted as “worse-case scenarios”.

Whereas embryo toxicity and cytotoxicity in permanent cell lines show parallels with respect

to acute toxicity, there is no way to extrapolate from acute toxicity to specific toxic effects

with different modes of action. Therefore, for a comprehensive estimation of sediment pollu-

tion, the use of a test battery covering not only acute toxicity, but also sub-lethal endpoints

such as those considered here, is indispensible. Moreover, since the sediment contact fish em-

bryo test provides an ecologically highly realistic and relevant exposure scenario, it should be

added in ecotoxicological sediment quality assessments.

Chapter VII

168

Final Conclusions

169

Final Conclusions

Final Conclusions

170

Final Conclusions

Zebrafish (Danio rerio) embryos offer a multitude of possibilities for the assessment of aqua-

tic ecotoxicology, general toxicity and teratogenicity. Various official test guidelines (e.g.

DIN, ISO, OECD) using fish, with special emphasis on zebrafish, are available. Within Euro-

pean chemical risk assessment, the acute fish test according to OECD TG 203 is mandatory.

Therefore, especially regarding animal welfare considerations, many efforts have been taken

to develop alternative test methods. The international OECD validation study for the zebrafish

embryo toxicity test (and further work) revealed excellent correlation of the fish embryo data

to adult fish data. Hence, chemical testing with the ZFET has been documented to be neither

better nor worse than using adult fish, independent of the respective fish species. The inter-

laboratory reproducibility of the ZFET is very high; the coefficients of variation for all chem-

icals tested in the validation study were acceptable, with most values below 30 %. Hence, the

results demonstrate that the ZFET is a highly reliable test, ready to use and to replace acute

fish testing.

Extensive studies with highly teratogenic substances proved that the ZFET is also well suited

for the assessment of teratogenesis. Exposure of zebrafish embryos to coumarin and warfarin

revealed strong teratogenesis within a concentration range relevant for human therapeutic

concentrations. For several years, zebrafish had been thought to be suited for the testing of

teratogens; however, there was concern that the effects after exposure to proteratogens would

not be reliable. Chapters II and III, however, proved that proteratogens can be reliably as-

sessed with zebrafish embryos. Bachmann (2002) had observed no significant teratogenic

effects on zebrafish embryos after exposure to thalidomide. Ten years later, Ito and co-

workers (2010, 2012) not only clearly demonstrated that zebrafish embryos are susceptible to

thalidomide as well, but also succeeded in revealing the previously unknown mode of action

of thalidomide. The malformations of the pectoral fins seen in zebrafish are clearly dose-

dependent and are definitely comparable to the limb malformations in humans. That indicates

that zebrafish embryos have also promise to be used as surrogates for the assessment of

proteratogens. In this context, a protocol for an in vitro zebrafish embryo teratogenicity test

was developed and 10 proteratogens were analyzed. The results led to the conclusion that

zebrafish embryos are able to activate proteratogens themselves; there is no need for addition

of an exogenous activation system. Zebrafish embryos are a useful alternative model for tradi-

tional teratogenicity testing with mammals with only a limited risk for false positive or false

negative results. After comparison of the EC20 values to the human therapeutic concentra-

Final Conclusions

171

tions, it is evident that teratogenic effects can already be observed at relevant concentration

close to the real situation.

As revealed in the present thesis, zebrafish embryos are suited for the assessment of very spe-

cific teratogenic endpoints such as the evaluation of skeletal malformations of the notochord

or the cartilage and bone elements in the head. The staining protocol according to Walker and

Kimmel (2007) for 6 day old larvae is fast and simple and allows the assessment of all indi-

vidual cartilages and bones. Six day old zebrafish embryos were exposed to one

dithiocarbamate and several hydrazines and hydrazides and the malformations of the single

cartilages and bones were assessed in a semi-quantitative manner. The teratogenic effects

were dose-dependent and very similar to those observed in other vertebrates as e.g. Xenopus

laevis (Schultz and Ranney, 1988). In this context more different chemicals have to be tested

and compared to higher vertebrates as chicken, mice and rat. Nevertheless, zebrafish embryos

seem to be an excellent model organism for the assessment of skeletal defects, at least as a

fast and cheap screening test before conducting tests with e.g. mice or rats.

Zebrafish embryos are not only used for the testing of chemicals, but also for the assessment

of the toxicity of waste waters and sediments. Given the occasionally high load with organics,

oxygen conditions/gradients within the water are of major importance. In Germany, the 48 h

fish embryo test has already been mandatory for the assessment within waste water treatment

since 2003 and replaced the acute fish test using the golden ide (Leuciscus idus). Zebrafish

embryos were able to adapt to a broad range of oxygen concentrations, but depending on the

sediment composition, at strong hypoxic/anoxic conditions the embryos were retarded in their

development with additional sublethal effects. Oxygen concentrations below approx. 1 mg/L

were 100 % lethal. However, oxygen conditions during the sediment contact test can be en-

hanced by placing the 24-well plates on a shaker, which mixes the water phase above the sed-

iment layer resulting in a better oxygen supply to the test embryos. This improved procedure

of the sediment contact test with zebrafish embryos was then successfully used for the testing

of complex sediment samples from the Tietê River (Brazil). An adapted sediment contact test

using zebrafish embryos proved to be a sensitive and powerful method for the detection of

toxicity in sediments and also considers bioavailability, providing an ecological highly realis-

tic exposure scenario.

Publications

172

Publications

The following chapters of this thesis are based on the following publications:

Chapter I

The final reports of the ZFET OECD validation study (No 157 and 179) are published via the

OECD homepage:

http://www.oecd.org/env/chemicalsafetyandbiosafety/testingofchemicals/seriesontestingandas

sessmentecotoxicitytesting.htm

Chapter II

Weigt, S., Huebler, N., Strecker, R., Braunbeck, T. and Broschard, T.H. (2011): Zebrafish

(Danio rerio) embryos as a model for testing proteratogens. Toxicology 281: 25-36.

Chapter III

Weigt, S., Huebler, N., Strecker, R., Braunbeck, T. & Broschard, T.H. (2011): Developmen-

tal effects of coumarin and the anticoagulant coumarin derivative warfarin on zebrafish

(Danio rerio) embryos. Reprod. Toxicol. 33(2): 133-141.

Chapter IV

Strecker, R., Weigt, S., Braunbeck, T., (2012): Cartilage and bone malformations in the head

of zebrafish (Danio rerio) embryos following exposure to disulfiram and acetic acid

hydrazide. Toxicol. Appl. Pharmacol. (accepted manuscript, January 2013)

Chapter V

Unpublished data

Publications

173

Chapter VI

Strecker, R., Seiler, T.-B., Hollert, H., Braunbeck, T. (2011): Oxygen requirements of

zebrafish (Danio rerio) embryos in embryo toxicity tests with environmental samples. Comp.

Biochem. Physiol. Part C 153: 318-327.

Chapter VII

Rocha, P., Bernecker, C., Strecker, R., Mariani, C., Pompêo, M., Storch, V., Hollert, H.,

Braunbeck, T. (2011): Sediment-contact fish embryo toxicity assay with Danio rerio to assess

particle-bound pollutants in the Tietê River Basin (São Paulo, Brazil). Ecotox. Environ. Safe.

74 (7): 1951-1959.

Publications

174

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175

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